Flavobacterium

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 409 - 16
Sejongia antarctica gen . nov., sp . nov . and Sejongia jeonii sp . nov., isolated from the Antarctic; Yi H et al.; Two yellow-pigmented, Gram-negative and aerobic bacterial strains, designated AT1013(T) and AT1047(T), were isolated from terrestrial samples of the Antarctic . On the basis of 16S rRNA gene sequence analyses, the two Antarctic strains shared 97.7 % sequence similarity and showed moderate relationships to the genera Chryseobacterium (92.5-95.3 %), Riemerella (92.3-93.5 %), Bergeyella (92.5-92.6 %) and Kaistella (92.5-93.3 %) . In phylogenetic analyses, the two isolates formed a robust monophyletic clade and represented a distinct phyletic line that equated to novel generic status . Cells were non-motile, non-gliding and psychrotolerant with an optimum growth temperature of about 20 degrees C . Flexirubins were absent . The major isoprenoid quinone was MK-6 . The predominant cellular fatty acids were 15 : 0 iso, 15 : 0 anteiso and 17 : 1 iso omega9c . DNA G+C contents were 34-36 mol% . The two isolates shared low genomic relatedness (27 %) and were differentiated from each other by several phenotypic characteristics . The polyphasic data presented in this study indicated that these isolates should be recognized as two separate novel species in a novel genus within the family Flavobacteriaceae . The name Sejongia gen . nov . is therefore proposed for the Antarctic isolates, with the type species Sejongia antarctica sp . nov . (type strain AT1013(T)=IMSNU 14040(T)=KCTC 12225(T)=JCM 12381(T)) and Sejongia jeonii sp . nov . (type strain AT1047(T)=IMSNU 14049(T)=KCTC 12226(T)=JCM 12382(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 375 - 8
Bizionia paragorgiae gen . nov., sp . nov., a novel member of the family Flavobacteriaceae isolated from the soft coral Paragorgia arborea; Nedashkovskaya OI et al.; A novel marine bacterium isolated from the soft coral Paragorgia arborea in the Sea of Okhotsk was studied by using a polyphasic taxonomic approach . The strain, KMM 6029(T), was strictly aerobic, heterotrophic, yellow-pigmented, non-motile by gliding, Gram-negative and oxidase-, catalase- and alkaline phosphatase-positive . From results of 16S rRNA gene sequence analysis, strain KMM 6029(T) occupied a distinct lineage within the family Flavobacteriaceae and showed 95.5 % similarity to its closest relative, Formosa algae . The DNA G+C content was 37.6 mol% . Major respiratory quinone was MK-6 . The predominant fatty acids were i15 : 0, a15 : 0, i15 : 1, a15 : 1, i16 : 1, i16 : 0, i16 : 0 3-OH and summed feature 3 (i15 : 0 2-OH and/or 16 : 1omega7c) . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics the novel bacterium has been assigned to Bizionia gen . nov., as Bizionia paragorgiae gen . nov., sp . nov . The type strain is KMM 6029(T) (=KCTC 12304(T)=LMG 22571(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 321 - 3
Gillisia mitskevichiae sp . nov., a novel bacterium of the family Flavobacteriaceae, isolated from sea water; Nedashkovskaya OI et al.; The taxonomic position of a novel marine, heterotrophic, aerobic, pigmented bacterium, non-motile by gliding, that was isolated from a sea-water sample collected in the Sea of Japan, was determined . 16S rRNA gene sequence analysis revealed that strain KMM 6034(T) is a member of the genus Gillisia . The phenotypic and chemotaxonomic data showed that the isolate represents a novel species of the genus Gillisia, for which the name Gillisia mitskevichiae sp . nov . is proposed . The type strain is KMM 6034(T) (=KCTC 12261(T)=NBRC 100590(T)=LMG 22575(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 225 - 9
Description of Aquimarina muelleri gen . nov., sp . nov., and proposal of the reclassification of {Cytophaga} latercula Lewin 1969 as Stanierella latercula gen . nov., comb . nov; Nedashkovskaya OI et al.; The taxonomic position of three novel sea-water isolates was determined . The strains studied were strictly aerobic, heterotrophic, pigmented, motile by gliding, Gram-negative and oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive . 16S rRNA gene sequence phylogenetic analysis indicated that the strains KMM 6020(T), KMM 6021 and KMM 6028 occupied a distinct lineage within the family Flavobacteriaceae . The major respiratory quinone was MK-6 . The predominant fatty acids were i15 : 0, i15 : 1, i15 : 0 3-OH, i17 : 1omega9c and i17 : 0 3-OH . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacteria were assigned to the genus Aquimarina gen . nov., as Aquimarina muelleri gen . nov., sp . nov . The type strain is KMM 6020(T) (=KCTC 12285(T)=LMG 22569(T)) . From the results of the 16S rRNA gene sequence analysis and phenotypic features, the species {Cytophaga} latercula Lewin 1969 is proposed to be reclassified in the new genus Stanierella as Stanierella latercula gen . nov., comb . nov., with type strain CIP 104806(T) (=ATCC 23177(T)=NCIMB 1399(T)=LMG 1343(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 177 - 81
Pibocella ponti gen . nov., sp . nov., a novel marine bacterium of the family Flavobacteriaceae isolated from the green alga Acrosiphonia sonderi; Nedashkovskaya OI et al.; A marine, heterotrophic, Gram-negative, aerobic, yellow-pigmented, bacterium that was motile by gliding, isolated from the green alga Acrosiphonia sonderi, was studied by polyphasic taxonomic methods . 16S rRNA gene sequence analysis indicated that strain KMM 6031(T) formed a distinct lineage within the family Flavobacteriaceae . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic analyses, the novel bacterium was classified as Pibocella ponti gen . nov., sp . nov . The type strain is KMM 6031(T) (=KCTC 12262(T)=NBRC 100591(T)=LMG 22573(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 49 - 55
Winogradskyella thalassocola gen . nov., sp . nov., Winogradskyella epiphytica sp . nov . and Winogradskyella eximia sp . nov., marine bacteria of the family Flavobacteriaceae; Nedashkovskaya OI et al.; Three novel heterotrophic, Gram-negative, yellow-pigmented, aerobic, gliding, oxidase- and catalase-positive bacteria were isolated from algae collected in the Gulf of Peter the Great, Sea of Japan . 16S rRNA gene sequence analysis revealed that the strains studied represented members of the family Flavobacteriaceae and showed 93.5-93.8 % similarity with their closest relative, Psychroserpens burtonensis . The DNA G+C content of the strains was 34-37 mol% . The major respiratory quinone was MK-6 . The predominant fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(15 : 1), iso-C(16 : 0)-3OH and iso-C(17 : 0)-3OH . On the basis of their phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the newly described bacteria have been assigned to the new genus Winogradskyella gen . nov., as Winogradskyella thalassocola sp . nov . (type strain, KMM 3907(T)=KCTC 12221(T)=LMG 22492(T)=DSM 15363(T)), Winogradskyella epiphytica sp . nov . (type strain, KMM 3906(T)=KCTC 12220(T)=LMG 22491(T)=CCUG 47091(T)) and Winogradskyella eximia sp . nov . (type strain, KMM 3944(T) (=KCTC 12219(T)=LMG 22474(T)).

Lett Appl Microbiol, 2005, 40(2), 123 - 7
Production, characterization and evaluation of virulence of an adhesion defective mutant of Flavobacterium columnare produced by beta-lactam selection; Bader JA et al.; Abstract j.a . bader, c.a . shoemaker and p.h . klesius . 2004.Aims: The aim of this study was to develop and evaluate mutants of Flavobacterium columnare, the causative agent of columnaris disease in fish . Methods and Results: Serial passage on ampicillin (beta-lactam) enriched modified Hsu-Shotts medium resulted in a F . columnare mutant that differed from the parent strain in colony morphology, whole cell proteins, adhesion and virulence . The mutant differed from its parent in virulence during immersion challenge, but not during injection challenge or generation of antibodies . Conclusion: Flavobacterium columnare exposure to ampicillin produces both resistance to that antibiotic and produces mutants that lack or have reduced adhesion characteristics and modified ability to adhere to fish tissue . Significance and Impact of the Study: This is the first description of an adhesion-defective mutant of F . columnare and the effects of altered adhesion on columnaris disease . This mutant has considerable potential as a tool to study the role of adhesion in columnaris disease.

J Biochem Mol Biol, 2004 Nov 30, 37(6), 684 - 90
Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15; Kim WS et al.; Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography . This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate . The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF . The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C . It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C . The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol . The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg . The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate . It was inactive in the cleavage of N-desulfated heparin and acharan sulfate . In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.

J Ind Microbiol Biotechnol . 2004 Dec 11; {Epub ahead of print}
beta-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea; Bhosale P et al.; Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3' R-beta, beta-carotene-3, 3' diol) as its main carotenoid . The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of beta-carotene (beta, beta-carotene), beta-cryptoxanthin (beta, beta-caroten-3-ol), and zeaxanthin produced by F . multivorum was investigated . Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold . Urea and sodium carbonate had an unexpectedly strong positive effect on beta-carotene production at the expense of zeaxanthin formation . The effect was found to be independent of incubation time, and beta-carotene represented 70% (w/w) of the total carotenoid content . The cumulative effect of urea and sodium carbonate was further studied using response surface methodology . An optimum medium was found to contain 4,000 and 4,070 mg l(-1) urea and sodium carbonate, respectively . The maximum beta-carotene level was 7.85 mug ml(-1) culture broth, which represented 80% (w/w) of the total carotenoid produced . Optimization resulted in 77- and 88-fold improvements in the volumetric and specific beta-carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium . The carotenoid production profile in the optimized medium indicated that beta-carotene was produced maximally during the late exponential phase at 0.41 mug ml(-1) h(-1) . It is possible that this organism could be an excellent commercial source of either beta-carotene or zeaxanthin, depending on initial culture conditions.

J Fish Dis, 2004 Dec, 27(12), 719 - 29
Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G4; Xie HX et al.; In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs . Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods . Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper . The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa) . Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH approximately 32 aa approximately E motif of the F . columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F . columnare G(4) represents a new family of zincins . The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa . Sequence homology analysis revealed that the Pop is also novel as it has < 50% identity with other reported prolyl oligopeptidase family proteins . The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F . columnare.

Microb Ecol, 2004 Aug, 48(2), 200 - 8 Epub 2004 May 13.
Biological soil crusts of sand dunes in Cape Cod National Seashore, Massachusetts, USA; Smith SM et al.; Biological soil crusts cover hundreds of hectares of sand dunes at the northern tip of Cape Cod National Seashore (Massachusetts, USA) . Although the presence of crusts in this habitat has long been recognized, neither the organisms nor their ecological roles have been described . In this study, we report on the microbial community composition of crusts from this region and describe several of their physical and chemical attributes that bear on their environmental role . Microscopic and molecular analyses revealed that eukaryotic green algae belonging to the genera Klebsormidium or Geminella formed the bulk of the material sampled . Phylogenetic reconstruction of partial 16S rDNA sequences obtained from denaturing gradient gel electrophoresis (DGGE) fingerprints also revealed the presence of bacterial populations related to the subclass of the Proteobacteria, the newly described phylum Geothrix/ Holophaga/ Acidobacterium, the Cytophaga/ Flavobacterium/ Bacteroides group, and spirochetes . The presence of these crusts had significant effects on the hydric properties and nutrient status of the natural substrate . Although biological soil crusts are known to occur in dune environments around the world, this study enhances our knowledge of their geographic distribution and suggests a potential ecological role for crust communities in this landscape.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2335 - 41
Aquiflexum balticum gen . nov., sp . nov., a novel marine bacterium of the Cytophaga-Flavobacterium-Bacteroides group isolated from surface water of the central Baltic Sea; Brettar I et al.; A bacterial isolate from the Baltic Sea, BA160(T), was characterized for its physiological and biochemical features, fatty acid profile, G+C content and phylogenetic position based on 16S rRNA gene sequences . The strain was isolated from the surface water of the central Baltic Sea during the decay of a plankton bloom . Phylogenetic analyses of the 16S rRNA gene sequence revealed a clear affiliation with the family 'Flexibacteraceae', and showed the closest phylogenetic relationship with the species Belliella baltica and Cyclobacterium marinum . The G+C content of the DNA was 38.4 mol% . The strain was red-coloured due to carotenoids, Gram-negative, rod-shaped, and catalase- and oxidase-positive . Growth was observed at salinities from 0 to 6 %, with an optimum around 1.5 % . Temperature for growth ranged from 4 to 40 degrees C, with an optimum around 30 degrees C . The fatty acids were dominated by branched-chain fatty acids (>87 %), with a high abundance of iso-C(15 : 0) (23 %) and anteiso-C(15 : 0) (19 %) . According to its morphology, physiology, fatty acid composition, G+C content and 16S rRNA gene sequence, strain BA160(T) is considered to represent a new genus of the family 'Flexibacteraceae' . Due to its aquatic origin, the name Aquiflexum balticum gen . nov, sp . nov . is suggested for the type species (type strain, BA160(T)=DSM 16537(T)=LMG 22565(T)=CIP 108445(T)) of the new genus.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2319 - 24
Kaistella koreensis gen . nov., sp . nov., a novel member of the Chryseobacterium-Bergeyella-Riemerella branch; Kim MK et al.; Gram-negative, non-spore-forming, rod-shaped, yellow-pigmented bacteria isolated from a freshwater stream in Korea were investigated to determine their taxonomic position . Complete 16S rRNA gene sequence analysis indicated that the organisms should be placed in the Chryseobacterium-Bergeyella-Riemerella branch in the family Flavobacteriaceae . Phylogenetically, the strains were most closely related to Chryseobacterium balustinum ATCC 33487(T) and Chryseobacterium scophthalmum LMG 13028(T) (94.3 and 94.1 % 16S rRNA gene sequence similarity, respectively) and they clustered on a separate well-supported branch . The strains contained menaquinone MK-6 as the predominant respiratory quinone and showed higher G+C contents (41.7 mol%) than other species in the Chryseobacterium-Bergeyella-Riemerella branch and i-C(15 : 0) as a major fatty acid (47-52 %) . The phylogenetic distances from any species with validly published names and their phenotypic properties confirmed that the strains constitute a separate species in a new genus, for which the name Kaistella koreensis gen . nov., sp . nov . is proposed (type strain Chj707(T)=KCTC 12107(T)=IAM 15050(T)).

Appl Environ Microbiol, 2004 Nov, 70(11), 6753 - 66
Changes in bacterioplankton composition under different phytoplankton regimens; Pinhassi J et al.; The results of empirical studies have revealed links between phytoplankton and bacterioplankton, such as the frequent correlation between chlorophyll a and bulk bacterial abundance and production . Nevertheless, little is known about possible links at the level of specific taxonomic groups . To investigate this issue, seawater microcosm experiments were performed in the northwestern Mediterranean Sea . Turbulence was used as a noninvasive means to induce phytoplankton blooms dominated by different algae . Microcosms exposed to turbulence became dominated by diatoms, while small phytoflagellates gained importance under still conditions . Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments showed that changes in phytoplankton community composition were followed by shifts in bacterioplankton community composition, both as changes in the presence or absence of distinct bacterial phylotypes and as differences in the relative abundance of ubiquitous phylotypes . Sequencing of DGGE bands showed that four Roseobacter phylotypes were present in all microcosms . The microcosms with a higher proportion of phytoflagellates were characterized by four phylotypes of the Bacteroidetes phylum: two affiliated with the family Cryomorphaceae and two with the family Flavobacteriaceae . Two other Flavobacteriaceae phylotypes were characteristic of the diatom-dominated microcosms, together with one Alphaproteobacteria phylotype (Roseobacter) and one Gammaproteobacteria phylotype (Methylophaga) . Phylogenetic analyses of published Bacteroidetes 16S rRNA gene sequences confirmed that members of the Flavobacteriaceae are remarkably responsive to phytoplankton blooms, indicating these bacteria could be particularly important in the processing of organic matter during such events . Our data suggest that quantitative and qualitative differences in phytoplankton species composition may lead to pronounced differences in bacterioplankton species composition.

Biodegradation, 2004 Oct, 15(5), 337 - 46
Biotransformation and dissolution of petroleum hydrocarbons in natural flowing seawater at low temperature; Brakstad OG et al.; The objective of this study was to establish methods for controlled studies of hydrocarbon depletion from thin oil films in cold natural seawater, and to determine biotransformation in relation to other essential depletion processes . Mineral oil was immobilized on the surface of hydrophobic Fluortex fabrics and used for studies of microbial biodegradation in an experimental seawater flow-through system at low temperatures (5.9-7.4 degrees C) during a test period of 42 days . The seawater was collected from a depth of 90 m, and microbial characterization by epifluorescence microscopy, fluorescence in situ hybridization, and most-probable number analysis showed relatively larger fractions of archaea and oil-degrading microbes than in the corresponding surface water . Chemical analysis of hydrocarbons attached to the fabrics during the test period showed that n-alkanes (C10-C36) were decreased by 98% after 21 days, while naphthalenes were depleted by 99-100% during the same period . At the end of the period 4-5 ring polyaromatic hydrocarbon (PAH) compounds were removed by 82% from the fabrics . Analysis of the recalcitrant pentacyclic triterpane C30 17alpha(H),21beta(H)-hopane showed that the oil remained adsorbed to the fabrics during the test period . Comparison of depletion analysis with calculation of hydrocarbon dissolution in a flow-through system indicated that naphthalenes and smaller PAH compounds (alkylated 2-ring and 3-ring compounds) were removed from the fabrics by dissolution . The data further implied that depletion of n-alkanes and 4-5 ring PAH hydrocarbons were the result of biotransformation processes . PCR amplification of bacterial 16S rRNA genes from microbes adhering on the immobilized oil surfaces showed the dominance of a few bands when analysed in denaturing gradient gel electrophoresis (DGGE) . Sequence analysis of DGGE bands revealed phylogenetic affiliation to the alpha- and gamma-subdivisions of proteobacteria and to the Chloroflexus-Flavobacterium-Bacteroides group.

Mol Cell Probes, 2004 Dec, 18(6), 421 - 7
Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae; Darwish AM et al.; Species-specific polymerase chain reaction (PCR) primers have been designed to identify the causative agent of columnaris disease, Flavobacterium columnare . The 16S rRNA gene sequences of F . columnare (eight sequences representing the different genotypes of the species) and related species (18 sequences) were aligned and compared to choose specific regions that are unique to F . columnare and do not have significant intraspecies variability . The species-specific regions in the 16S rRNA gene were used to design a pair of species-specific PCR primers, ColF and ColR . The PCR technique produced a specific amplicon of about 675 base pairs (bp) in 27 isolates of F . columnare and there was no amplification in the closely related species . The specificity of the amplified product was confirmed by digesting with HhaI . The PCR primers did not produce a 675 bp product with F . columnare ATCC43622 strain . This ATCC43622 strain was characterized by biochemical and ribotyping methods and renamed Flavobacterium johnsoniae . The American Type Culture Collection has confirmed these findings and made the change.

J Fish Dis, 2004 Oct, 27(10), 573 - 81
Systemic and mucosal antibody response in tilapia, Oreochromis niloticus (L.), following immunization with Flavobacterium columnare; Grabowski LD et al.; Specific antibody responses to Flavobacterium columnare (isolate ATCC 23463T) were characterized in plasma and mucus of tilapia following intraperitoneal (i.p.) injection or immersion immunization with formalin-killed sonicated or whole cell preparations . Fish (30 per treatment) received a primary immunization and were booster immunized 4 weeks later . An enzyme-linked immunosorbent assay was developed for detection and quantification of specific anti-F . columnare antibody, and it was found that formalin-killed sonicated cells in Freund's complete adjuvant (FCA) injected i.p . stimulated a significant systemic antibody response within 2 weeks (mean titre 11,200) which increased to 30,600 following secondary immunization . At 10 weeks post-immunization, the mean titre remained significantly elevated above the controls . Antibodies were also observed in cutaneous mucus of fish immunized i.p . with formalin-killed sonicated cells in FCA at 6 and 8 weeks post-immunization (mean titres 67 and 33, respectively) . Although some individual fish responded, mean plasma and cutaneous mucus antibody titres were not significantly greater than controls in any of the other treatment groups . The results of this study demonstrate that tilapia can mount a significant humoral response in plasma and cutaneous mucus to F . columnare, but i.p . immunization with FCA is required to elicit this response.

Appl Environ Microbiol, 2004 Oct, 70(10), 6210 - 9
Flow sorting of marine bacterioplankton after fluorescence in situ hybridization; Sekar R et al.; We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH) . In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338) . Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a) . A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry . The purity of sorted cells was >95% . 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library) . Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%) . No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library . These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes . This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1845 - 8
Hongiella marincola sp . nov., isolated from sea water of the East Sea in Korea; Yoon JH et al.; Two Gram-negative, non-motile, non-spore-forming, rod-shaped strains, SW-2T and SW-26, were isolated from sea water of the East Sea in Korea . These organisms grew optimally at 37 degrees C and in the presence of 2-3 % (w/v) NaCl . They did not grow without NaCl or in the presence of >9 % (w/v) NaCl . Strains SW-2T and SW-26 were characterized chemotaxonomically as having MK-7 as the predominant isoprenoid quinone and iso-C(15 : 0) as the major fatty acid . The DNA G + C content of strains SW-2T and SW-26 was 43 mol% . A neighbour-joining tree based on 16S rRNA gene sequences showed that strains SW-2T and SW-26 fell within the Cytophaga-Flavobacterium-Bacteroides group and formed a coherent cluster with Hongiella species . Strains SW-2T and SW-26 showed a 16S rRNA gene sequence similarity value of 99.9 % and a mean DNA-DNA relatedness level of 87 % to each other . Levels of 16S rRNA gene sequence similarity between strains SW-2T and SW-26 and the type strains of two Hongiella species ranged from 94.2 to 96.6 % . On the basis of phenotypic and chemotaxonomic properties and phylogenetic distinctiveness, strains SW-2T and SW-26 should be placed in the genus Hongiella as members of a novel species, for which the name Hongiella marincola sp . nov . is proposed . The type strain is SW-2T (= KCTC 12180T = DSM 16067T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1643 - 8
Zobellia amurskyensis sp . nov., Zobellia laminariae sp . nov . and Zobellia russellii sp . nov., novel marine bacteria of the family Flavobacteriaceae; Nedashkovskaya OI et al.; The taxonomic position of four newly isolated marine, heterotrophic, gliding, Gram-negative, aerobic, pigmented, agarolytic bacteria was established . 16S rRNA gene sequence analysis indicated affiliation of the isolates to the genus Zobellia in the family Flavobacteriaceae . DNA-DNA hybridization experiments revealed that the strains studied represent three distinct and novel species, for which the names Zobellia amurskyensis sp . nov., Zobellia laminariae sp . nov . and Zobellia russellii sp . nov . are proposed, with KMM 3526T (= LMG 22069T = CCUG 47080T), KMM 3676T (= LMG 22070T = CCUG 47083T) and KMM 3677T (= LMG 22071T = CCUG 47084T), respectively, as the type strains.

Dis Aquat Organ, 2004 Jul 5, 60(1), 31 - 9
Impact of PCB on resistance to Flavobacterium psychrophilum after experimental infection of rainbow trout Oncorhynchus mykiss eggs by nanoinjection; Ekman E et al.; The effects of sublethal exposure of a commercial blend of polychlorinated biphenyls (PCB), i.e . Clophen A50, on disease resistance to the aetiological agent of rainbow trout fry syndrome, Flavobacterium psychrophilum, were investigated . Newly fertilised rainbow trout Oncorhynchus mykiss eggs were nanoinjected with 2 doses of Clophen A50 (0.4 or 2 microg egg(-1)) and/or 100 colony forming units of F . psychrophilum . The mean cumulative mortality in control groups, and groups exposed to the lower dose of Clophen A50 (0.4 microg egg(-1)) was below 5.0% . The mean cumulative mortality in groups exposed to the higher dose of Clophen A50 (2.0 microg egg(-1)) was 5.8%, which was not significantly different from the control groups . In all groups infected with F . psychrophilum, with or without exposure to Clophen A50, significantly higher cumulative mortalities compared with control groups were recorded . No differences in mortality were recorded between groups exposed to bacteria alone or bacteria in combination with the higher dose of Clophen A50 (21.6 and 20.4%, respectively) . Decreased disease resistance was recorded in groups exposed to F . psychrophilum and the lower dose of Clophen A50, with a mean cumulative mortality of 56.0% . These results could be due to non dose-dependent effects on the immune system, or toxic effects of PCB or their metabolites on the bacteria in groups exposed to the higher dose of Clophen A50 . The present study indicates that maternal transfer of PCB might affect disease resistance to vertically transmitted F . psychrophilum.

Arch Microbiol, 2004 Oct, 182(2-3), 244 - 53 Epub 2004 Aug 31.
Alkaliflexus imshenetskii gen . nov . sp . nov., a new alkaliphilic gliding carbohydrate-fermenting bacterium with propionate formation from a soda lake; Zhilina TN et al.; Anaerobic saccharolytic bacteria thriving at high pH values were studied in a cellulose-degrading enrichment culture originating from the alkaline lake, Verkhneye Beloye (Central Asia) . In situ hybridization of the enrichment culture with 16S rRNA-targeted probes revealed that abundant, long, thin, rod-shaped cells were related to Cytophaga . Bacteria of this type were isolated with cellobiose and five isolates were characterized . Isolates were thin, flexible, gliding rods . They formed a spherical cyst-like structure at one cell end during the late growth phase . The pH range for growth was 7.5-10.2, with an optimum around pH 8.5 . Cultures produced a pinkish pigment tentatively identified as a carotenoid . Isolates did not degrade cellulose, indicating that they utilized soluble products formed by so far uncultured hydrolytic cellulose degraders . Besides cellobiose, the isolates utilized other carbohydrates, including xylose, maltose, xylan, starch, and pectin . The main organic fermentation products were propionate, acetate, and succinate . Oxygen, which was not used as electron acceptor, impaired growth . A representative isolate, strain Z-7010, with Marinilabilia salmonicolor as the closest relative, is described as a new genus and species, Alkaliflexus imshenetskii . This is the first cultivated alkaliphilic anaerobic member of the Cytophaga/ Flavobacterium/ Bacteroides phylum.

Acta Crystallogr D Biol Crystallogr, 2004 Sep, 60(Pt 9), 1644 - 6 Epub 2004 Aug 26.
Crystallization and preliminary X-ray analysis of heparinase II from Pedobacter heparinus; Shaya D et al.; Heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), which acts on both heparin and heparan sulfate, is one of several glycosaminoglycan-degrading enzymes produced by this organism . This enzyme, with a molecular weight of 84 kDa, utilizes a lytic mechanism to cleave the alpha(1-4) glycosidic bond between hexosamine (D-glucosamine) and L-iduronic or D-glucuronic acid, resulting in a product with an unsaturated sugar ring at the non-reducing end . The enzyme was crystallized by the hanging-drop vapour-diffusion method . The crystals belong to orthorhombic space group P2(1)2(1)2(1) and diffract to 2 A resolution . There are two molecules in the asymmetric unit, consistent with the finding that recombinant heparinase II functions as a dimer in solution.

Parassitologia, 2004 Jun, 46(1-2), 19 - 24
{Ultrastructural basis of interactions between prokaryotes and eukaryotes in different symbiotic models}; Sacchi L; This paper reviews the Author's contribution to the knowledge of the ultrastructural basis of the prokaryote-eukaryote interactions in different models assessed by an ultrastructural approach . In agreement with the hypothesis of the origin of eukaryotic cells, which are chimeras of several prokaryotes with different morpho-functional specializations, symbiosis had major consequence for evolution of life . In Arthropods, one of the most successful lifestyles, the presence of endosymbiotic prokaryotes, plays an important role in their metabolism . In some cases, genome integration has occurred in the endosymbiotic relationships with the host, proving that intracellular symbiosis is not merely a nutritional supplement . Intracellular symbiotic bacteria are also described in nematodes . In particular, the presence of intracellular Wolbachia in filariae, even if its function is not yet completely known, influences positively the reproductive biology and the survival of the host, as proved by antibiotic treatment against this bacterium . The ultrastructural images reported in this review were obtained using different species of cockroaches, termites, ticks and filarial nematodes . The traditional methods of transmission (TEM), scansion (SEM) and immuno electron microscopy were used . In addition, also freeze-fracture and deep-etching techniques were employed . The cockroaches and the primitive termite Mastotermes darwiniensis host symbiotic bacteria in the ovary and in specialized cells (bacteriocytes) of the fat body . These bacteria have the typical cell boundary profile of gram-negative bacteria and are enveloped in a vacuolar membrane produced by the host cell . Molecular sequence data of 16S rDNA of endosymbionts of five species of cockroaches and M . darwiniensis indicate that they are members of the Flavobacteria-bacteroides group and that the infection occurred in an ancestor common to cockroaches and termites probably after the end of the Paleozoic (250 Ma BP) . The symbiotic bacteria are transmitted transovarially and, during embryogenesis, they are integrated into the morphogenetic processes . In particular, we were able to demonstrate that the origin of the bacteriocyte should be looked for in the cells of the haemocyte line (embryonic plasmatocytes) . The eggs are infected by the bacteria emerging from the bacteriocytes of the ovaric fat body and, at the end of the vitellogenesis, they are actively phagocytized by the egg membrane . In filarial nematodes, intracellular bacteria belonging to the genus Wolbachia have been described: they have evolved an obligatory mutualistic association with their host . In fact, antibiotic treatments lead to the clearance of bacteria and this loss produces a negative impact on reproduction and survival of the filarial host . We evidenced, by TEM, the degenerative events occurring during the embriogenesis of Brugia pahangi and Dirofilaria immitis after tetracycline treatment . The data suggest that the Wolbachia play a direct role in worm metabolism . Finally, a new additional model of the prokaryote-eukaryote interaction has been described: we have recently discovered a new intracellular alpha-proteobacterium, named Iric ES1, which resides in the ovarian tissues of the tick Ixodes ricinus . The intriguing characteristic of this bacterium is its ability to invade and consume the ovaric mitochondria . From an evolutionary perspective, it is interesting to note that Iric ES1 enters mitochondria in a similar way to that employed by the "predatory" bacterium Bdellovibrio bacteriovorus.

Acta Crystallogr D Biol Crystallogr, 1994 Jul, 50(Pt 4), 380 - 4
Enzymatic deglycosylation as a tool for crystallization of mamalian binding proteins; Baker HM; Enzymatic deglycosylation has been used in attempts to crystallize several glycoproteins with the aim of overcoming the problems resulting from heterogeneity and flexibility of the attached glycan chains . An endoglycosidase preparation from Flavobacterium meningosepticum, comprising the enzymes endo F and PNGase-F, was used in experiments on the mammalian binding proteins lactoferrin and haemopexin . Significant differences were found in the susceptibility of different proteins to deglycosylation . For human lactoferrin (Lf) and its recombinant N-terminal half-molecule (Lf(N)), deglycosylation was rapid and complete, and was essential for obtaining high-quality crystals of both apo-Lf and Lf(N); for bovine Lf, however, complete deglycosylation did not occur . Similarly, for rabbit haemopexin the carbohydrate chain on the C-terminal domain was easily removed, but the three chains on the N-terminal domain proved more resistant and their removal led to some fragmentation of the protein . Nevertheless, this approach provided the only means of crystallizing the C-terminal domain and is likely to be useful for other glycoproteins.

Appl Environ Microbiol, 2004 Aug, 70(8), 4648 - 57
Use of microautoradiography combined with fluorescence in situ hybridization to determine dimethylsulfoniopropionate incorporation by marine bacterioplankton taxa; Vila M et al.; The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU) . In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from {(35)S}DMSP ranged between 27 and 51% of 4',6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating {(3)H}leucine . In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from {(35)S}DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom . At the same time, the proportion of cells incorporating {(3)H}leucine increased from 21 to 40% . The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (alpha-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating {(35)S}DMSP at both sampling sites . Significant uptake of sulfur from DMSP was also found among members of the gamma-proteobacteria and Cytophaga-Flavobacterium groups . Roseobacter and gamma-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating (35)S from DMSP (around 50%) . Altogether, the application of AU with {(35)S}DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization . These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.

J Fish Dis, 2004 Aug, 27(8), 483 - 92
Salmonid gill bacteria and their relationship to amoebic gill disease; Bowman JP et al.; 16S ribosomal RNA gene analysis was used to assess the bacterial community associated with Atlantic salmon, Salmo salar L., gills which were either affected by amoebic gill disease (AGD) or were AGD-negative, in order to determine the role that bacteria may play in the development of AGD . AGD-positive specimens were either infected in the laboratory with Neoparamoeba pemaquidensis, the causative agent of AGD, or were obtained from commercial salmon cages . Samples from laboratory fish maintained in sea water possessed a marine-type community while field samples which had been treated by a series of freshwater baths possessed a more diverse community which included variable proportions of different bacterial ecotypes, including groups typically associated with soil, skin surfaces and faeces . Samples from fish infected with AGD in the laboratory and a sample from one of two salmon cage fish specimens were dominated by a phylotype belonging to the strictly marine bacterial genus Psychroserpens (family Flavobacteriaceae, phylum Bacteroidetes) . The phylotype was not detected in any of the AGD-negative samples or in one of two AGD-positive samples obtained from fish subjected to temporary freshwater immersion . The possibility of certain Psychroserpens species as potential opportunistic pathogens associated with salmonid AGD is proposed.

Trends Biochem Sci, 2004 Aug, 29(8), 396 - 9
GIFT domains: linking eukaryotic intraflagellar transport and glycosylation to bacterial gliding; Beatson S et al.; We describe GIFT {for GldG, intraflagellar transport (IFT)} domains in the flavobacterial gliding protein GldG and eukaryotic IFT-52 . In eukaryotes, domain homologues are also found in the eukaryotic oligosaccharyltransferase complex and in subtilisin kexin isozyme-1 (SKI-1 or S1P) . A distant evolutionary relationship to periplasmic-binding proteins hints that GIFT domains might possess oligosaccharide-binding functions.

J Appl Microbiol, 2004, 97(3), 574 - 80
Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum; Massias B et al.; AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues . METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl . psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions . Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass . The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl . psychrophilum tested here . CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e . S-layer), one of the most common surface structures on bacteria . SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface .

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1257 - 61
Algibacter lectus gen . nov., sp . nov., a novel member of the family Flavobacteriaceae isolated from green algae; Nedashkovskaya OI et al.; Three strains of the marine, gliding, pigmented, facultatively anaerobic, heterotrophic, Gram-negative bacteria were isolated from the green algae Acrosiphonia sonderi (Kutz) Kornm and Ulva fenestrata Ruprecht inhabiting the Sea of Japan . 16S rDNA sequence analysis indicated that the strains were members of the family Flavobacteriaceae, in which they occupied separate lineages . The predominant cellular fatty acids were i15 : 0, a15 : 0, i15 : 1, 15 : 0, 15 : 1omega6c, i15 : 0 3-OH and i17 : 0 3-OH . The DNA base compositions were 31-33 mol% G+C . Based on the phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, the novel bacteria should be placed in a novel taxon as Algibacter lectus gen . nov., sp . nov . with type strain KMM 3902G (=KCTC 12103T=DSM 15365T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1101 - 6
Robiginitalea biformata gen . nov., sp . nov., a novel marine bacterium in the family Flavobacteriaceae with a higher G+C content; Cho JC et al.; Two Gram-negative, chemoheterotrophic, non-motile, rust-coloured, marine strains were isolated from the western Sargasso Sea by high-throughput culturing . Characterization of the two strains by polyphasic approaches indicated that they are members of the same species . Phylogenetic analyses based on 16S rRNA gene sequences using three treeing algorithms revealed that the strains formed a coherent and novel genus-level lineage within the family Flavobacteriaceae . The dominant fatty acids were branched or hydroxy acids, i15 : 0, i15 : 1 and 3-OH i17 : 0 being the most abundant . The higher DNA G+C content of the strains (55-56 mol%) clearly differentiated them from other genera of the family Flavobacteriaceae (27-44 mol%) . It is proposed, from the polyphasic evidence, that the strains be placed into a novel genus and a novel species named Robiginitalea biformata gen . nov., sp . nov., with strain HTCC2501T (=ATCC BAA-864T=KCTC 12146T) as the type strain.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1017 - 23
Maribacter gen . nov., a new member of the family Flavobacteriaceae, isolated from marine habitats, containing the species Maribacter sedimenticola sp . nov., Maribacter aquivivus sp . nov., Maribacter orientalis sp . nov . and Maribacter ulvicola sp . nov; Nedashkovskaya OI et al.; Six novel gliding, heterotrophic, Gram-negative, yellow-pigmented, aerobic, oxidase- and catalase-positive bacteria were isolated from the green alga Ulva fenestrata, sea water and a bottom sediment sample collected in the Gulf of Peter the Great, Sea of Japan . 16S rRNA gene sequence analysis revealed that the strains studied were members of the family Flavobacteriaceae . On the basis of their phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacteria have been assigned to the new genus Maribacter gen . nov., as Maribacter sedimenticola sp . nov., Maribacter orientalis sp . nov., Maribacter aquivivus sp . nov . and Maribacter ulvicola sp . nov., with the type strains KMM 3903T (=KCTC 12966T=CCUG 47098T), KMM 3947T (=KCTC 12967T=CCUG 48008T), KMM 3949T (=KCTC 12968T=CCUG 48009T) and KMM 3951T (=KCTC 12969T=DSM 15366T), respectively.

Proc R Soc Lond B Biol Sci, 2004 May 7, 271 Suppl 4, S193 - 5
Increased fecundity associated with infection by a cytophaga-like intracellular bacterium in the predatory mite, Metaseiulus occidentalis; Weeks AR et al.; The endosymbiont Wolbachia has gained widespread notoriety over the past decade because of its high infection frequency among arthropods, and the unique heterogeneity of the host reproductive effects that it has been implicated as causing to enhance its own spread . Recently, another endosymbiotic bacterium from the Cytophaga-Flavobacterium-Bacteroides phylum has been shown to be widespread among arthropods and manipulate its hosts' reproduction to enhance its own spread . We show that infection by this Cytophaga-like organism (CLO) in the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae) is associated with a significant increase in the fecundity of infected females . This adds to the growing list of phenotypes that the CLO can induce in its hosts, which now include feminization, parthenogenesis induction, cytoplasmic incompatibility and fecundity enhancement, rivalling Wolbachia for overall diversity of host reproductive manipulations.

Biochem J, 2004 Oct 15, 383(Pt 2), 311 - 8
Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue; Shan L et al.; Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye . We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX) . These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis . In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes . All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa . Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity . SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF . In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX . Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites . While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.

Appl Environ Microbiol, 2004 Jul, 70(7), 3968 - 72
Relationship between gyrA mutations and quinolone resistance in Flavobacterium psychrophilum isolates; Izumi S et al.; Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome . It has been reported that some isolates of F . psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained . In this study, we examined the quinolone susceptibility patterns of 27 F . psychrophilum strains isolated in Japan and the United States . Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA . When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA {Escherichia coli numbering} and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates . These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F . psychrophilum.

J Appl Microbiol, 2004, 97(2), 421 - 8
Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish; Arias CR et al.; AIMS: To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare . METHODS AND RESULTS: Genetic variability among Fl . columnare isolates was characterized using restriction fragment length polymorphism analysis of the 16S rDNA gene, intergenic spacer region (ISR) sequencing, and amplified fragment length polymorphism (AFLP) fingerprinting . Thirty Fl . columnare cultures isolated from different fish species and geographical origins as well as reference strains were included in the study . Fifteen isolates belonged to genomovar I while eleven were ascribed to genomovar II . Analysis of the ISR sequence confirmed the genetic differences between both genomovars but revealed a higher diversity among genomovar I isolates . The maximum resolution was provided by AFLP fingerprinting, as up to 22 AFLP profiles could be defined within the species . CONCLUSIONS: We confirmed the division of Fl . columnare isolates from cultured fish into different genogroups . We showed that both genomovars I and II are present in channel catfish from the US . We described a unique genetic group represented by four Fl . columnare isolates from tilapia in Brazil which appears to be related to both genomovars . We were able to further subdivide the species by analysing the ISR . Finally, the use of AFLP allowed us to fingerprint the species at clone level without losing the higher genetic hierarchy of genomovar division . SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the fish pathogen Fl . columnare.

Dis Aquat Organ, 2004 Apr 21, 59(1), 79 - 84
Experimental infection of Flavobacterium psychrophilum in fins of Atlantic salmon Salmo salar revealed by scanning electron microscopy; Martinez JL et al.; Infections caused by Flavobacterium psychrophilum include 'bacterial coldwater disease' (BCWD) and 'rainbow trout fry syndrome' (RTFS), which are severe diseases that can cause high mortality and significant losses in hatchery-reared salmonids worldwide . Usually, these conditions start with necrosis along the edge of the fins . As the infection progresses, both the fish surface and the internal organs can be involved . The aetiological agent produces a Ca-dependent protease that can be responsible for some of the pathogenic responses, although the precise nature of the response remains to be elucidated . Atlantic salmon Salmo salar were experimentally infected by F . psychrophilum in order to investigate the bacterial invasion in the fin tissues by scanning electron microscopy . The images showed numerous bacteria embedded in the mucous layer when this remained on the tegument . In other zones without mucus, it was observed that bacteria were present on the axis of fin rays, but not on the epidermal surface . The material on these axes was largely eroded by tubular boreholes, and bacterial rods could be seen in these perforations . EDX (Energy Dispersive X-ray) microanalysis of the axis of the fin rays showed significant amounts of P and Ca, revealing the ossification of the ray axis . The protease activity could explain the formation of the tubular boreholes, allowing the bacteria the necessary Ca for the activation of the enzyme . The erosion pattern suggests that the gliding motility of F . psychrophilum could be involved in this burrowing ability.

Dis Aquat Organ, 2004 Apr 21, 59(1), 17 - 26
Protective immunity in rainbow trout Oncorhynchus mykiss following immunization with distinct molecular mass fractions isolated from Flavobacterium psychrophilum; LaFrentz BR et al.; Vaccine development for coldwater disease (CWD), also known as rainbow trout fry syndrome (RTFS), has been based primarily on whole-cell bacterins or outer membrane fractions of Flavobacterium psychrophilum . In the present study, immunogenic regions of the bacterium corresponding to 18-28, 41-49, and 70-100 kDa were identified by western blot analysis using rainbow trout Oncorhynchus mykiss immune sera . Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), antigens within these regions were isolated by electro-elution and used in immunization trials . Groups of rainbow trout fry were immunized with these regions emulsified with Freund's complete adjuvant (FCA) and a formalin-killed bacterin emulsified with FCA . It was demonstrated that the 70-100 and 41-49 kDa regions and F . psychrophilum treatments elicited significant protection when compared to the saline control following subcutaneous challenge with 2 doses of a virulent strain of F . psychrophilum . Immunization with the 70-100 kDa region resulted in near complete protection in fish with mean cumulative percent mortality (CPM) of 6% and mean relative percent survival (RPS) of 94% at the lower challenge dose (6.25 x 10(6) colony forming units fish(-1)) . This preparation also stimulated a high level of specific antibody to F . psychrophilum, as detected by an enzyme-linked immunosorbent assay (ELISA) . Western blot analysis using sera from fish immunized with the 70-100 kDa region demonstrated that high molecular weight proteins and the O-polysaccharide component of lipopolysaccharide (LPS) are recognized by serum antibodies . This suggests that these antigens may be involved in eliciting a highly protective immune response, and could serve as vaccine candidates.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 273 - 9
Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis; Escalante A et al.; The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples . Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L . kefir, L . acetotolerans, L . hilgardii, L . plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque . Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%) . Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.

Int Microbiol, 2004 Mar, 7(1), 13 - 8
Competition for polymers among heterotrophic bacteria, isolated from particles of the Equatorial Atlantic; Berkenheger I et al.; Three heterotrophic bacterial strains, isolated from organic particles of the upper water column of the Equatorial Atlantic, taken during a cruise on the R/V METEOR (1997), were investigated concerning their physiological and phylogenetic properties using classic microbiological and modern molecular-biological methods . All isolates are gram-negative rods able to use polymers such as cellulose, chitin or starch as sole carbon source . The phylogeny of these isolates was investigated by fluorescence in situ hybridization (FISH) and 16S rDNA sequencing . The three isolated strains belong to the Cytophaga/Flavobacteria, gamma-Proteobacteria (Marinobacter sp.), and alpha-Proteobacteria (Sulfitobacter pontiacus) . In order to study succession during growth on polymers naturally occurring in marine habitats, FISH was used as a new approach to detect cells from different phylogenetic clusters in the course of a single growth experiment . Mixed cultures consisting of the isolated strains in equal amounts were incubated with cellulose, chitin or starch . Isolate 4301-10/2, a member of the gamma-Proteobacteria, dominated in mixed cultures growing on cellulose, chitin, or starch after only 10 days, with 55, 60, and 95%, respectively, of cells hybridizing with 4,6-diamidino-2-phenylindole (DAPI).

J Mol Microbiol Biotechnol, 2004, 7(1-2), 63 - 71
Cytophaga-flavobacterium gliding motility; McBride MJ; Flavobacterium johnsoniae, like many other members of the Cytophaga-Flavobacterium-Bacteroides group, displays rapid gliding motility . Cells of F . johnsoniae glide over surfaces at rates of up to 10 microm/s . Latex spheres added to F . johnsoniae bind to and are rapidly propelled along cells, suggesting that adhesive molecules move laterally along the cell surface during gliding . Genetic analyses have identified a number of gld genes that are required for gliding . Three Gld proteins are thought to be components of an ATP-binding-cassette transporter . Five other Gld proteins are lipoproteins that localize to the cytoplasmic membrane or outer membrane . Disruption of gld genes results not only in loss of motility, but also in resistance to bacteriophages that infect wild-type cells, and loss of the ability to digest the insoluble polysaccharide chitin . Two models that attempt to incorporate the available data to explain the mechanism of F . johnsoniae gliding are presented .

J Mol Microbiol Biotechnol, 2003, 6(3-4), 182 - 90
Lipopolysaccharide O-antigen antibody-based detection of the fish pathogen Flavobacterium psychrophilum; Crump EM et al.; Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens . Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease . A unique antigen from F . psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test . LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests . Rabbit polyclonal anti-O-PS serum was highly specific for F . psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test . Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F . psychrophilum .

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 705 - 11
Formosa algae gen . nov., sp . nov., a novel member of the family Flavobacteriaceae; Ivanova EP et al.; Four light-yellow-pigmented, Gram-negative, short-rod-shaped, non-motile isolates were obtained from enrichment culture during degradation of the thallus of the brown alga Fucus evanescens . The isolates studied were chemo-organotrophic, alkalitolerant and mesophilic . Polar lipids were analysed and phosphatidylethanolamine was the only phospholipid identified . The predominant cellular fatty acids were 15 : 0, i15 : 0, ai15 : 0, i15 : 1 and 15 : 1(n-6) . The DNA G+C contents of the four strains were 34.0-34.4 mol% . The level of DNA relatedness of the four isolates was conspecific (88-98 %), indicating that they belong to the same species . The 16S rDNA sequence of strain KMM 3553(T) was determined . Phylogenetic analysis revealed that KMM 3553(T) formed a distinct phyletic line in the phylum Bacteroidetes, class Flavobacteria in the family Flavobacteriaceae and that, phylogenetically, this strain could be placed almost equidistant from the genera Gelidibacter and Psychroserpens (16S rRNA gene sequence similarities of 94 %) . On the basis of significant differences in phenotypic and chemotaxonomic characteristics, it is suggested that the isolates represent a novel species in a new genus; the name Formosa algae gen . nov., sp . nov . is proposed . The type strain is KMM 3553(T) (=CIP 107684(T)).

Environ Microbiol, 2004 Jun, 6(6), 568 - 73
Proposal to transfer 'Aegyptianella ranarum', an intracellular bacterium of frog red blood cells, to the family Flavobacteriaceae as 'Candidatus Hemobacterium ranarum' comb . nov; Zhang C et al.; 'Aegyptianella ranarum' (order Rickettsiales), an ultrastructurally defined small, Gram-negative rod, is known to replicate in the red blood cells of frogs . Heretofore, this bacterium has not been characterized genetically . We cloned and sequenced the 16S rRNA (1310 bp) and gyrB (718 bp) genes of 'A . ranarum' from a Canadian frog blood specimen . In situ hybridization (with an 'A . ranarum' 16S rRNA gene polymerase chain reaction product as probe) and electron microscopy confirmed that 'A . ranarum' forms cytoplasmic inclusions in frog erythrocytes . blast comparisons with GenBank 16S rRNA and gyrB sequences showed that both 'A . ranarum' genes were most similar (91% and 67% identity) to those of Chryseobacterium meningosepticum, a bacterium in the family Flavobacteriaceae . In contrast, 'A . ranarum' 16S rRNA shared only 61% identity with Aegyptianella pullorum . Phylogenetic analyses of these genes using phylip supported 'A . ranarum' as a member of Flavobacteriaceae, but suggested that its cladistic sibling may be Bergeyella zoohelcum or Weeksella virosa, rather than C . meningosepticum . We propose to classify 'Aegyptianella ranarum' as 'Candidatus Hemobacterium ranarum' in the family Flavobacteriaceae . Our results provide a starting point for studies of related intraerythrocytic bacterial infections in frogs.

Microb Ecol, 2004 Aug, 48(2), 263 - 73 Epub 2004 May 06.
A population survey of members of the phylum Bacteroidetes isolated from salt marsh sediments along the east coast of the United States; Lydell C et al.; The population diversity of cultured isolates of the phylum Bacteroidetes was investigated from salt-marsh sediments . A total of 44 isolates that belonged to this phylum were isolated either from high-dilution plates or from end-dilution most-probable-number (MPN) tubes . The majority of the isolates came from Virginia, with others isolated from salt marshes in Delaware and North Carolina . All the isolates were aerobic Gram-negative, catalase positive small rods that formed uniform colonies; most had either yellow or orange pigmentation . Riboprinting of 40 isolates revealed they were genotypically diverse, consisting of 33 different riboprint patterns; there were four riboprint groups with two or more members . The isolates could be divided into 23 different fatty acid methyl ester (FAME) profiles at the species level with 14 of the profiles being unique to single isolates . One group of 10 isolates was closely related, suggesting this group may be well adapted for life in salt marshes . Thirteen of the isolates were selected for sequencing of the small-subunit ribosomal RNA gene representing a diverse group of isolates that fell within the classes Sphingobacteria and Flavobacteria . Only one of the isolates was >97% similar at the 16S rDNA to a described species of Cytophaga marinoflava; the other isolates were 94 to 96.5% related to undescribed isolates mostly within the class Flavobacteria . There was good concordance between the FAME dendrogram and a phylogenetic tree based on comparison of 16S sequences . There were no obvious temporal or spatial distribution patterns to the isolates, suggesting that this group of bacteria is inherently diverse.

J Bacteriol, 2004 Apr, 186(8), 2295 - 302
GldI is a lipoprotein that is required for Flavobacterium johnsoniae gliding motility and chitin utilization; McBride MJ et al.; Cells of Flavobacterium johnsoniae glide rapidly over surfaces by an unknown mechanism . Seven genes (gldA, gldB, gldD, gldF, gldG, gldH, and ftsX) that are required for gliding motility have been described . Complementation of the nonmotile mutants UW102-41, UW102-85, and UW102-92 identified another gene, gldI, that is required for gliding motility . gldI mutants formed nonspreading colonies, and individual cells were completely nonmotile . They were also resistant to bacteriophages that infect wild-type cells, and they failed to digest chitin . Introduction of wild-type gldI on a plasmid restored colony spreading, cell motility, phage sensitivity, and the ability to digest chitin to the gldI mutants . gldI encodes a predicted 199-amino-acid protein that localized to the membrane fraction . Labeling studies with {(3)H}palmitate indicated that GldI is a lipoprotein . GldI is similar to peptidyl-prolyl cis/trans-isomerases of the FK506-binding protein family and may be involved in folding cell envelope protein components of the motility machinery.

Zhonghua Shao Shang Za Zhi, 2004 Feb, 20(1), 14 - 6
{Isolation and analysis of the drug resistance of the flavobacterium and its production of beta-lactamases}; Luo Y et al.; OBJECTIVE: To investigate the drug resistance of flavobacterium and its ability to produce BLA (beta-lactamases) and ESBLs (Extended-spectrum beta-lactamases) . METHODS: The production of BLA and ESBLs from 6 clinical isolated flavobacterium strains was determined by nitrocefin disc test and double-disc synergy method, respectively . The antibiotic susceptibilities of the strains were determined by Kirby-Bauer disc diffusion test and the agar dilution method and the MIC was assessed . RESULTS: All the six flavobacteria were BLA-producing strains and more than 80% of them were ESBLs-producing, and they were highly resistant to beta-lactamase antibiotics (MIC 32 - 256 mg/L), but susceptible to fluoroquinolones and cephalosporin with beta-lactamase inhibitors (MIC 0.125 - 8 mg/L) . CONCLUSION: Most of the flavobacteria in nosocomial infections were beta-lactamase-producing and were highly resistant to beta-lactamase antibiotics . Fluoroquinolones and beta-lactamase antibiotics with lactamase inhibitors should be the first choice for the management of infection caused by flavobacterium.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 609 - 13
Cellulophaga pacifica sp . nov; Nedashkovskaya OI et al.; Three marine, heterotrophic, aerobic, agarolytic, pigmented and gliding bacteria were isolated in June 2000 from a sea water sample that was collected in the Gulf of Peter the Great, Sea of Japan, and analysed in a polyphasic taxonomic study . 16S rDNA sequence analysis indicated that strains KMM 3664(T), KMM 3669 and KMM 3915 were members of the family Flavobacteriaceae . Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, the isolates were classified in the genus Cellulophaga as members of a novel species, Cellulophaga pacifica sp . nov . The type strain is KMM 3664(T) (=JCM 11735(T)=LMG 21938(T)).

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 519 - 24
Tenacibaculum skagerrakense sp . nov., a marine bacterium isolated from the pelagic zone in Skagerrak, Denmark; Frette L et al.; A number of bacteria were isolated from sea water in Skagerrak, Denmark, at 30 m depth . Two of the isolates, strains D28 and D30(T), belonged to the Flavobacteriaceae within the Cytophaga-Flavobacterium-Bacteroides group . Sequencing of 16S rRNA genes of the two strains indicated strongly that they belonged to the genus Tenacibaculum and that they showed greatest similarity to the species Tenacibaculum amylolyticum and Tenacibaculum mesophilum . DNA-DNA hybridization values, DNA base composition and phenotypic characteristics separated the Skagerrak strains from the other species within TENACIBACULUM: Thus, it is concluded that the strains belong to a novel species within the genus Tenacibaculum, for which the name Tenacibaculum skagerrakense sp . nov . is proposed, with strain D30(T) (=ATCC BAA-458(T)=DSM 14836(T)) as the type strain.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 445 - 8
Gillisia limnaea gen . nov., sp . nov., a new member of the family Flavobacteriaceae isolated from a microbial mat in Lake Fryxell, Antarctica; Van Trappen S et al.; A taxonomic study was performed on three strains isolated from microbial mats in Lake Fryxell, McMurdo Dry Valleys, Antarctica . Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belong to the family Flavobacteriaceae, in which they form a distinct lineage . The isolates are Gram-negative, chemoheterotrophic, aerobic, rod-shaped cells . They are psychrophilic and yellow-pigmented, with DNA G+C contents in the range 37.8-38.9 mol% . Whole-cell fatty acid profiles revealed mainly branched fatty acids and 17 : 0 2-OH . On the basis of genotypic, phenotypic, chemotaxonomic and phylogenetic results, it is proposed that the isolates represent a novel species in a new genus, Gillisia limnaea gen . nov., sp . nov . The type strain is LMG 21470(T) (=DSM 15749(T)).

Biochimie, 2004 Feb, 86(2), 151 - 6
Purification and characterization of ascorbic acid 2-kinase from Flavobacterium devorans ATCC 10829; Jung Ahn M et al.; Maximum activity for phosphorylating C(2)-OH of the ascorbic acid was observed at the time of 16 h incubation from the culture of Flavobacterium devorans ATCC 10829 . The enzyme was purified 1.178-fold, via ammonium sulfate fractionation, Fast Q anion exchange, and phenyl agarose chromatography . Gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a tetramer with subunit MW of 29 kDa . Among available second substrates, pyrophosphate showed the highest activity . Optimum temperature and pH were 45 degrees C and 5.5, respectively . The enzyme was chemically modified only by diethylpyrocarbonate and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), indicating that histidine and carboxylate are in the active site . pH studies showed that two histidines are involved in the binding of the substrates and a carboxylate in catalysis . Therefore, the chemical mechanism of the enzyme is likely that two histidines bind to pyrophosphate and carboxylate, respectively, and a carboxylate acts as a general base.

Fundam Clin Pharmacol, 2003 Dec, 17(6), 717 - 23
Study of the mechanism of Flavobacterium sp . for hydrolyzing organophosphate pesticides; Ortiz-Hernandez ML et al.; The biotransformation by Flavobacterium sp . of the following organophosphate pesticides was experimentally and theoretically studied: phorate, tetrachlorvinphos, methyl-parathion, terbufos, trichloronate, ethoprophos, phosphamidon, fenitrothion, dimethoate and DEF . The Flavobacterium sp . ATCC 27551 strain bearing the organophosphate-degradation gene was used . Bacteria were incubated in the presence of each pesticide for a duration of 7 days . Parent pesticides were identified and quantified by means of a gas-chromatography mass spectrum system . Activity was considered as the amount (micromol) of each pesticide degraded by Flavobacterium sp . Also, structural parameters obtained by means of the CAChe program package for biomolecules, the reactivity index of phosphorus, of oxygen at the P = O function and of sulfur at the P = S function, and lipophilicity (log Poct) (ALOGPS v . 2.0) were obtained for each pesticide . Pesticides were hydrolyzed at the bond between phosphorous and the heteroatom, producing phosphoric acid and three metabolites . Enzymatic activity was significantly explained by the following multiple linear relationship: Enzymatic activity = 162.2 - 9.5(dihedral angle energy) - 25.0(Total energy) - 0.51(Molecular weight) . Finally, a mechanism of Flavobacterium sp . to hydrolyze pesticides was proposed.

Carbohydr Res, 2004 Feb 25, 339(3), 699 - 703
The synthesis of a novel thio-linked disaccharide of chondroitin as a potential inhibitor of polysaccharide lyases; Rye CS et al.; A thio-linked disaccharide based on the structure of the glycosaminoglycan chondroitin was synthesized as a potential inhibitor of chondroitin AC lyase from Flavobacterium heparinum for structural analysis of the active site . Instead it was found to be a slow substrate, thereby demonstrating that lyases, in contrast to glycosidases, can cleave thioglycoside links between sugars.

Appl Environ Microbiol, 2004 Mar, 70(3), 1777 - 86
Bacterial succession in a petroleum land treatment unit; Kaplan CW et al.; Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C(10) to C(32) range . The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts . Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation . TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project . A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation . Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study . The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased . For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones . Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation . After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance . These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.

J Mol Biol, 2004 Mar 19, 337(2), 367 - 86
High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism; Lunin VV et al.; Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases . Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate . Like other chondroitin AC lyases, it is capable of cleaving hyaluronan . We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A . The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry . The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals . The electron density map for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation . This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group . Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.

Vet Res Commun, 2004 Feb, 28(2), 93 - 101
Comparison of the API 20E and BBL Crystal E/NF identification systems for differentiating bacterial isolates from apparently healthy reared sea bass (Dicentrarchus labrax); Topic Popovic N et al.; The ability of two commercial rapid identification systems, API 20E and BBL Crystal E/NF, to reliably identify bacterial isolates from the internal organs of reared sea bass were compared . The tests gave different results: API 20E identified bacteria as Pseudomonas spp . with 37% accuracy, while BBL Crystal E/NF identified them as Flavobacterium odoratum with 99% accuracy . Although F . odoratum is not a marine fish pathogen, conventional tests conducted with the same isolates were more indicative of them being Flavobacterium spp . than Pseudomonas spp., suggesting that BBL Crystal E/NF was more reliable in this identification . Both systems were found to be applicable for diagnostics of marine fish pathogens, but should be used with caution because of possible misinterpretation.

J Fish Dis, 2004 Jan, 27(1), 29 - 35
Morphological and genetic characteristics of Flavobacterium columnare isolates: correlations with virulence in fish; Thomas-Jinu S et al.; Variability in pathogenicity of Flavobacterium columnare makes disease treatment difficult because there is currently no way to easily recognize those strains that warrant aggressive treatments . In order to identify suitable virulence markers, 17 isolates of F . columnare were cultured from six different fish species . The DNA from all isolates was analysed using random amplified polymorphic DNA (RAPD) . Bootstrap analysis of the RAPD data produced a tree with three major groups supported by bootstrap scores of 80-100% . Virulence of the isolates was determined by bath exposure of channel catfish, Ictaluruspunctatus (Rafinesque), and golden shiners, Notemigonus crysoleucas (Mitchill), to broth cultures of F . columnare . In channel catfish, 13 of 17 isolates produced 100% mortality within 48 h post-exposure . All isolates of cyprinid fish origin clustered in a single RAPD group . At least two of the four isolates that were not virulent in channel catfish were of cyprinid fish origin . There was a wide variation in cell morphology between isolates with lengths of cells or cell chains ranging from 3 to 11 microm, even under identical culture conditions . Most of the shorter or single cell isolates fell into a single RAPD group . No clear association was identified between virulence and any other characteristic, including RAPD group.

J Fish Dis, 2004 Jan, 27(1), 23 - 8
Acute columnaris infection in channel catfish, Ictalurus punctatus (Rafinesque): efficacy of practical treatments for warmwater aquaculture ponds; Thomas-Jinu S et al.; Columnaris disease was induced in channel catfish, Ictalurus punctatus (Rafinesque), by bath exposure to four highly virulent isolates of Flavobacterium columnare . In untreated controls, mortality began 20 h after exposure and reached 100% by 48 h . Mortality in channel catfish given antibiotic treatments with oxytetracycline or a combination of sulphadimethoxine and ormetoprim in feed prior to bacterial challenge was zero with all four strains of F . columnare . Diquat (Zeneca Agricultural Products, Wilmington, DE, USA) was the most effective bath treatment; mortality with all four strains was zero . With potassium permanganate, chloramine-T, hydrogen peroxide and copper sulphate, bath treatment efficacy varied significantly among strains (P = 0.0346) and among treatments (P = 0.0033) . Bath treatments with chloramine-T and potassium permanganate significantly reduced (P < 0.05) mortality from 100 to 75 and 69%, respectively, but copper sulphate and hydrogen peroxide treatments were not effective . Based on our results, oral antibiotics prevented columnaris disease but, of the bath treatments, only Diquat produced a dramatic reduction in the mortality of acutely infected fish . Diquat is labelled for aquatic use as an herbicide in the USA but in large ponds it is prohibitively expensive.

Water Sci Technol, 2004, 49(2), 255 - 61
Microbial community structure of membrane fouling film in an intermittently and continuously aerated submerged membrane bioreactor treating domestic wastewater; Lim BR et al.; There was an observable difference in microbial community structure between suspended microorganisms and membrane biofouling film in intermittently and continuously aerated SMBRs . The dominant quinone type of membrane biofouling film in an intermittently aerated SMBR was ubiquinone (UQs)-8, -10 followed by menaquinone (MKs)-8(H4) and -8(H2) . But that of the continuously aerated SMBR was UQs-10, -8 followed by MKs-6 and -8(H4) . The experimental results also showed that the conditions of an intermittently aerated SMBR may contribute to biofouling by Pseudomonas, Moraxella, Vibrio (quinone type UQ-8), Staphylococcus warneri (quinone type MK-7), Micrococcus sp . (quinone type MK-8(H2)) and Nocardia sp . (quinone type MK-8(H4)), but biofouling in a continuously aerated SMBR may be due to Paracoccus sp . (quinone type: UQ-10) and Flavobacterium species (quinone type: MK-6) . The microbial diversities in the intermittently aerated SMBR were 10.9 and 9.4 for biofouling film and suspended microorganisms, respectively . For the continuously aerated SMBR, the results were 10.4 and 10.5 for biofouling film and suspended microorganisms, respectively.

J Appl Microbiol, 2004, 96(3), 623 - 9
Factorial analysis of tricarboxylic acid cycle intermediates for optimization of zeaxanthin production from Flavobacterium multivorum; Bhosale P et al.; AIMS: To study the effect of intermediates of the tricarboxylic acid (TCA) cycle on the production of zeaxanthin from Flavobacterium multivorum in order to optimize production of this xanthophyll carotenoid . METHODS AND RESULTS: The concentration of selected TCA cycle intermediates (malic acid, isocitric acid and alpha-ketoglutarate) was optimized in shake flask culture, using a statistical two-level, three-variable factorial approach . The carotenoid production profile was also studied in the optimized medium at various growth phases . Optimized medium resulted in a sixfold increase in volumetric production of zeaxanthin (10.65 +/- 0.63 microg ml-1) using malic acid (6.02 mm), isocitric acid (6.20 mm) and alpha-ketoglutarate (0.02 mm) . The majority of zeaxanthin was produced in the late logarithmic growth phase whereas a substantial amount of beta-cryptoxanthin and beta-carotene were observed in the early logarithmic phase . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates improvement of zeaxanthin production from F . multivorum which might aid in the commercialization of zeaxanthin production from this microbe.

J Appl Microbiol, 2004, 96(3), 560 - 8
Identification of the culturable and nonculturable bacterial population in ground water of a municipal water supply in Germany; Ultee A et al.; AIMS: The identification of the culturable and nonculturable bacterial population in ground water of a municipal water supply in Mainz (Germany) during the year 2002 . METHODS AND RESULTS: Total counts varied between 3.5 x 103 and 2.2 x 104 cells ml-1, viable counts were approximately between 8.1 x 102 and 3.3 x 103 cells ml-1 . After cultivation on different nutrient media (R2A, DEV, PCA, Endo, Standard) <1% appeared to be culturable on the media used . After denaturating gradient gel electrophoreses, up to 24 different bacterial species were detected in the ground water . With the aid of 16S rDNA isolation, amplification and sequencing, the isolated organisms and clones could be identified . CONCLUSIONS: The isolated and cultured organisms mainly belonged to the Proteobacteria (alpha, beta and gamma), Flavobacteria or Actinobacteria . However, most of the noncultured micro-organisms were beta-Proteobacteria . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in which the identification of all culturable and nonculturable bacteria in a ground water has been attempted.

Mar Biotechnol (NY), 2002 Sep, 4(4), 399 - 405
A marine strain of flavobacteriaceae utilizes brown seaweed fucoidan; Sakai T et al.; Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) with a yield of about 3.8% dry weight . To isolate enzymes that degrade fucoidan, we first screened marine bacteria for their ability to utilize fucoidan, and isolated one strain of Flavobacteriaceae from seawater that could do this . Phylogenetic analysis of the 16S ribosomal DNA sequence suggested that this strain appeared to belong to a new genus, and was tentatively named Fucobacter marina . The strain utilized L-fucose (17%), D-mannose (91%), D-galactose (46%), and D-glucuronic acid (66%) in the fucoidan from K . crassifolia . The strain partially utilized fucoidan from 2 other seaweeds that belong to the order Laminariales, Undaria pinnatifida (10%) and Lessonia nigrescens (48%).

Environ Microbiol, 2004 Mar, 6(3), 288 - 300
Diverse microbial communities inhabit Antarctic sponges; Webster NS et al.; Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus . The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments . Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales . Of the 150 archaeal clones from L . apicalis, K . varialosa and M . acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota . Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones . Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity . Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group . Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species . Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives . In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K . varialosa.

Environ Microbiol, 2004 Mar, 6(3), 227 - 41
Early steps in microbial colonization processes at deep-sea hydrothermal vents; Alain K et al.; A pluri-disciplinary in situ colonization experiment was performed to study early stages of colonization in deep-sea vent Alvinella spp . worm habitats . Four colonization devices were deployed onto Alvinella spp . colonies of different chimneys of the East-Pacific Rise (EPR 13 degrees N), for two different periods: a short (less than a week) and a longer one (3 weeks) . Video imagery and monitoring of the thermal and physico-chemical conditions were performed during the colonization experiments . Numerous microorganisms bearing specialized adhesion-appendages and/or high amounts of polymeric extracellular matrix were observed on devices, which may efficiently contribute to the colonization of new surfaces . The microbial cohorts preceding and accompanying Alvinella spp . settlement were identified . In all cases, Archaea could not be detected and the microbial mats were essentially composed of e-Proteobacteria . Within this group, one phylotype (AlviH2) was found to dominate the libraries of three colonization devices . Dominance of e-Proteobacteria in the libraries may reflect the wide physiological variety encountered within this group or an adaptability of these microorganisms towards their changing environment . Bacteria affiliated to the Cytophaga-Flavobacterium-Bacteroides group or to the e-Proteobacteria, that grow either chemo-organoheterotrophically by fermentation or chemolithoautotrophically with H2 as an electron donor and S degrees /S2O32- or NO3- as a terminal electron acceptor, were isolated from one of the microbial mat formed in 20 days.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 119 - 23
Ulvibacter litoralis gen . nov., sp . nov., a novel member of the family Flavobacteriaceae isolated from the green alga Ulva fenestrata; Nedashkovskaya OI et al.; Two heterotrophic, aerobic, Gram-negative, pigmented and non-motile marine bacteria that were isolated from the green alga Ulva fenestrata were studied by polyphasic taxonomic methods . 16S rDNA sequence analysis indicated that strain KMM 3912T formed a distinct lineage within the family Flavobacteriaceae . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic analyses, the novel bacteria were classified as Ulvibacter litoralis gen . nov., sp . nov . The type strain is KMM 3912T (=KCTC 12104T=CCUG 47093T).

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 85 - 92
Flavobacterium degerlachei sp . nov., Flavobacterium frigoris sp . nov . and Flavobacterium micromati sp . nov., novel psychrophilic bacteria isolated from microbial mats in Antarctic lakes; Van Trappen S et al.; Taxonomic studies were performed on 36 strains that were isolated from microbial mats in Antarctic lakes of the Vestfold Hills, the Larsemann Hills and the McMurdo Dry Valleys . Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains are related to members of the genus Flavobacterium; sequence similarity values with their nearest phylogenetic neighbours ranged from 96.8 to 98.5% . Results of DNA-DNA hybridization and comparison of repetitive extragenic palindromic DNA-PCR fingerprinting patterns revealed that these strains are members of three distinct species . Genotypic results, together with phenotypic characteristics, allowed the differentiation of these species from related Flavobacterium species with validly published names . The isolates are Gram-negative, chemoheterotrophic, rod-shaped cells that are psychrophilic and moderately halotolerant; their DNA G+C contents range from 33.1 to 34.5 mol% . Their whole-cell fatty acid profiles are similar and include C(15:0), anteiso-C(15:0), iso-C(15:0), C(15:1)omega6c, iso-C(16:0), iso-C(16:0) 3-OH and summed feature 3 (which comprises iso-C(15:0) 2-OH, C(16:1)omega7c or both) as major fatty acid components . On the basis of these results, three novel species are proposed, namely Flavobacterium degerlachei sp . nov . (consisting of 14 strains, with LMG 21915T=DSM 15718T as the type strain), Flavobacterium micromati sp . nov . (consisting of three strains, with LMG 21919T=CIP 108161T as the type strain) and Flavobacterium frigoris sp . nov . (consisting of 19 strains, with LMG 21922T=DSM 15719T as the type strain).

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 65 - 70
Belliella baltica gen . nov., sp . nov., a novel marine bacterium of the Cytophaga-Flavobacterium-Bacteroides group isolated from surface water of the central Baltic Sea; Brettar I et al.; Two bacterial isolates from the Baltic Sea, BA1 and BA134T, were characterized for their physiological and biochemical features, fatty acid profiles and phylogenetic position based on 16S rRNA gene sequences . The strains were isolated from surface water of the central Baltic Sea during the decay of a plankton bloom . Phylogenetic analysis of their 16S rRNA gene sequences revealed a clear affiliation to the family 'Flexibacteriaceae' and showed highest sequence similarity (91%) to Cyclobacterium marinum . The G+C content of the DNA was 35.4 mol% . The strains were pink-coloured due to carotinoids, Gram-negative, rod-shaped and catalase- and oxidase-positive . Growth was observed at 0-6% salinity, with good growth at 0-3% . Temperature for growth was 4-37 degrees C, with an optimum around 25 degrees C . The fatty acid profiles were dominated by branched-chain fatty acids (70%), with a high abundance of iso-C(15:0) (29-33%), iso-C(17:1)omega9c (7-10%) and C(17:1)omega6c (5-10%) . According to their morphology, physiology, fatty acid composition, 16S rRNA gene sequences and DNA-DNA similarity, on one hand, the described bacteria are considered to be members of the same novel species; on the other hand, they are suggested as a novel genus of the family 'Flexibacteriaceae' . To honour the late aquatic microbiologist Russell T . Bell, the name Belliella baltica gen . nov, sp . nov . is suggested for the Baltic Sea isolates, for which the type strain is BA134T (=DSM 15883T=LMG 21964T=CIP 108006T).

Appl Environ Microbiol, 2004 Jan, 70(1), 616 - 20
Intracellular symbionts and other bacteria associated with deer ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts; Benson MJ et al.; The diversity of bacteria associated with the deer tick (Ixodes scapularis) was assessed using PCR amplification, cloning, and sequencing of 16S rRNA genes originating from seven ticks collected from Nantucket Island and Wellfleet, Cape Cod, Mass . The majority of sequences obtained originated from gram-negative proteobacteria . Four intracellular bacteria were detected including strains of Ehrlichia, Rickettsia, and Wolbachia and an organism related to intracellular insect symbionts from the Cytophaga-Flavobacterium-Bacteroides group . Several strains of members of the Sphingomonadaceae were also detected in all but one tick . The results provide a view of the diversity of bacteria associated with I . scapularis ticks in the field.

Appl Environ Microbiol, 2004 Jan, 70(1), 581 - 7
Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum; Alvarez B et al.; Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish . Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure . Here we describe the development of techniques for the genetic manipulation of F . psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen . The antibiotic resistance genes ermF, cfxA, and tetQ function in F . psychrophilum . Cloning vectors based on the F . psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E . coli, resulting in antibiotic-resistant colonies of F . psychrophilum . Conjugative transfer of DNA into F . psychrophilum was strain dependent . Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90) . E . coli lacZY functioned in F . psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression . Plasmids isolated from F . psychrophilum were efficiently introduced into F . psychrophilum by electroporation, but plasmids isolated from E . coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier . DNA isolated from F . psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier . Tn4351 was introduced into F . psychrophilum from E . coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies . The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.

Appl Environ Microbiol, 2004 Jan, 70(1), 550 - 7
Bacterial Activity at -2 to -20 degrees C in Arctic wintertime sea ice; Junge K et al.; Arctic wintertime sea-ice cores, characterized by a temperature gradient of -2 to -20 degrees C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures . With the fluorescent stains 4',6'-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O(2)-based respiration), the abundances of total, particle-associated (>3- micro m), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (-2 to -20 degrees C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt) . Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and ARCHAEA: Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice . For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries) . CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (-20 degrees C), where virtually all active cells were particle associated . The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells) . These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures . Measuring activity down to -20 degrees C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.

Appl Environ Microbiol, 2004 Jan, 70(1), 202 - 13
Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core; Miteva VI et al.; We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago . Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments . Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study . The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group . The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library . The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species . The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations . A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.

Appl Environ Microbiol, 2004 Jan, 70(1), 114 - 20
Structure and characterization of flavolipids, a novel class of biosurfactants produced by Flavobacterium sp . strain MTN11; Bodour AA et al.; Herein we report the structure and selected properties of a new class of biosurfactants that we have named the flavolipids . The flavolipids exhibit a unique polar moiety that features citric acid and two cadaverine molecules . Flavolipids were produced by a soil isolate, Flavobacterium sp . strain MTN11 (accession number AY162137), during growth in mineral salts medium, with 2% glucose as the sole carbon and energy source . MTN11 produced a mixture of at least 37 flavolipids ranging from 584 to 686 in molecular weight (MW) . The structure of the major component (23%; MW = 668) was determined to be 4-{{5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl}amino}-2-{2-{{5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl}amino}-2-oxoethyl}-2-hydroxy-4-oxobutanoic acid . The partially purified flavolipid mixture isolated from strain MTN11 exhibited a critical micelle concentration of 300 mg/liter and reduced surface tension to 26.0 mN/m, indicating strong surfactant activity . The flavolipid mixture was a strong and stable emulsifier even at concentrations as low as 19 mg/liter . It was also an effective solubilizing agent, and in a biodegradation study, it enhanced hexadecane mineralization by two isolates, MTN11 (100-fold) and Pseudomonas aeruginosa ATCC 9027 (2.5-fold), over an 8-day period . The flavolipid-cadmium stability constant was measured to be 3.61, which is comparable to that for organic ligands such as oxalic acid and acetic acid . In summary, the flavolipids represent a new class of biosurfactants that have potential for use in a variety of biotechnological and industrial applications.

Microb Ecol, 2003 Aug, 46(2), 279 - 88
Dynamics of microcystin-degrading bacteria in mucilage of Microcystis; Maruyama T et al.; To reveal the process of degradation of hepatotoxic microcystin produced in Microcystis cells during the Microcystis bloom period, we used fluorescence in situ hybridization (FISH) to analyze the population dynamics of microcystin-degrading bacteria in Microcystis mucilage . We designed and applied an oligonucleotide probe targeted to the 16S rRNA sequence of strain Y2 of a microcystin-degrading bacterium (MCD-bacterium), which was isolated from Lake Suwa, Japan . In both the 1998 and 1999 tests, FISH clearly showed that MCD-bacteria existed in the mucilage and that, when a high concentration of cell-bound microcystin was detected, MCD-bacteria exceeded 10% of the sum of bacteria hybridized with group-specific probes . The concentration of MCD-bacteria was highest in summer 1998, when a toxic species, M . viridis, was dominant . There was a high correlation between the number of MCD-bacteria in the mucilage and the concentration of cell-bound microcystin in the lake . Our results suggest that MCD-bacteria responded to changes in the concentration of microcystin and degraded the microcystin when it was released from Microcystis cells . We also analyzed changes in the bacterial community structure associated with the Microcystis colonies by using domain- and group-specific oligonucleotide probes . Changes in the concentrations of the Cytophaga/Flavobacterium group and delta-Proteobacteria, which can degrade macromolecules derived from Microcystis cells, were synchronized with changes in the concentration of Microcystis . The results not only suggest the significant role of MCD-bacteria in detoxification, but also demonstrate a possible sequence of degradation from Microcystis cells to microcystin maintained in the cell, which is then carried out by bacterial consortia in the mucilage.

Carbohydr Res, 2003 Nov 14, 338(23), 2653 - 8
The structure of the glycopeptides from the fish pathogen Flavobacterium columnare; Vinogradov E et al.; Proteolytic digestion of the phenol-water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4-O-methyl-2-acetamido-2-deoxy-D-glucuronic acid (GlcNAcA), D-glucuronic acid (D-GlcA), 2,3-di-O-acetyl-D-xylose (D-Xyl), 2-O-methyl-D-glucuronic acid (D-GlcA), D-mannose (D-Man), and 2-O-methyl-L-rhamnose (L-Rha) . By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: {carbohydrate structure--see text} where all monosaccharides have the D-configuration except for 2-O-methyl-L-rhamnose; and were in the pyranose form . Only one carbohydrate structure was found . The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.

Dis Aquat Organ, 2003 Oct 24, 56(3), 207 - 14
Genotyping of Flavobacterium psychrophilum using PCR-RFLP analysis; Izumi S et al.; Genetic variability among 242 strains of Flavobacterium psychrophilum was characterized using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis . Universal Primers GYR-1 and GYR-1R, which were designed to amplify the gyrase subunit B gene (gyrB), yielded a 1178 bp PCR product encoding gyrB and a 290 bp PCR product of anonymous DNA from all F . psychrophilum strains tested . In the RFLP analysis of the anonymous 290 bp DNA marker, the restriction enzyme HinfI generated 2 cleavage patterns (Genotypes A and B) . Genotype A was found only in isolates from ayu (n = 109), while Genotype B was found in isolates from coho salmon (n = 11), ayu (n = 35), rainbow trout (n = 43) and other fishes (n = 44) . In the second experiment, Primers PSY-G1F and PSY-G1R specific for F . psychrophilum, were used to amplify gyrB . The specific primer pair amplified the expected size (1017 bp) PCR product from all F . psychrophilum strains . In the RFLP analysis of the gyrB, the restriction enzyme RsaI produced 2 genotypes, R and S . Genotype R was found in isolates from coho salmon (n = 6), ayu (n = 27), rainbow trout (n = 39) and other fishes (n = 4) . Genotype S was found in isolates from coho salmon (n = 5), ayu (n = 117), rainbow trout (n = 4) and other fishes (n = 40).

Syst Appl Microbiol, 2003 Nov, 26(4), 523 - 8
Chryseobacterium miricola sp . nov., a novel species isolated from condensation water of space station Mir; Li Y et al.; Classification of strain W3-B1, which was isolated from condensation water in the Russian space laboratory Mir, was investigated by a polyphasic taxonomic approach . Cells of strain W3-B1 were nonmotile, asporogenous, gram-negative slender rods with rounded ends . 16S rRNA gene sequence analysis indicated that organism should be placed in the genus Chryseobacterium . This organism contains menaquinone MK-6 as the predominent isoprenoid quinone and 3-OH iso 17:0 (40%), iso 15:0 (33%) as the major fatty acids . Phylogenetically, the nearest relative of strain W3-B1 is Chryseobacterium meningosepticum with sequence similarity of 98.4%, but DNA-DNA hybridization resulted in similarity values of only 52.3% . The G+C mol% is 34.6 mol% . Based upon results obtained by morphological, biochemical, chemotaxonomic, and molecular methods, strain W3-B1 was clearly distinguishable from other Chryseobacterium species . For these reasons, a novel species of family Flavobacteriaceae is proposed; strain W3-B1(T) (= GTC 862(T) = JCM 11413(T) = DSM 14571(T)) is the type strain.

Appl Environ Microbiol, 2003 Dec, 69(12), 7035 - 43
Role of soil pH in the development of enhanced biodegradation of fenamiphos; Singh BK et al.; Repeated treatment with fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate) resulted in enhanced biodegradation of this nematicide in two United Kingdom soils with a high pH (>/= 7.7) . In contrast, degradation of fenamiphos was slow in three acidic United Kingdom soils (pH 4.7 to 6.7), and repeated treatments did not result in enhanced biodegradation . Rapid degradation of fenamiphos was observed in two Australian soils (pH 6.7 to 6.8) in which it was no longer biologically active against plant nematodes . Enhanced degrading capability was readily transferred from Australian soil to United Kingdom soils, but only those with a high pH were able to maintain this capability for extended periods of time . This result was confirmed by fingerprinting bacterial communities by 16S rRNA gene profiling of extracted DNA . Only United Kingdom soils with a high pH retained bacterial DNA bands originating from the fenamiphos-degrading Australian soil . A degrading consortium was enriched from the Australian soil that utilized fenamiphos as a sole source of carbon . The 16S rRNA banding pattern (determined by denaturing gradient gel electrophoresis) from the isolated consortium migrated to the same position as the bands from the Australian soil and those from the enhanced United Kingdom soils in which the Australian soil had been added . When the bands from the consortium and the soil were sequenced and compared they showed between 97 and 100% sequence identity, confirming that these groups of bacteria were involved in degrading fenamiphos in the soils . The sequences obtained showed similarity to those from the genera Pseudomonas, Flavobacterium, and CAULOBACTER: In the Australian soils, two different degradative pathways operated simultaneously: fenamiphos was converted to fenamiphos sulfoxide (FSO), which was hydrolyzed to the corresponding phenol (FSO-OH) or was hydrolyzed directly to fenamiphos phenol . In the United Kingdom soils in which enhanced degradation had been induced, fenamiphos was oxidized to FSO and then hydrolyzed to FSO-OH, but direct conversion to fenamiphos phenol did not occur.

J Fish Dis, 2003 Oct, 26(10), 563 - 74
Flavobacterium psychrophilum infections in salmonid fish; Nematollahi A et al.; Flavobacterium psychrophilum is the causative agent of bacterial cold water disease and rainbow trout fry syndrome, disease entities responsible for substantial economic losses in salmonid aquaculture . Problems associated with epizootics include high mortality rate, increased susceptibility to other diseases, high labour costs of treatment and the enormous expenditure on chemotherapy . Despite the increasing significance of the disease, the pathogenesis of F . psychrophilum infections has only been partially elucidated, hampering the development of preventive measures to efficiently combat this disease condition . This literature review discusses the agent and the disease it causes, with emphasis on the bacterium-host interactions.

J Appl Microbiol, 2003, 95(5), 1008 - 15
Chloramphenicol and florfenicol susceptibility of fish-pathogenic bacteria isolated in France: comparison of minimum inhibitory concentration, using recommended provisory standards for fish bacteria; Michel C et al.; AIM: To investigate the distribution of antimicrobial resistance to phenicols in the fish pathogenic bacteria Aeromonas salmonicida, motile Aeromonas, Yersinia ruckeri, lactic bacteria and the nutritionally fastidious Flavobacterium psychrophilum . The last species was screened on two media (diluted Mueller-Hinton and peptone-enriched Anacker and Ordal), both supplemented with horse serum . METHODS AND RESULTS: Minimal inhibitory concentration (MIC) assessment, using the agar dilution method according to proposed standards, confirmed that chloramphenicol resistance was more frequent and expressed at higher levels than florfenicol resistance . A significant resistant population, highlighted by the bimodal distribution of MICs, was detected only for chloramphenicol in A . salmonicida . No link could be found with the geographical origin of the isolates or fish species . Other cases of resistance appeared randomly distributed or related to the natural properties of the bacterial species . Although the two media used for testing F . psychrophilum resulted in comparable performances in dilution methods, Anacker and Ordal was more adapted to disc diffusion tests . CONCLUSION: Despite wide use, resistance to florfenicol does not seem to occur frequently in French fish farms . SIGNIFICANCE AND IMPACT OF THE STUDY: It is important to maintain a surveillance, as development of florfenicol resistance has occasionally been documented . For this purpose, and for the species studied in this work, the recently proposed standards appear generally well-adapted.

Appl Environ Microbiol, 2003 Nov, 69(11), 6659 - 68
Microbial community structure in midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae); Egert M et al.; The guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter . In a companion paper (T . Lemke, U . Stingl, M . Egert, M . W . Friedrich, and A . Brune, Appl . Environ . Microbiol . 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle Pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and the presence of a diverse, yet unstudied microbial community . Here, we report on the community structure of bacteria and archaea in the midgut, hindgut, and food soil of P . ephippiata larvae, determined with cultivation-independent techniques . Clone libraries and terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed that the intestines of P . ephippiata larvae contain a complex gut microbiota that differs markedly between midgut and hindgut and that is clearly distinct from the microbiota in the food soil . The bacterial community is dominated by phylogenetic groups with a fermentative metabolism (Lactobacillales, Clostridiales, Bacillales, and Cytophaga-Flavobacterium-Bacteroides {CFB} phylum), which is corroborated by high lactate and acetate concentrations in the midgut and hindgut and by the large numbers of lactogenic and acetogenic bacteria in both gut compartments reported in the companion paper . Based on 16S rRNA gene frequencies, Actinobacteria dominate the alkaline midgut, while the hindgut is dominated by members of the CFB phylum . The archaeal community, however, is less diverse . 16S rRNA genes affiliated with mesophilic Crenarchaeota, probably stemming from the ingested soil, were most frequent in the midgut, whereas Methanobacteriaceae-related 16S rRNA genes were most frequent in the hindgut . These findings agree with the reported restriction of methanogenesis to the hindgut of Pachnoda larvae.

Appl Environ Microbiol, 2003 Nov, 69(11), 6610 - 9
Diversity and structure of bacterial communities in Arctic versus Antarctic pack ice; Brinkmeyer R et al.; A comprehensive assessment of bacterial diversity and community composition in arctic and antarctic pack ice was conducted through cultivation and cultivation-independent molecular techniques . We sequenced 16S rRNA genes from 115 and 87 pure cultures of bacteria isolated from arctic and antarctic pack ice, respectively . Most of the 33 arctic phylotypes were >97% identical to previously described antarctic species or to our own antarctic isolates . At both poles, the alpha- and gamma-proteobacteria and the Cytophaga-Flavobacterium group were the dominant taxonomic bacterial groups identified by cultivation as well as by molecular methods . The analysis of 16S rRNA gene clone libraries from multiple arctic and antarctic pack ice samples revealed a high incidence of closely overlapping 16S rRNA gene clone and isolate sequences . Simultaneous analysis of environmental samples with fluorescence in situ hybridization (FISH) showed that approximately 95% of 4',6'-diamidino-2-phenylindole (DAPI)-stained cells hybridized with the general bacterial probe EUB338 . More than 90% of those were further assignable . Approximately 50 and 36% were identified as gamma-proteobacteria in arctic and antarctic samples,respectively . Approximately 25% were identified as alpha-proteobacteria, and 25% were identified as belonging to the Cytophaga-Flavobacterium group . For the quantification of specific members of the sea ice community, new oligonucleotide probes were developed which target the genera Octadecabacter, Glaciecola, Psychrobacter, Marinobacter, Shewanella, and Polaribacter: High FISH detection rates of these groups as well as high viable counts corroborated the overlap of clone and isolate sequences . A terrestrial influence on the arctic pack ice community was suggested by the presence of limnic phylotypes.

Appl Environ Microbiol, 2003 Nov, 69(11), 6587 - 96
Diversity and abundance of uncultured cytophaga-like bacteria in the Delaware estuary; Kirchman DL et al.; The Cytophaga-Flavobacterium group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material . However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes . To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the Cytophaga-Flavobacterium group and was used to construct a library of these genes from the Delaware Estuary . This library had several novel Cytophaga-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments . An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays . Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean) . The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries . The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea . DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.

Dis Aquat Organ, 2003 Sep 24, 56(2), 115 - 26
Starvation of Flavobacterium psychrophilum in broth, stream water and distilled water; Vatsos IN et al.; Physical changes in Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS), were examined over a 19 wk period of starvation . Bacteria were maintained in either Cytophaga broth, filtered stream water, or filtered distilled water, or were maintained in broth after disinfection as a negative control for dead bacteria . Culturability and viability of the bacterium were assessed using colony-forming units (CFUs) and a commercially available live/dead kit . Antigenic profiles and general morphology of the bacterium were also examined using Western blot analysis and electron microscopy, respectively . The bacterium appeared to stop multiplying and became smaller and rounded when maintained in stream water . Its culturability declined until it was no longer possible to obtain colonies on agar plates at the end of the trial at 19 wk, and results from the live/dead kit did not correspond with the viability obtained as CFUs in culture . However, it was still possible to obtain growth of the bacterium after 36 wk with a resuscitation step in Cytophaga broth . Bacteria maintained in distilled water or treated with a disinfectant appeared non-viable using the live/dead kit and were unable to grow on agar 1 h after setting up the experiment; no morphological changes were observed in the bacteria maintained under these conditions . Bacteria maintained in broth were present as long, slim rods, some of which developed into 'ring' formations . Small differences were observed in the antigen profiles of the bacteria maintained under the different treatments, possibly due to a reduction in the size and metabolism of the bacteria . There was also a marked decline in the sensitivity of the PCR with bacteria maintained under the different treatments 14 wk from the onset of the study.

Vet Parasitol, 2003 Nov 3, 117(1-2), 117 - 22
Concomitant exposure of rainbow trout fry to Gyrodactylus derjavini and Flavobacterium psychrophilum: effects on infection and mortality of host; Busch S et al.; Although rainbow trout fry mortality syndrome caused by the bacterium Flavobacterium psychrophilum is widespread in fish farms it is difficult to reproduce infection of rainbow trout in the laboratory using immersion exposure with bacterial suspensions . It has therefore been speculated that ectoparasites could act as enhancers of bacterial infections under natural conditions . In the present study rainbow trout fry were exposed to infections with F . psychrophilum (immersion for 30 min or 10 h) alone, exposed to the ectoparasitic monogenean Gyrodactylus derjavini alone or exposed to both pathogens in combination . Infection levels and host mortality were subsequently monitored to elucidate if the ectoparasitic monogeneans could enhance infection of fish with the bacterium . Immersion of fish in bacterial suspensions alone did not result in infection . Only one fish became infected with the bacterium and this fish belonged to the combination exposure group . The parasite populations increased differently in the various groups and it was found that host mortality was correlated to gyrodactylid infection levels (r=0.94) but not to bacterial exposure . The results emphasise the pathogenicity of the parasite G . derjavini, the relative resistance of intact fish to direct exposure to F . psychrophilum but provide only a weak indication of a possible enhancement of bacterial invasion due to ectoparasitic infections . It cannot be excluded that higher parasite burdens and/or prolonged immersion (more than 10 h) in bacterial suspensions may result in bacterial invasion.

J Bacteriol, 2003 Nov, 185(22), 6648 - 57
Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization; McBride MJ et al.; Cells of Flavobacterium johnsoniae move rapidly over surfaces by gliding motility . The mechanism of this form of motility is not known . Six genes (gldA, gldB, gldD, gldF, gldG, and ftsX) that are required for gliding have been described . Tn4351 mutagenesis was used to identify another gene, gldH, which is required for cell movement . GldH mutants formed nonspreading colonies, and individual cells lacked the cell movements and ability to propel latex spheres along their surfaces that are characteristic of wild-type cells . gldH mutants also failed to digest chitin and were resistant to bacteriophages that infect wild-type cells . Introduction of pMM293, which carries wild-type gldH, restored to the gldH mutants colony spreading, cell motility, the ability to move latex spheres, phage sensitivity, and the ability to digest chitin . gldH encodes a predicted 141-amino-acid protein that localized to the membrane fraction . Labeling studies with {3H}palmitate demonstrated that GldH is a lipoprotein . GldB and GldD, which were previously described, also appear to be lipoproteins . GldH does not exhibit significant amino acid similarity to proteins of known function in the databases . Putative homologs of gldH of unknown function are found in motile (Cytophaga hutchinsonii) and apparently nonmotile (Bacteroides thetaiotaomicron, Bacteroides fragilis, Tannerella forsythensis, Porphyromonas gingivalis, and Prevotella intermedia) members of the Cytophaga-Flavobacterium-Bacteroides group.

J Endotoxin Res, 2003, 9(5), 301 - 7
Molecular basis for lipopolysaccharide mimetic action of Taxol and flavolipin; Kawasaki K et al.; We previously reported that Taxol, which mimics the action of LPS on murine macrophages, induces signals via mouse TLR4/MD-2, but not via human TLR4/MD-2 . Here we investigated the molecular basis for this species-specific action of Taxol . Expression of mouse MD-2 conferred both LPS and Taxol responsiveness on HEK293 cells expressing mouse TLR4, whereas expression of human MD-2 conferred LPS responsiveness alone, suggesting that MD-2 is responsible for the species-specificity of Taxol responsiveness . Furthermore, mouse MD-2 mutants, in which Gln-22 was changed to other amino acids, showed dramatically reduced ability to confer Taxol responsiveness, although their ability to confer LPS responsiveness was not affected . These results indicated that Gln-22 of mouse MD-2 is essential for Taxol signaling, but not for LPS signaling . In this study, we also found that the TLR4/MD-2 complex, together with CD14, mediated signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum.

J Fish Dis, 2003 Sep, 26(9), 553 - 61
Feed deprivation of channel catfish, Ictalurus punctatus (Rafinesque), influences organosomatic indices, chemical composition and susceptibility to Flavobacterium columnare; Shoemaker CA et al.; Withholding feed has been suggested as a strategy to manage infectious disease of channel catfish, Ictalurus punctatus (Rafinesque) . In a previous study, we demonstrated that deprivation of feed for as little as 7 days reduced innate resistance of catfish to Flavobacterium columnare . This study was conducted to evaluate the effect of feeding regimens {no feeding (NF), fed once every other day to satiation (FEOD) and fed once daily to satiation (FD)} on organosomatic indices, physiological changes and susceptibility of channel catfish to F . columnare . Fish that were not fed for 2 and 4 weeks had a significant increase (P < 0.05) in gutted weight:-wet weight ratio and decrease in other organosomatic indices {gut index (GI), mesenteric fat index (MFI) and hepatosomatic index (HSI)} . Haematology was not effected by feeding regimen except at week 4, when a significantly higher haemoglobin level was observed in the NF fish . Serum protein did not differ at week 2, but the level at week 4 of the NF fish (35.91 mg mL(-1)) was significantly lower than that of the FD fish (41.77 mg mL(-1)) . Significantly lower (P < 0.05) blood glucose (39.5 and 40.3 mg dL(-1)) and liver glycogen (1.7 and 1.8 mg g(-1)) were seen in the NF fish at weeks 2 and 4, respectively, as compared with blood glucose and liver glycogen levels of FD fish (67.5 and 92.8 mg dL(-1) and 46.5 and 52.6 mg g(-1) at weeks 2 and 4, respectively) and FEOD (82.8 and 85.5 mg dL(-1) and 45.1 and 51.4 mg g(-1) at weeks 2 and 4, respectively) . Mortality in the NF fish caused by F . columnare (78%) was significantly higher (P < 0.05) than mortality in the FD and FEOD treatments (0.0 and 1.7%, respectively) . Blood glucose and liver glycogen showed the same trend of low values for NF fish following challenge (week 6) . Blood glucose, liver glycogen, GI and HSI are sensitive indicators for channel catfish deprived of feed (NF) for 4 weeks . Blood glucose and liver glycogen levels around 40 mg dL(-1) and 2 mg g(-1), respectively, are indicative of starvation in juvenile channel catfish . Moreover, NF fish were susceptible to F . columnare infection . Thus, it is suggested that in the absence of natural food, juvenile channel catfish should be fed at least once every other day to apparent satiation to maintain normal physiological function and improve resistance to F . columnare.

J Fish Dis, 2003 Sep, 26(9), 529 - 38
Pathology and immunohistochemistry in three species of salmonids after experimental infection with Flavobacterium psychrophilum; Ekman E et al.; Rainbow trout, Oncorhynchus mykiss (Walbaum), sea trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., were experimentally infected with Flavobacterium psychrophilum in order to evaluate any species differences in susceptibility to the bacterium . Furthermore, differences in pathological changes and distribution of the bacteria in internal organs were studied . The bacteria were injected intraperitoneally in two doses, high dose (Hd) 1 x 10(7) colony forming units (CFU) fish(-1) and low dose (Ld) 1 x 10(6) CFU fish(-1) . The mortalities in the Ld groups varied between 0 and 7.5% and in the Hd groups between 55-70% . No significant differences in mortality between the species were recorded . Clinical signs and pathological findings were similar in the three species and in accordance with those of rainbow trout fry syndrome . Rainbow trout showed more pronounced lesions in the spleen compared with the other species . Necrosis of renal tubular epithelium and haematopoietic tissue was most prominent in rainbow trout and Atlantic salmon . Intracellular eosinophilic droplets in the kidney tubular epithelium were a prominent finding in rainbow trout and sea trout surviving the infection . The distribution of the bacteria in internal organs was similar in the three species, as studied with immunohistochemistry.

J Biochem (Tokyo), 2003 Sep, 134(3), 365 - 71
Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium; Yapeng C et al.; A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock . It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold . MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme . Peptide mass spectrum showed poor homogeneity with the database in the peptide bank . Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity . K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively . The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung . The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4% . Thus modification of N-site in heparin structure was favorable for heparinase activity . On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150% . Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources.

FEMS Microbiol Lett, 2003 Sep 26, 226(2), 273 - 9
Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum; Secades P et al.; To go further into the characterization of the proteolysis exocellular system of the salmonid pathogen Flavobacterium psychrophilum, the purification and characterization of a novel protease designated Fpp2 (F . psychrophilum protease 2) was undertaken . A protease (Fpp2) hydrolyzing azocasein was purified . The Fpp2 can be defined as a metalloprotease, it had an estimated molecular mass of 62 kDa with calcium playing an important role in the thermostability of the enzyme . Proteolytic activity was optimal at pH 6.0-7.0 and 24 degrees C and activation energy for the hydrolysis of azocasein was determined to be 5.4 kcal mol(-1), being inactive at temperatures above 42 degrees C . All these results are characteristic of 'cold adapted enzymes' . Fpp2 proved to be a broad range hydrolytic enzyme because in optimal conditions it was able to hydrolyze matrix and muscular proteins . It can be concluded that the Fpp1, a previously characterized 55 kDa metalloprotease, and the Fpp2 protease were produced under different physiological conditions and were immunologically as well as biochemically different.

Fish Shellfish Immunol, 2003 Nov, 15(5), 387 - 95
Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro; Wiklund T et al.; The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro . The results showed that F . psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined . These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes . A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F . psychrophilum to phagocytes . A significant dose dependent inhibition of the association was observed with sialic acid . Treatment of F . psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes . The results indicate that the binding of F . psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion . All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F . psychrophilum

Vet Res Commun, 2003 Sep, 27 Suppl 1, 471 - 9
Emerging pathologies in aquaculture: effects on production and food safety; Ghittino C et al.; Infectious diseases represent a limiting factor for the further development of Italian aquaculture . The recent introduction and spreading of new pathogens, along with the global climatic change, has contributed to a considerable decrease in trout production . Emerging pathologies in rainbow trout culture include viral diseases, e.g . infectious haematopoietic necrosis (IHN), bacterial diseases, such as lactococcosis and visceral flavobacteriosis, and parasitical diseases, e.g . proliferative kidney disease (PKD) . Higher mortality rates in trout fry and fingerlings are generally induced by visceral flavobacteriosis and IHN, while the main losses in large trout during the warm season are due to lactococcosis and PKD . Mariculture has at present a better sanitary status compared to trout culture, but a rapid dissemination of pathogens, including zoonosis agents, is envisaged also for seabass and seabream . Emerging pathologies in sea bass include VNN, pseudotuberculosis, streptococcosis and tuberculosis . Seabream is much more resistant and is mainly affected by novel Vibrio infections and enteromyxidiosis . A good sanitary management of fish farms is essential for avoiding or limiting losses caused by emerging pathologies . Transmission of zoonosis agents to man, through the consumption of cultured fish, is very remote in Italy . On the contrary, transmission of Streptococcus iniae, Vibrio vulnificus and Mycobacterium marinum by means of improper manipulation of infected fish, could represent a potential hazard for fish farmers and fish processors, as well as for people preparing fish meals.

J Fish Dis, 2003 Aug, 26(8), 461 - 7
Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque); Bader JA et al.; The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product . Evaluations were conducted following an abrasion bath immersion challenge with F . columnare . Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS) . The FvpF1-FvpR1 primer set was useful in detecting the early entry of F . columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C . Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney . These differences were not significant, except in the case of blood . Mucus, skin and gill tissues were positive for F . columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish) . CPS following challenge with F . columnare was significantly affected by abrasion, which supports the use of abrasion for the F . columnare challenge model for channel catfish.

Environ Microbiol, 2003 Oct, 5(10), 961 - 76
Bacterial diversity in hydrothermal sediment and epsilonproteobacterial dominance in experimental microcolonizers at the Mid-Atlantic Ridge; Lopez-Garcia P et al.; We report here a molecular survey based on 16S rRNA genes of the bacterial diversity found in two deep-sea vent niches at the Mid-Atlantic Ridge: hydrothermal sediment (Rainbow site), and microcolonizers made of three different substrates (organic-rich, iron-rich and pumice) that were exposed for 15 days to a vent emission . Bacterial diversity in sediment samples was scattered through many bacterial divisions . The most abundant and diverse environmental sequences (phylotypes) in our libraries corresponded to the Gammaproteobacteria, followed by the Acidobacteria . We detected members of all the subdivisions within the Proteobacteria . Myxobacterial lineages were the most represented within the delta subdivision . Phylotypes ascribing to the Cytophaga-Flavobacterium-Bacteroides, Planctomycetales, high and low G + C Gram-positives, Nitrospirae, and the candidate division TM7 were also identified . Compared to this broad taxonomic coverage, microcolonizers were almost exclusively colonized by epsilonproteobacteria, although these exhibited considerable morphological and phylogenetic in-group diversity . No specificity for any of the substrates tested was seen . This observation further supports the idea of the ecological dominance of epsilonproteobacteria in the fluid-seawater interface environment . Because oxidation of reduced S species and/or sulphur-reduction is thought to be essential for their energetic metabolism in these areas, we mapped different oxidation states of S in individual bacterial filaments from the iron-rich microcolonizer . For this, we used high-resolution, non-destructive synchrotron micro-X-ray Absorption Near-Edge Spectroscopy (micro-XANES), which revealed the co-existence of different S oxidation states, from sulphide to sulphate, at the level of individual cells . This suggests that these cells were metabolizing sulphur in situ.

Environ Microbiol, 2003 Oct, 5(10), 896 - 907
Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil; Gremion F et al.; Bacterial diversity in 16S ribosomal DNA and reverse-transcribed 16S rRNA clone libraries originating from the heavy metal-contaminated rhizosphere of the metal-hyperaccumulating plant Thlaspi caerulescens was analysed and compared with that of contaminated bulk soil . Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Acidobacteria and the Planctomycetales . Only 14.7% of all phylotypes (sequences with similarities> 97%), but 45% of all clones, were common in the rhizosphere and the bulk soil clone libraries . The combined use of rDNA and rRNA libraries indicated which taxa might be metabolically active in this soil . All dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries compared with the rDNA libraries . Clones belonging to the Verrucomicrobiales, Firmicutes, Cytophaga-Flavobacterium-Bacteroides and OP10 were found only in rDNA clone libraries, indicating that they might not represent active constituents in our samples . The most remarkable result was that sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA (50% and 60% of all phylotypes respectively) . Seventy per cent of these clone sequences were related to the Rubrobacteria subgroups 2 and 3, thus providing for the first time evidence that this group of bacteria is probably metabolically active in heavy metal-contaminated soil.

Carbohydr Res, 2003 Sep 26, 338(20), 2101 - 4
Chondroitin O-methyl ester: an unusual substrate for chondroitin AC lyase; Avci FY et al.; Chondroitin O-methyl ester was depolymerized by chondroitin AC lyase (EC 4.2.2.5) from Flavobacterium heparinum . The major product isolated from the depolymerization reaction was found to be methyl alpha-L-threo-hex-4-enopyranosyluronate-(1-->4)-2-acetamido-2-deoxy-alpha,beta-D-galactopyranoside.

J Burn Care Rehabil, 2003 Sep-Oct, 24(5), 285 - 8
Fusarium infections in burn patients: a case report and review of the literature; Latenser BA; Fusarium species, or saprophytic molds, are important plant pathogens and recognized as agents of human mycotic infections . Frequently superficial, deep-tissue involvement, and dissemination occurs in immunocompromised hosts with hematologic malignancies, aplastic anemia, and chemotherapy treatment . Presented in this work is a burn patient with a fatal disseminated infection in addition to a review of the literature . A 40-year-old white male acquired a 73% grease scald injury at work . His hospital course was interspersed with multiple episodes of Flavobacterium, Fusarium, Candida, Proteus, Enterococcus, coagulase-negative Staphylococcus, and Serratia infections . He underwent nine operative procedures for debridement, excision, and skin grafting . The last operative procedure included bilateral below knee amputations to halt an invasive Fusarium infection that was invading normal unburned skin . The patient died 55 days after injury . Fusarium and Aspergillus infections are frequently confused and do not have characteristic clinical features . With the prolonged survival of severely burned patients, better fungal diagnostic and treatment modalities are needed to improve outcome.

Dis Aquat Organ, 2003 Aug 4, 55(3), 261 - 4
Efficacy of oral vaccine against bacterial coldwater disease in ayu Plecoglossus altivelis; Kondo M et al.; The development of a practical vaccination method against bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis and the efficacy of oral administration of formalin-killed cells (FKCs) of Flavobacterium psychrophilum was investigated . The FKC was administrated at a dose of 0.1-0.2 g kg(-1) body weight to juvenile ayu (0.5 g body weight) every day for 2 wk or on 5 days over 2 wk . Experimental immersion challenge at 3 and 7 wk after vaccination showed significantly higher survival rates than the controls . The results show the effectiveness of oral vaccination against BCWD in ayu.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1343 - 55
Algoriphagus ratkowskyi gen . nov., sp . nov., Brumimicrobium glaciale gen . nov., sp . nov., Cryomorpha ignava gen . nov., sp . nov . and Crocinitomix catalasitica gen . nov., sp . nov., novel flavobacteria isolated from various polar habitats; Bowman JP et al.; Several cold-adapted strains isolated from a variety of algal-rich Antarctic and Southern Ocean samples formed three distinct groups within the class Flavobacteria, phylogenetically distant from other cultivated species . The first taxon, designated Algoriphagus ratkowskyi gen . nov., sp . nov., was isolated from sea ice and from saline lake cyanobacterial mats and includes non-motile, strictly aerobic, saccharolytic rod-like or serpentine strains that were most closely related to the genus Cyclobacterium according to 16S rDNA sequence analysis (sequence similarity 0.85) . The second taxon, designated Brumimicrobium glaciale gen . nov., sp . nov., isolated from sea ice and from continental shelf sediment, formed gliding, rod-like cells that were facultatively anaerobic with a fermentative metabolism . The third taxon, designated Cryomorpha ignava gen . nov., sp . nov., isolated from Southern Ocean particulates and from quartz stone subliths, included strictly aerobic, pleomorphic rod-like cells . Brumimicrobium glaciale and Cryomorpha ignava were most closely allied with 'Microscilla aggregans var . catalatica', which, on the basis of its distinctive taxonomic traits, is also proposed as a new genus and species, Crocinitomix catalasitica gen . nov., sp . nov . It is proposed that the three genera Brumimicrobium, Cryomorpha and Crocinitomix belong to a new family, Cryomorphaceae fam . nov . (type genus Cryomorpha), as they possess generally similar morphological and ecophysiological characteristics and form a common and distinct clade within class FLAVOBACTERIA:

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1287 - 90
Arenibacter troitsensis sp . nov., isolated from marine bottom sediment; Nedashkovskaya OI et al.; A novel marine, heterotrophic, aerobic, pigmented, non-motile bacterium was isolated from a bottom sediment sample collected from Troitsa Bay in the Gulf of Peter the Great, Sea of Japan, during June 2000 . 16S rDNA sequence analysis revealed that this bacterium was a member of the family FLAVOBACTERIACEAE: On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic analyses, the bacterium was shown to belong to a novel species of the genus Arenibacter, for which the name Arenibacter troitsensis sp . nov . is proposed . The type strain is KMM 3674(T) (=JCM 11736(T)).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1281 - 6
Vitellibacter vladivostokensis gen . nov., sp . nov., a new member of the phylum Cytophaga-Flavobacterium-Bacteroides; Nedashkovskaya OI et al.; A novel heterotrophic, yellow-orange-pigmented, non-motile, asporogenic, strictly aerobic, Gram-negative, oxidase and catalase-positive bacterium KMM 3516(T) was isolated from the holothurian Apostichopus japonicus collected from Troitsa Bay in the Gulf of Peter the Great (Sea of Japan) during November 1997 . 16S rDNA sequence analysis revealed that strain KMM 3516(T) was a member of the family FLAVOBACTERIACEAE: The DNA G+C content of KMM 3516(T) was 41.3 mol% . Major respiratory quinone was MK-6 . Predominant fatty acids were i15 : 0 and alpha15 : 0 (68.8 and 8.4 %, respectively) . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium has been designated Vitellibacter vladivostokensis gen . nov., sp . nov . The type strain is KMM 3516(T) (=NBRC 16718(T)).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1241 - 5
Flavobacterium gelidilacus sp . nov., isolated from microbial mats in Antarctic lakes; Van Trappen S et al.; Twenty-two isolates from microbial mats in eastern Antarctic lakes showed similar fatty acid compositions and were investigated further using a polyphasic taxonomic approach . Repetitive extragenic palindromic DNA-PCR fingerprinting of the 22 strains revealed three groups, and DNA-DNA hybridizations between representatives showed more than 87 % DNA-DNA reassociation with each other . 16S rRNA gene sequence analysis placed two representative strains, LMG 21477(T) and LMG 21619, within the genus Flavobacterium, with 95.1 % sequence similarity to Flavobacterium flevense, 95.0 % to Flavobacterium tegetincola, less than 95 % to other Flavobacterium species and less than 90 % to representatives of other genera . The name Flavobacterium gelidilacus sp . nov . is proposed, with LMG 21477(T) (=DSM 15343(T)) as the type strain, and a description of the species is given on the basis of morphological, biochemical and physiological characteristics and fatty acid composition . The G+C content of the genomic DNA is 30.0-30.4 mol%.

Appl Environ Microbiol, 2003 Sep, 69(9), 5275 - 80
Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum; Moller JD et al.; Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells . Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties . None of the strains was able to cause agglutination of yeast cells . Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains . Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains . HA activities of F . psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin . The supernatant from washed bacterial cells also showed some HA properties . All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen . These results indicate that the aggregation of F . psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F . psychrophilum strains and absent on HA-negative strains . This lectin reacts specifically with sialic acid . The adhesion differences observed for F . psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F . psychrophilum and rainbow trout.

J Bacteriol, 2003 Sep, 185(18), 5591 - 601
Complete genome sequence of the oral pathogenic Bacterium porphyromonas gingivalis strain W83; Nelson KE et al.; The complete 2,343,479-bp genome sequence of the gram-negative, pathogenic oral bacterium Porphyromonas gingivalis strain W83, a major contributor to periodontal disease, was determined . Whole-genome comparative analysis with other available complete genome sequences confirms the close relationship between the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum and the green-sulfur bacteria . Within the CFB phyla, the genomes most similar to that of P . gingivalis are those of Bacteroides thetaiotaomicron and B . fragilis . Outside of the CFB phyla the most similar genome to P . gingivalis is that of Chlorobium tepidum, supporting the previous phylogenetic studies that indicated that the Chlorobia and CFB phyla are related, albeit distantly . Genome analysis of strain W83 reveals a range of pathways and virulence determinants that relate to the novel biology of this oral pathogen . Among these determinants are at least six putative hemagglutinin-like genes and 36 previously unidentified peptidases . Genome analysis also reveals that P . gingivalis can metabolize a range of amino acids and generate a number of metabolic end products that are toxic to the human host or human gingival tissue and contribute to the development of periodontal disease.

J Fish Dis, 2003 Jul, 26(7), 371 - 84
Passive immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), against Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease and rainbow trout fry syndrome; LaFrentz BR et al.; Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS), causes high mortality in cultured salmonids . The present study was designed to determine the role antibody plays in conferring protection to rainbow trout fry, Oncorhynchus mykiss (Walbaum), by passive immunization with convalescent serum or serum from adult rainbow trout immunized with F . psychrophilum, and goat anti-F . psychrophilum serum . In each experiment, rainbow trout fry were injected intraperitoneally with antiserum and challenged by subcutaneous injection with a virulent strain (CSF-259-93) of F . psychrophilum 24-h post-immunization . Relative percentage survival (RPS) ranged from 9-42% when rainbow trout fry (mean weight 1.3 g) were injected with a 1:2 dilution of 25 microL of convalescent serum ranging in enzyme-linked immunosorbent assay antibody titres from 1600-102400 . Rainbow trout fry (mean weight 1.0 g) passively immunized with 25 microL of serum from immunized adult fish exhibited RPS values of up to 57% . In each of these experiments, RPS increased with increasing antibody titres against F . psychrophilum . Passive immunization with 25 or 50 microL goat anti-F . psychrophilum serum, however, did not confer protection to fry (mean weight 1.3 g) . These results suggest that trout antibody plays a role in conferring protection to F . psychrophilum, but antibody alone is unable to provide complete protection.

Mol Biol (Mosk), 2003 Jul-Aug, 37(4), 619 - 24
{The unique FauI restriction-modification system: cloning and comparative analysis of protein structure}; Abdurashitov MA et al.; The nucleotide sequence was established for the full-length Flavobacterium aquatile operon coding for the FauI restriction-modification system . The operon is unusual in structure and has the gene order control protein gene-DNA methyltransferase A gene-restriction endonuclease gene-DNA methyltransferase B gene, other than in the known analogs . The genes are similarly oriented and overlap . On evidence of sequence analysis, both methyltransferases are C5 enzymes, the control protein is similar to that of other restriction-modification systems, and restriction endonuclease is low-homologous to other enzymes cleaving the DNA upper strand in position 4 or 5 relative to the recognition site.

Leg Med (Tokyo), 2001 Dec, 3(4), 193 - 204
The mechanism of human adipocere formation; Takatori T; In adipocere, some specific fatty acids possessing higher melting points, together with soap, play an important role in the formation and stabilization of adipocere . These fatty acids were shown to be mainly 10-hydroxy stearic and 10-hydroxy palmitic acids . Slight amounts of 10-oxo stearic and 10-oxo palmitic acids, which have higher melting points than those of hydroxy fatty acids (OHFAs), exist in the adipocere as well . The substantial adipocere is formed and stabilized mainly by these specific fatty acids . The OHFA and oxo fatty acid (OXOFA) are biosynthesized by some bacterial enzymes . Various aerobic and anaerobic bacteria are involved in the formation of adipocere . For instance, microbial conversion of various unsaturated fatty acids to 10-OHFA by Micrococcus luteus was investigated . It turned out that 10-OHFA was synthesized only from fatty acids possessing cis-9-unsaturatin . It was also shown that 10-OHFAs were converted to the corresponding 10-OXOFAs but 10-OXO compounds were inactive as substrates . It was further found that the enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxy stearic acid, but also dehydrogenation of this product in the presence of deuterium.On the other hand, we found out that there was 10-hydroxy-12-octadecenoic acid (10-OHODA) in the linoleic acid in human adipocere and that there were 9-chloro-10-methoxy (9-methoxy-10-chloro) palmitic acid and 9-chloro-10-methoxy (9-methoxy-10-chloro) stearic acid in human neonate adipocere.

Dis Aquat Organ, 2003 Jul 8, 55(2), 101 - 7
Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow trout Oncorhynchus mykiss; Nematollahi A et al.; The ability of Flavobacterium psychrophilum to adhere to the gill tissue of rainbow trout Oncorhynchus mykiss was evaluated . A gill perfusion model was adopted, offering a number of advantages compared to other in vitro as well as in vivo models . A comparison between the adhesion capacity of a high and low virulence F . psychrophilum strain was made . Experiments were additionally carried out to assess the influence of water quality (organic material, nitrite) and temperature on the adhesion process of the bacterial cells . The high virulence strain attached more readily to the gill tissue than did the low virulence strain . Moreover, the adherence of the high virulence strain of F . psychrophilum was influenced by a number of factors . These were immersion of the gill arches in water to which organic material or nitrite were added, and elevated temperature . The former 2 increased the adhesion ability, while the latter had a negative influence on the adherence process.

Dis Aquat Organ, 2003 Jul 8, 55(2), 93 - 9
Nanoinjection as a tool to mimic vertical transmission of Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss; Ekman E et al.; Newly fertilised eggs of rainbow trout Oncorhynchus mykiss were nanoinjected with Flavobacterium psychrophilum in order to mimic vertical transmission . Two bacterial isolates with different elastin-degrading capacity were used . All infected groups (10, 100 and 1000 colony forming units egg(-1)) showed significantly higher cumulative mortalities than the control groups at the end of the experiment, 70 d post-hatching . The total mortalities in the control groups were below 2.5% . In the high-dose groups, 95 to 100% of the eggs died during the eyed stage . In the intermediate group infected with the elastin-negative isolate, the major mortality occurred during the eyed stage of the egg, with a total cumulative mortality of 83% at the end of the experiment . In the intermediate group infected with the elastin-positive isolate, a total mortality of 63% was recorded . In this group, diseased fry showed clinical signs of disease and morphological changes similar to those described in connection with rainbow trout fry syndrome (RTFS) shortly after the beginning of feeding . In the low dose groups, the mortality in the elastin-negative group was 14% and in the elastin-positive group 11% . The bacterium was isolated from dead eggs and fry in infected groups and demonstrated in internal organs of dead and moribund fry by immunohistochemistry . The nanoinjection method used in this study may be a useful method to study pathogens, like F . psychrophilum, that can be vertically transmitted.

Extremophiles, 2003 Aug, 7(4), 275 - 82 Epub 2003 Apr 09.
High 16S rDNA bacterial diversity in glacial meltwater lake sediment, Bratina Island, Antarctica; Sjoling S et al.; The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny . Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out . Results revealed the presence of a highly diverse bacterial population and a significantly depth-related composition . Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'G/CGC3'and Msp I . 5'C/CGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units) . However, only seven archaeal OTUs were detected, indicating low archaeal diversity . Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the alpha, gamma, and delta subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides, the Spirochaetaceae, and the Actinobacteria . All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.

Eur J Biochem, 2003 Aug, 270(16), 3440 - 6
Structural characterization of the lipopolysaccharide O-polysaccharide antigen produced by Flavobacterium columnare ATCC 43622; MacLean LL et al.; The structure of the antigenic O-chain polysaccharide of Flavobacterium columnare ATCC 43622, a Gram-negative bacterium that causes columnaris disease in warm water fish, was determined by high-field 1D and 2D NMR techniques, MS, and chemical analyses . The O-chain was shown to be an unbranched linear polymer of a trisaccharide repeating unit composed of 2-acetamido-2-deoxy-d-glucuronic acid (d-GlcNAcA), 2-acetamidino-2,6-dideoxy-l-galactose (l-FucNAm) and 2-acetamido-2,6-dideoxy-d-xylo-hexos-4-ulose (d-Sug) (1 : 1 : 1), having the structure: {structure: see text}.

Biotechnol Lett, 2003 Jul, 25(13), 1007 - 11
Resonant Raman quantification of zeaxanthin production from Flavobacterium multivorum; Bhosale P et al.; Resonant Raman scattering was used as a novel, rapid, non-destructive optical technique to measure zeaxanthin levels in Flavobacterium multivorum ATCC 55238 . Culture broth, after bacterial growth for 40 h, exhibited characteristic resonance Raman vibrational modes at 1159 cm(-1) (C-C stretch) and 1525 cm(-1) (C=C stretch) upon excitation at 488 nm . A striking correlation was observed between the carotenoid level as estimated by HPLC and by resonance Raman spectroscopy.

Appl Environ Microbiol, 2003 Jul, 69(7), 3701 - 9
Heterotrophic bacterial growth efficiency and community structure at different natural organic carbon concentrations; Eiler A et al.; Batch cultures of aquatic bacteria and dissolved organic matter were used to examine the impact of carbon source concentration on bacterial growth, biomass, growth efficiency, and community composition . An aged concentrate of dissolved organic matter from a humic lake was diluted with organic compound-free artificial lake water to obtain concentrations of dissolved organic carbon (DOC) ranging from 0.04 to 2.53 mM . The bacterial biomass produced in the cultures increased linearly with the DOC concentration, indicating that bacterial biomass production was limited by the supply of carbon . The bacterial growth rate in the exponential growth phase exhibited a hyperbolic response to the DOC concentration, suggesting that the maximum growth rate was constrained by the substrate concentration at low DOC concentrations . Likewise, the bacterial growth efficiency calculated from the production of biomass and CO(2) increased asymptotically from 0.4 to 10.4% with increasing DOC concentration . The compositions of the microbial communities that emerged in the cultures were assessed by separation of PCR-amplified 16S rRNA fragments by denaturing gradient gel electrophoresis . Nonmetric multidimensional scaling of the gel profiles showed that there was a gradual change in the community composition along the DOC gradient; members of the beta subclass of the class Proteobacteria and members of the Cytophaga-Flavobacterium group were well represented at all concentrations, whereas members of the alpha subclass of the Proteobacteria were found exclusively at the lowest carbon concentration . The shift in community composition along the DOC gradient was similar to the patterns of growth efficiency and growth rate . The results suggest that the bacterial growth efficiencies, the rates of bacterial growth, and the compositions of bacterial communities are not constrained by substrate concentrations in most natural waters, with the possible exception of the most oligotrophic environments.

Water Sci Technol, 2003, 47(9), 123 - 8
Biodegradation of MTBE and BTEX in an aerobic fluidized bed reactor; Pruden A et al.; An aerobic fluidized bed reactor (FBR) was operated for the removal of methyl tert-butyl (MBE) and benzene, toluene, ethylbenzene, and p-xylene (BTEX) from water . The reactor was seeded with a mixed culture adapted to MTBE . Granular activated carbon (GAC) was used as the biological attachment medium . Influent MTBE to the reactor was 7.8 mg/L MTBE, with a flow rate of 22.7 L/day, and an empty bed contact time of 1 hour . The acclimation period required was relatively short, about 30 days before reaching an average stable effluent concentration of 18.5 +/- 10 microg/L . BTEX was introduced to the feed at an equivalent chemical oxygen demand (COD) as the MTBE at day 225 and was biodegraded spontaneously with no apparent acclimation period required . The average influent of each of the four BTEX compounds was about 2 mg/L, and the range of the average effluent concentrations wae 1.4-2.2 microg/L . After achieving 180 days of stable performance with BTEX addition, the total low rate to the reactor was gradually increased by 20% increments to 160% of the original flow (36.4 L/day) . Increases by 20% and 40% had no apparent effect on reactor performance, but increase by 60% required 30 days before effluent quality returned to previous values . Composition of the culture was monitored throughout operation of the reactor using denaturing gradient gel electrophoresis (DGGE) . The culture consisted of Flavobacteria-Cytophaga and organisms with high similarity to the known MTBE degrader PM1.

Extremophiles, 2003 Oct, 7(5), 377 - 84 Epub 2003 Jun 19.
Isolation and characterization of marine psychrophilic phage-host systems from Arctic sea ice; Borriss M et al.; Phage-host systems from extreme cold environments have rarely been surveyed . This study is concerned with the isolation and characterization of three different phage-host systems from Arctic sea ice and melt pond samples collected north-west of Svalbard (Arctic) . On the basis of 16S rDNA sequences, the three bacterial phage hosts exhibited the greatest similarity to the species Shewanella frigidimarina (96.0%), Flavobacterium hibernum (94.0%), and Colwellia psychrerythraea (98.4%), respectively . The host bacteria are psychrophilic with good growth at 0 degrees C, resulting in a rapid formation of visible colonies at this temperature . The phages showed an even more pronounced adaptation to cold temperatures than the bacteria, with growth maxima below 14 degrees C and good plaque formation at 0 degrees C . Transmission electron microscopy (TEM) examinations revealed that the bacteriophages belonged to the tailed, double-stranded DNA phage families Siphoviridae and Myoviridae . All three phages were host-specific.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 853 - 7
Flavobacterium xinjiangense sp . nov . and Flavobacterium omnivorum sp . nov., novel psychrophiles from the China No . 1 glacier; Zhu F et al.; Two novel psychrophilic bacterial strains (ZF-6(T) and ZF-8(T)) were isolated from the China No . 1 glacier . Polyphasic taxonomy using physiological and biochemical properties and phylogenetic analysis based on 18S rRNA gene sequences showed that the two isolates belonged to the genus Flavobacterium, and that they were distinct from each other and also from the known species of this genus . Strains ZF-6(T) and ZF-8(T) are Gram-negative and both have an optimal growth temperature of 11 degrees C . Strain ZF-6(T) is able to grow at 0-20 degrees C, the G + C content of its genomic DNA is 34.4 mol% and the major fatty acids of ZF-6(T) are C(16 : 1)omega7c (17.7%) and C15 : 1)omega6c (12.7%) . Strain ZF-8(T) showed a strong ability to degrade organic macromolecules such as starch, CM-cellulose, pectin and chitin . Its DNA G + C content is 35.1 mol%, and the major fatty acids are C(16 : 1)omega7C (18.2%) and C(15 : 0) (9.9%) . Phylogenetic analysis based on 16S rDNA sequences indicated that ZF-6(T) and ZF-8(T) belong to the genus Flavobacterium and represent two novel species . DNA-DNA hybridization also supported the status of the two new isolates . The names Flavobacterium xinjiangense sp . nov . (type strain, ZF-6(T) = AS 1.2749(T) = JCM 11314(T)) and Flavobacterium omnivorum sp . nov . (type strain, ZF-8(T) = AS 1.2747(T) = JCM 11313(T)) are proposed for the two new isolates.

Appl Environ Microbiol, 2003 Jun, 69(6), 3607 - 16
Combining culture-dependent and -independent methodologies for estimation of richness of estuarine bacterioplankton consuming riverine dissolved organic matter; Kisand V et al.; Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM) . We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample . Forty-two different cultivatable strains were isolated from rich and poor solid media . In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector . Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group . The different methods gave similar distributions of taxa at the genus level and higher . However, the match at the species level among the methods was poor, and only one species was identified by all three methods . Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system . The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species . Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied . Culture-independent methods also suggested several not-yet-cultivated beta-proteobacteria to be RDOM consumers.

Appl Environ Microbiol, 2003 Jun, 69(6), 3280 - 7
Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid Southwestern soils; Bodour AA et al.; Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications in the remediation of organic- and metal-contaminated sites, in the enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control . However, little is known about the distribution of biosurfactant-producing bacteria in the environment . The goal of this study was to determine how common culturable surfactant-producing bacteria are in undisturbed and contaminated sites . A series of 20 contaminated (i.e., with metals and/or hydrocarbons) and undisturbed soils were collected and plated on R(2)A agar . The 1,305 colonies obtained were screened for biosurfactant production in mineral salts medium containing 2% glucose . Forty-five of the isolates were positive for biosurfactant production, representing most of the soils tested . The 45 isolates were grouped by using repetitive extragenic palindromic (REP)-PCR analysis, which yielded 16 unique isolates . Phylogenetic relationships were determined by comparing the 16S rRNA gene sequence of each unique isolate with known sequences, revealing one new biosurfactant-producing microbe, a Flavobacterium sp . Sequencing results indicated only 10 unique isolates (in comparison to the REP analysis, which indicated 16 unique isolates) . Surface tension results demonstrated that isolates that were similar according to sequence analysis but unique according to REP analysis in fact produced different surfactant mixtures under identical growth conditions . These results suggest that the 16S rRNA gene database commonly used for determining phylogenetic relationships may miss diversity in microbial products (e.g., biosurfactants and antibiotics) that are made by closely related isolates . In summary, biosurfactant-producing microorganisms were found in most soils even by using a relatively limited screening assay . Distribution was dependent on soil conditions, with gram-positive biosurfactant-producing isolates tending to be from heavy metal-contaminated or uncontaminated soils and gram-negative isolates tending to be from hydrocarbon-contaminated or cocontaminated soils.

J Microbiol Methods, 2003 Aug, 54(2), 183 - 91
Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences; Kisand V et al.; The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method . The occurrence of multiple melting domains and poor coupling between Tm and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities . We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus . Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers.

J Appl Microbiol, 2003, 94(6), 1120 - 7
Purification and characterization of a membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum; Merle C et al.; AIMS: The cell envelope of the fish pathogen Flavobacterium psychrophilum contains more than 50 polypeptides resolved by sodium dodecyl sulphate-polyacrylaminde gel electrophoresis analysis including a major component named P60 . Here, we have developed a simple and efficient procedure for the purification of P60 and therefore permitting its biochemical characterization . METHODS AND RESULTS: Membrane proteins were selectively extracted from isolated cell envelopes with the mild non-ionic detergent Triton X-100 . About 10 polypeptides were identified from the detergent fraction, including P60 . The P60-enriched fraction was thereafter subjected to an anion exchange chromatographic step in the presence of Triton X-100 . The molecule was purified at the milligram level (yield, about 75%; purification factor, 6.2) . Analyses performed by charge shift electrophoresis, Triton X-114 phase separation and by detection of sugar-modified components showed that P60 is a true amphiphilic membrane-associated glycoprotein . CONCLUSIONS: The method described in this paper provides pure and non-denaturated P60 and should prove to be easily scaled-up . As sugar-modified protein, P60 should be included in the growing list of glycosylated prokaryotic proteins . SIGNIFICANCE AND IMPACT OF THE STUDY: It offers the possibility of obtaining P60 in amounts allowing the testing of the potential of P60 as a candidate for anti-flavobacteria subunit vaccines, as P60 is one of the major antigens.

Eur J Biochem, 2003 May, 270(10), 2332 - 41
Covalent and three-dimensional structure of the cyclodextrinase from Flavobacterium sp . no . 92; Fritzsche HB et al.; Starting with oligopeptide sequences and using PCR, the gene of the cyclodextrinase from Flavobacterium sp . no . 92 was derived from the genomic DNA . The gene was sequenced and expressed in Escherichia coli; the gene product was purified and crystallized . An X-ray diffraction analysis using seleno-methionines with multiwavelength anomalous diffraction techniques yielded the refined 3D structure at 2.1 A resolution . The enzyme hydrolyzes alpha(1,4)-glycosidic bonds of cyclodextrins and linear malto-oligosaccharides . It belongs to the glycosylhydrolase family no . 13 and has a chain fold similar to that of alpha-amylases, cyclodextrin glycosyltransferases, and other cyclodextrinases . In contrast with most family members but in agreement with other cyclodextrinases, the enzyme contains an additional characteristic N-terminal domain of about 100 residues . This domain participates in the formation of a putative D2-symmetric tetramer but not in cyclodextrin binding at the active center as observed with the other cyclodextrinases . Moreover, the domain is located at a position quite different from that of the other cyclodextrinases . Whether oligomerization facilitates the cyclodextrin deformation required for hydrolysis is discussed.

Syst Appl Microbiol, 2003 Mar, 26(1), 76 - 83
Croceibacter atlanticus gen . nov., sp . nov., a novel marine bacterium in the family Flavobacteriaceae; Cho JC et al.; A bright, saffron-colored marine bacterium HTCC2559T was isolated from the Bermuda Atlantic Time Series station in the western Sargasso Sea, Atlantic Ocean by high throughput culturing methods and characterized by polyphasic approaches . Phenotypic data and phylogenetic analyses showed that the strain is a member of the family Flavobacteriaceae . The strain was gram-negative, non-motile, chemoheterotrophic, strictly aerobic, NaCl-requiring, rod-shaped cells that contain carotenoid pigments but not flexirubin . Several kinds of macromolecules (gelatin, DNA, starch, casein, and elastin) were degraded and carbohydrates, sugar alcohols, organic acids, and amino acids were utilized as sole carbon sources . The dominant fatty acids were branched or hydroxy acids, and 3-OH i17:0, i15:0, i15:1, and i17:1 omega9c were abundant . The DNA G+C content of the strain is 34.8 mol% . Phylogenetic analyses using three treeing algorithms based on 16S rRNA gene sequences revealed that the strain formed a very distinct lineage that is allied closely with several seawater environmental clones in the family Flavobacteriaceae . Therefore, it is proposed from the polyphasic studies that strain HTCC2559T (=ATCC BAA-628T = KCTC 12090T) belongs to a new genus and species named Croceibacter atlanticus gen . nov., sp . nov.

Microb Ecol, 2003 Jul, 46(1), 92 - 105 Epub 2003 May 13.
Bacterioplankton community structure in a maritime antarctic oligotrophic lake during a period of holomixis, as determined by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH); Pearce DA; The bacterioplankton community structure in Moss Lake, a maritime Antarctic oligotrophic lake, was determined with vertical depth in the water column, during the ice-free period on Signy Island in the South Orkney Islands . Bacterioplankton community structure was determined using a combination of direct counting of 4',6-diamidino-2-phenylindole (DAPI) stained cells, PCR amplification of 16S rRNA gene fragments, denaturing gradient gel electrophoresis (DGGE) and in situ hybridization with group-specific, fluorescently labeled oligonucleotide probes . Using PCR amplification of 16S rRNA gene fragments and DGGE, the bacterioplankton community composition was shown to be constant with vertical depth in the water column . Specific bacterioplankton species identified through cloning and sequencing the DGGE products obtained were Flavobacterium xinjiangensis (a Flavobacterium), Leptothrix discophora (a beta-Proteobacterium), and a number of uncultured groups: two beta-Proteobacteria, an unclassified Proteobacterium, three sequences from Actinobacteria, and a Cyanobacterium . Fluorescence in situ hybridization (FISH), however, demonstrated that there were minor but significant fluctuations in different groups of bacteria with vertical depth in the water column . It showed that the beta-Proteobacteria accounted for between 26.4 and 71.5%, the alpha-Proteobacteria 2.3-10.6%, the gamma-Proteobacteria 0-29.6%, and the Cytophaga-Flavobacterium group 1.8-23.5% of cells hybridizing to a universal probe . This study reports the first description of the community structure of an oligotrophic Antarctic freshwater lake as determined by PCR-dependent and PCR-independent molecular techniques . It also suggests that the bacterioplankton community of Moss Lake contains classes of bacteria known to be important in freshwater systems elsewhere in the world.

Appl Environ Microbiol, 2003 May, 69(5), 2533 - 9
Transposon-like organization of the plasmid-borne organophosphate degradation (opd) gene cluster found in Flavobacterium sp; Siddavattam D et al.; Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions . All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (opd) located on a large indigenous plasmid . These plasmids show considerable genetic diversity, but the region containing the opd gene is highly conserved . Two opd plasmids, pPDL2 from Flavobacterium sp . and pCMS1 from Pseudomonas diminuta, are well characterized, and in each of them a region of about 5.1 kb containing the opd gene shows an identical restriction pattern . We now report the complete sequence of the conserved region of plasmid pPDL2 . The opd gene is flanked upstream by an insertion sequence, ISFlsp1, that is a member of the IS21 family, and downstream by a Tn3-like element encoding a transposase and a resolvase . Adjacent to opd but transcribed in the opposite direction is an open reading frame (orf243) with the potential to encode an aromatic hydrolase somewhat similar to Pseudomonas putida TodF . We have shown that orf243 encodes a polypeptide of 27 kDa, which plays a role in the degradation of p-nitrophenol and is likely to act in concert with opd in the degradation of parathion . The linkage of opd and orf243, the organization of the genes flanking opd, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.

Appl Environ Microbiol, 2003 May, 69(5), 2448 - 62
Prokaryotic metabolic activity and community structure in Antarctic continental shelf sediments; Bowman JP et al.; The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66 degrees S, 143 degrees E; depth range, 709 to 964 m) were studied . Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity . Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm . Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured . The median optimal growth temperature for the sediment isolates was 15 degrees C . Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species . Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community . Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident . Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea . rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 519 - 26
Flavobacterium limicola sp . nov., a psychrophilic, organic-polymer-degrading bacterium isolated from freshwater sediments; Tamaki H et al.; Three novel strains of cold-adapted bacteria, ST-82T, ST-10 and ST-92, were isolated from freshwater sediments . These three isolates were very similar to each other in phenotypic and chemotaxonomic traits, as well as in 16S rDNA sequence . The strains were Gram-negative, elongated filament-like rods that formed bright yellow colonies . They showed neither flexirubin pigments nor gliding motility . The strains were able to hydrolyse casein, gelatin, starch, agar, aesculin, urea, uric acid and tyrosine . They also lysed cells of Escherichia coil and Pseudomonas putida . The temperature range for growth was 0-25 degrees C, with optimum growth occurring at 15-20 degrees C . For all isolates, protease secretion increased as temperature decreased . Sodium chloride inhibited their growth, although the strains tolerated up to 1.5% (w/v) NaCl . Menaquinone-6 was the major respiratory quinone . The major cellular fatty acids were C15 : 0, iso-C15 : 0, anteiso-C15 : 0, C15:1, iso-C15:1, C16 : 1omega7cis, iso-C16 : 1, iso-C17 : 1, iso-C15 : 3-OH and iso-C16 : 0 3-OH . The DNA G + C content was 34.0-34.8 mol% . Phylogenetic analysis based on 16S rDNA sequences suggested that the strains belonged to the genus Flavobacterium and were closely related to Flavobacterium xanthum and Flavobacterium frigidarium, with sequence similarities of 96.9 and 96.3%, respectively . In physiological and biochemical analyses, the isolates were differentiated from all known members of the genus Flavobacterium . The name Flavobacterium limicola is proposed for these novel strains, and the type strain is ST-82T (=JCM 11473T =DSM 15094T).

Biotechnol Bioeng, 2003 Jun 30, 82(7), 843 - 50
Flocculation behavior of Sphingobium chlorophenolicum in degrading pentachlorophenol at different life stages; Chang YI et al.; The cell flocculation behavior of degrading pentachlorophenol (PCP) by using Sphingobium chlorophenolicum (Flavobacterium sp., ATCC 39723) is investigated in the present paper . It is found that these Sphingobium cells can efficiently degrade PCP when the concentration of this toxic compound is below 150 ppm . These degradation rates of PCP can be facilitated with the additions of three supplementary carbons: glutamate (4.0 g/L), glucose (3.2 g/L), and cellobiose (3.05 g/L), among which the highest specific growth rate of cells is obtained when glucose is added . More importantly, in these biodegradation experiments described herein, in order to investigate the cell flocculation behavior, the temporal variations of cell size, zeta potential, and stability ratio of the cell suspension are also measured . It is found that, no matter what kind of supplementary carbon source is added, the highest stability ratio of the cell suspension can be always obtained at the end of the exponential growth phase, which can be well explained by using the results of cell size and zeta potential measurements according to the DLVO theory .

Appl Environ Microbiol, 2003 Apr, 69(4), 2253 - 68
Bacterioplankton community shifts in an arctic lake correlate with seasonal changes in organic matter source; Crump BC et al.; Seasonal shifts in bacterioplankton community composition in Toolik Lake, a tundra lake on the North Slope of Alaska, were related to shifts in the source (terrestrial versus phytoplankton) and lability of dissolved organic matter (DOM) . A shift in community composition, measured by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes, occurred at 4 degrees C in near-surface waters beneath seasonal ice and snow cover in spring . This shift was associated with an annual peak in bacterial productivity ({(14)C}leucine incorporation) driven by the large influx of labile terrestrial DOM associated with snow meltwater . A second shift occurred after the flux of terrestrial DOM had ended in early summer as ice left the lake and as the phytoplankton community developed . Bacterioplankton communities were composed of persistent populations present throughout the year and transient populations that appeared and disappeared . Most of the transient populations could be divided into those that were advected into the lake with terrestrial DOM in spring and those that grew up from low concentrations during the development of the phytoplankton community in early summer . Sequencing of DNA in DGGE bands demonstrated that most bands represented single ribotypes and that matching bands from different samples represented identical ribotypes . Bacteria were identified as members of globally distributed freshwater phylogenetic clusters within the alpha- and beta-Proteobacteria, the Cytophaga-Flavobacteria-Bacteroides group, and the ACTINOBACTERIA:

Can J Microbiol, 2003 Jan, 49(1), 1 - 8
Genetic profiling of noncultivated bacteria from the rhizospheres of sugar beet (Beta vulgaris) reveal field and annual variability but no effect of a transgenic herbicide resistance; Schmalenberger A et al.; In this field study, we compared the bacterial communities inhabiting the rhizosphere of a transgenic, herbicide-resistant sugar beet (Beta vulgaris) cultivar with those of its nonengineered counterpart, using a genetic profiling technique based on PCR amplifications of partial 16S rRNA gene sequences and single-strand conformation polymorphism (SSCP) . As a control for the plasticity of the bacterial community, we also analyzed the influence of herbicides, the field heterogeneity, and the annual variation . DNA was isolated from bacterial cell consortia that were directly collected from root material . PCR was carried out with primers that hybridized to evolutionarily conserved regions flanking variable regions 4 and 5 of the 16S rRNA gene . SSCP patterns of these PCR products were composed of approximately 50 distinguishable bands, as detected by silver staining of the gels after electrophoresis . Patterns of the replicates and the different treatments were highly similar, but digital image and similarity analyses revealed differences that corresponded to the positions of the replicates in the field . In addition, communities collected from sugar beet in two successive growing seasons could be distinguished . In contrast, no effect of the transgenic herbicide resistance was detectable . Sequencing of 24 dominant products of the SSCP profiles indicated the presence of bacteria from different phylogenetic groups, with Proteobacteria and members of the Cytophaga-Flavobacterium-Bacteroides group being most abundant.

Microb Ecol, 2003 Mar, 45(3), 282 - 90 Epub 2003 Mar 28.
High diversity among feather-degrading bacteria from a dry meadow soil; Lucas FS et al.; The aim of this study was to determine the diversity of cultivable bacteria able to degrade feathers and present in soil under temperate climate . We obtained 33 isolates from soil samples, which clustered in 13 ARDRA groups . These isolates were able to grow on solid medium with pigeon feathers as sole carbon and nitrogen source . One representative isolate of each ARDRA group was selected for identification and feather degradation tests . The phylogenetic analysis of 16S rDNA gene fragments revealed that only 4 isolates were gram positives . Two other isolates belonged to the Cytophaga-Flavobacterium group, and the remaining to Proteobacteria . High keratinolysis activity was found for strains related to Bacillus, Cytophagales, Actinomycetales, and Proteobacteria . The 13 selected strains showed variable efficiency in degrading whole feathers and 5 strains were able to degrade maximum 40% to 98% of the whole feathers . After 4 weeks incubation, five strains grown on milled feathers produced more than 0.5 U keratinase per mL . Keratinase activities across the 13 strains were positively correlated with the percentage of feather fragmentation and protein concentration.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 231 - 8
Paracoccus zeaxanthinifaciens sp . nov., a zeaxanthin-producing bacterium; Berry A et al.; A comprehensive taxonomic re-evaluation was performed on the marine, zeaxanthin-producing bacterium formerly classified as {Favobacterium} sp . strain R-1 512 (ATCC 21588) . This strain, together with two other previously described marine isolates, {Flavobacterium} strain R-1506 and Paracoccus sp . strain MBIC 3966, were found to comprise a new species of the genus Paracoccus . The name Paracoccus zeaxanthinifaciens sp . nov . is proposed, with ATCC 21588T (= R-1512T =LMG 21293T) designated as the type strain.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 81 - 5
Reichenbachia agariperforans gen . nov., sp . nov., a novel marine bacterium in the phylum Cytophaga-Flavobacterium-Bacteroides; Nedashkovskaya OI et al.; A heterotrophic, pigmented, agarolytic, gliding bacterium was isolated from a seawater sample collected from the Gulf of Peter the Great, Sea of Japan, during June 2000 . 16S rDNA sequence analysis indicated that the novel bacterium, strain KMM 3525T, was a member of the phlyum Cytophaga-Flavobacterium-Bacteroides . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic data, it is proposed that the marine bacterium represents the sole species of a novel genus, Reichenbachia, the type species of which is Reichenbachia agariperforans (KMM 3525T =IFO 16625T =JCM 11238T).

Verh K Acad Geneeskd Belg, 2002, 64(6), 421 - 30
Flavobacterium columnare infections in fish: the agent and its adhesion to the gill tissue; Decostere A; Flavobacterium columnare is the cause of columnaris disease, a serious condition affecting numerous freshwater fish species all over the world . Hitherto, only very scarce information is available on the pathogenesis of this bacterial disease, making it difficult to adopt a preventive approach to combat this pathogen . This study ambiates to shed more light on the way F . columnare interacts with its host . Since a number of difficulties occur in trying to isolate F . columnare from diseased fish, first of all, a selective medium was developed which enabled to recover various isolates from diseased live-bearing aquarium fish . In experimental infection studies in which these isolates were adopted, differences in virulence were noted . In a further stage, a clear correlation was made between virulence and the ability to adhere to the gill tissue, documenting the importance of the gills in the pathogenesis of columnaris disease . In this respect, a gill perfusion model was developed, by which means the adhesion of F . columnare to the gill tissue was partly pinpointed and various environmental factors influencing the adherence process elucidated . It may hence be stated that an important milestone has been achieved in the realisation of one of the most frequently used ways to prevent disease, that is through vaccination . Columnaris disease may also be opposed by ensuring good management practices eliciting adequate environmental circumstances.

Indian J Med Sci, 2002 Aug, 56(8), 391 - 6
Bacteraemia in high-risk patients; Seetha KS et al.; In this study, we noticed a high incidence of bacteraemia in high-risk patients especially due to nonfermenter gram negative bacilli (NFGNB) and coagulase negative staphylococci (CoNS) . Bacteraemia caused by some rare bacteria such as Moraxella spp., Aeromonas spp., Flavobacterium meningosepticum was also noted during the study . Antibiotic resistance pattern showed that many isolates were Multi Drug Resistant (MDR) . This can be attributed to nosocomial-infection, which may occur due to more and more invasive procedures for diagnosis and therapy during long stay of patients in the hospital . Also, the MDR strains and the wide spread oxacillin resistant CoNS (OR-CoNS) and slowly emerging vancomycin resistant CoNS (VR-CoNS) associated with nosocomial infections pose a great threat to the clinicians . We recommend the usage of commonly used antibiotics along with cephalosporins for the patients admitted to these high-risk units, before the antibiotic susceptibility test findings.

Biotechnol Appl Biochem, 2003 Apr, 37(Pt 2), 115 - 27
An improved methodology to produce Flavobacterium heparinum chondroitinases, important instruments for diagnosis of diseases; Aguiar JA et al.; Chondroitinases are very important tools for the identification and structural analysis of proteoglycans . Enzymic analysis with Flavobacterium heparinum chondroitinases has shown that chondroitin sulphate and dermatan sulphate structures are modified in many human diseases, suggesting a diagnostic value for these enzymes . Furthermore, it was recently shown that F . heparinum chondroitinases AC and B inhibit tumoural cell growth, invasion and angiogenesis . Due to the increasing importance of F . heparinum chondroitinases, we investigated optimized conditions for preparation and assay of chondroitinases AC, B and C . The Dimethylmethylene Blue assay was modified and fully developed to measure the chondroitinase activities of crude extracts of F . heparinum . This method estimates chondroitin sulphate or dermatan sulphate depolymerization upon the digestion of chondroitinase, and was compared with A (232), which measures the unsaturated products formed . Trypticase was the best culture medium, both for bacterial growth and enzyme induction . The chondroitinases were solubilized by ultrasound under conditions that do not completely disrupt the cells, suggesting that they are located at the periplasmic space . Maximum chondroitinase induction occurred in the presence of 0.2-1.0 g/l chondroitin sulphate . Chondroitin sulphate-degradation products were also inducers, but heparin and heparan sulphate were not . Chondroitinases AC, B and C were separated from each other by hydrophobic-interaction chromatography on Phenyl-Sepharose HP . When contaminant proteins were first removed from crude extract by Q-Sepharose, the chondroitinases could be purified to homogeneity in this phenyl-Sepharose chromatographic step.

Indian J Exp Biol, 2002 Jul, 40(7), 774 - 9
Localisation of identical organophosphorus pesticide degrading (opd) genes on genetically dissimilar indigenous plasmids of soil bacteria: PCR amplification, cloning and sequencing of opd gene from Flavobacterium balustinum; Somara S et al.; Plasmid borne organophosphorus pesticide degrading (opd) gene of Flavobacterium balustinum has been amplified using polymerase chain reaction (PCR) and the resulting PCR product (1.25 Kb) was cloned in pUC18 . Further, a detailed restriction map was determined to PCR product and subcloned as overlapping restriction fragments . The nucleotide sequence was determined for all subclones to obtain complete sequence of PCR amplified fragment . The sequence showed 98% similarity to opd genes cloned from other soil bacteria isolated from diversified geographical regions . The protein sequence predicted from the nucleotide sequence was almost identical to parathion hydrolase, a triesterase involved in hydrolysis of triester bond found in variety of op-pesticides . The signal sequence of parathion hydrolase contained recently discovered twin arginine transport (tat) motif . It appears that tat motif plays a critical role in membrane targeting of parathion hydrolase.

Vet Res, 2003 Jan-Feb, 34(1), 127 - 32
Virulence stability in Flavobacterium psychrophilum after storage and preservation according to different procedures; Michel C et al.; Experimental infections and lethal dose 50% (LD 50) evaluation were conducted in rainbow trout fingerlings, using a virulent strain of Flavobacterium psychrophilum processed and stored or maintained in different ways; lyophilisation, freezing at -80 degrees C, maintenance in enriched Anacker and Ordal (EAO) medium at 4 degrees C, revival and subsequent in vivo passages in fish . Experiments were performed 1, 8 and 23 months after storing the bacteria . Out of a total of 12 cultures revived for experimentation, one failed to grow and another was found to express modified properties including decreased virulence in spite of in vivo passages . In all other cases, whatever the conditions of preservation, virulence was fairly well maintained after 1 and 8 months of storage . In the last test, after 23 months, the bacteria maintained in the EAO medium at 4 degrees C were found significantly attenuated . Conversely, lyophilised and frozen bacteria only expressed a slight increase in LD0 . It was concluded that virulent strains of F . psychrophilum were likely to retain their properties without special provisions within limited periods of time, and that both lyophilisation and freezing at -80 degrees C were reliable methods for long-term preservation of virulence.

Syst Appl Microbiol, 2002 Dec, 25(4), 603 - 10
Diversity of 746 heterotrophic bacteria isolated from microbial mats from ten Antarctic lakes; Van Trappen S et al.; Microbial mats, growing in Antarctic lakes constitute unique and very diverse habitats . In these mats microorganisms are confronted with extreme life conditions . We isolated 746 bacterial strains from mats collected from ten lakes in the Dry Valleys (lakes Hoare and Fryxell), the Vestfold Hills (lakes Ace, Druzhby, Grace, Highway, Pendant, Organic and Watts) and the Larsemann Hills (lake Reid), using heterotrophic growth conditions . These strains were investigated by fatty acid analysis, and by numerical analysis, 41 clusters, containing 2 to 77 strains, could be delineated, whereas 31 strains formed single branches . Several fatty acid groups consisted of strains from different lakes from the same region, or from different regions . The 16S rRNA genes from 40 strains, representing 35 different fatty acid groups were sequenced . The strains belonged to the alpha, beta and gamma subclasses of the Proteobacteria, the high and low percent G+C Gram-positives, and to the Cytophaga-Flavobacterium-Bacteroides branch . For strains representing 16 fatty acid clusters, validly named nearest phylogenetic neighbours showed pairwise sequence similarities of less than 97% . This indicates that the clusters they represent, belong to taxa that have not been sequenced yet or as yet unnamed new taxa, related to Alteromonas, Bacillus, Clavibacter, Cyclobacterium, Flavobacterium, Marinobacter, Mesorhizobium, Microbacterium, Pseudomonas, Saligentibacter, Sphingomonas and Sulfitobacter.

J Antimicrob Chemother, 2003 Feb, 51(2), 267 - 73
Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae; Naas T et al.; Flavobacterium johnsoniae CIP100931 is resistant to most beta-lactam antibiotics and has a decreased susceptibility to carbapenems . A beta-lactamase gene was cloned and expressed in Escherichia coli DH10B . The purified beta-lactamase, JOHN-1, with a pI value of 9.0 and with a determined relative molecular mass of approximately 27 kDa was found to be a monomeric zinc-dependent enzyme that hydrolyses penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, but not monobactams . Sequence analysis revealed that JOHN-1 is a molecular class B beta-lactamase that is most closely related to BlaB from Chryseobacterium meningosepticum and IND-1 from Chryseobacterium indologenes (47% and 41% amino acid identity, respectively) . JOHN-1 is a new member of the highly divergent subclass B1 lineage of metallo-enzymes . Although F . johnsoniae and Chryseobacterium spp . are phylogenetically related bacteria, this report further underlines the heterogeneity of class B beta-lactamases that are naturally produced by environmental Gram-negative aerobes and that are now recognized as the most important reservoir for these beta-lactamase genes.

Clin Chim Acta, 2003 Feb, 328(1-2), 163 - 71
Determination of urinary myo-inositol concentration by an improved enzymatic cycling method using myo-inositol dehydrogenase from Flavobacterium sp; Yamakoshi M et al.; BACKGROUND: To determine myo-inositol more accurately, we improved the enzymatic cycling method . METHODS: We screened myo-inositol dehydrogenase (MIDH; EC.1.1.1.18) from Flavobacterium sp., which was highly specific to myo-inositol . We measured urinary myo-inositol/creatinine ratio 2 h after 75-g oral glucose tolerance test (2 h MI) of 71 volunteers, and investigated the relationship between diabetes and urinary myo-inositol concentration . RESULTS: The calibration curve was linear (r = 1.00) up to 2000 micromol/l, and the detection limit was 10 micromol/l . Within-run and between-run CVs were 0.5-1.1% and 0.4-1.3%, respectively . The 2 h MI of impaired fasting glycemia (IFG; 65.1 +/- 46.6 mg/g Cr, P < 0.005), impaired glucose tolerance (IGT; 85.0 +/- 73.7 mg/g Cr, P < 0.001) and diabetes (163.4 +/- 73.7 mg/g Cr, P < 0.0001) increased significantly compared with that of normal glucose tolerance (NGT; 24.0 +/- 14.4 mg/g Cr) . From receiver operating characteristic analyses on 2 h MI, with 50 mg/g Cr as a tentative cutoff value to detect diabetes, the sensitivity and specificity were 100% and 77%, respectively . With 40 mg/g Cr as a tentative cutoff value to detect NGT, the sensitivity and specificity were 74% and 85%, respectively . CONCLUSIONS: The myo-inositol measurement method demonstrated high specificity and yielded accurate results . The results of clinical trials suggested that 2 h MI could not only determine diabetes but also distinguish IFG and IGT from NGT .

Wei Sheng Wu Xue Bao, 1998 Dec, 38(6), 454 - 60
{Study of the physiological characteristics of a strain which can change the color of red lead on Dunhuang mural}; Feng Q et al.; A strain which was isolated from Dunhuang Murual was investigated . It belongs to Flavobacterium . In media added in Pb3O4, colonies show brown and black . After identified pigments, it showed that the brown was caused by the oxidation of Pb3 O4 into PbO2 by the strain . The optimal oxidative condition is at pH 9.8, 37 degrees C and dark environment . The genes for oxidation is on plasmid and the oxidation was inhibited under pure O2 and N2 conditions, too 5 x 10(-3) mol/ml NaN3 is enough . The strain was able to absorb lead initiative . The transmission electron microscope showed that lead locate in protoplasts.

Environ Microbiol, 2003 Jan, 5(1), 25 - 35
Isolation and phylogenetic characterization of bacteria capable of inducing differentiation in the green alga Monostroma oxyspermum; Matsuo Y et al.; Many green algae cannot develop normally when they are grown under axenic conditions . Monostroma oxyspermum, for example, proliferates unicellularly in an aseptic culture, but develops into a normal foliaceous gametophyte in the presence of some marine bacteria . More than 1000 bacterial strains were isolated from marine algae and sponges and assayed for their ability to induce the morphogenesis of unicellular M . oxyspermum . Fifty bacterial strains exhibiting morphogenesis-inducing activity against unicellular M . oxyspermum were isolated . The partial gyrB (approximately 1.2 kbp) and 16S rDNA (approximately 1.4 kbp) sequences of about 40 active strains were determined, and their phylogenetic relationships were analysed . All these strains were located within the Cytophaga-Flavobacterium-Bacteroides (CFB) complex, and most of these strains were clustered in a clade comprising Zobellia uliginosa . On the other hand, these bacteria also exhibited morphogenetic activity against germ-free spores of Ulva pertusa, Ulva conglobata and Enteromorpha intestinalis . Moreover, these bacteria induced the release of spores from the leafy young gametophyte of M . oxyspermum . These results indicate that strains belonging to several groups in the CFB complex play an important role in the normal development of green algae in the marine coastal environment.

Dis Aquat Organ, 2002 Nov 22, 52(2), 109 - 18
Occurrence of Flavobacterium psychrophilum in fish-farming environments; Madetoj J et al.; Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR . Characteristics of 64 F . psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically . Virulence of F . psychrophilum isolates from different sources was compared by injection into rainbow trout . Additionally, the number of F . psychrophilum cells shed by naturally infected rainbow trout was determined . F . psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease . The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish . Isolates were biochemically homogenous, excluding the capability to degrade elastin . Five different agglutination patterns with different antisera against F . psychrophilum were found among the isolates studied . Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant . Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F . psychrophilum in ovarian fluids . Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F . psychrophilum into the surrounding water.

J Biol Chem, 2003 Apr 4, 278(14), 12157 - 66 Epub 2003 Jan 07.
The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum . Molecular cloning, recombinant expression, and biochemical characterization; Myette JR et al.; Heparan sulfate glycosaminoglycans are structurally complex polysaccharides critically engaged in a wide range of cell and tissue functions . Any structure-based approach to study their respective biological functions is facilitated by the use of select heparan sulfate glycosaminoglycan-degrading enzymes with unique substrate specificities . We recently reported of one such enzyme, the Delta4,5-glycuronidase cloned from Flavobacterium heparinum and recombinantly expressed in Escherichia coli (Myette, J . R., Shriver, Z., Kiziltepe, T., McLean, M . W., Venkataraman, G., and Sasisekharan, R . (2002) Biochemistry 41, 7424-7434) . In this study, we likewise report the molecular cloning of the 2-O-sulfatase from the same bacterium and its recombinant expression as a soluble, highly active enzyme . At the protein level, the flavobacterial 2-O-sulfatase possesses considerable sequence homology to other members of a large sulfatase family, especially within its amino terminus, where the highly conserved sulfatase domain is located . Within this domain, we have identified by sequence homology the critical active site cysteine predicted to be chemically modified as a formylglycine in vivo . We also present a characterization of the biochemical properties of the enzyme as it relates to optimal in vitro reaction conditions and a kinetic description of its substrate specificity . In particular, we demonstrate that in addition to the fact that the enzyme exclusively hydrolyzes the sulfate at the 2-O-position of the uronic acid, it also exhibits a kinetic preference for highly sulfated glucosamines within each disaccharide unit, especially those possessing a 6-O-sulfate . The sulfatase also displays a clear kinetic preference for disaccharides with beta1-->4 linkages but is able, nevertheless, to hydrolyze unsaturated, 2-O-sulfated chondroitin disaccharides . Finally, we describe the substrate-product relationship of the 2-O-sulfatase to the Delta4,5-glycuronidase and the analytical value of using both of these enzymes in tandem for elucidating heparin/heparan sulfate composition.

J Biol Chem, 2003 Apr 4, 278(14), 12167 - 74 Epub 2003 Jan 07.
The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum . A structural and biochemical study of the enzyme active site and saccharide substrate specificity; Raman R et al.; In the previous paper (Myette, J . R., Shriver, Z., Claycamp, C., McLean, M . W., Venkataraman, G., and Sasisekharan, R . (2003) J . Biol . Chem . 278, 12157-12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum . In this paper, we extend our structure-function investigation of the 2-O-sulfatase . First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases . In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity . By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture . These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Delta4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme . The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data . Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site . This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.

J Gen Appl Microbiol, 2002 Oct, 48(5), 251 - 9
A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures; Kurahashi M et al.; Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied . Marine creatures were collected at the coasts of the Kanto area in Japan . A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata . Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group . The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database . Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group . Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria . Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria . Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage . From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.

J Chem Ecol, 2002 Oct, 28(10), 2045 - 56
Influence of Myriophyllum spicatum-derived tannins on gut microbiota of its herbivore Acentria ephemerella; Walenciak O et al.; The submerged living larvae of Acentria ephemerella were fed in the laboratory with either M . spicatum or Potamogeton perfoliatus, two of their host plants . Larvae exhibited a reduced growth when fed M . spicatum, a freshwater angiosperm that contains high concentrations of tannins, secondary metabolites known for their herbivore-deterrent and antimicrobial properties . In this study, we investigated the influence of food-derived tannins on gut microbiota . Bacterial densities in the guts did not differ between the food regimes, ranging from 2.8 to 13.3 x 10(6) cells per gut . Gut bacteria were characterized with cultivation techniques and subsequent identification of the strains by molecular methods . We isolated 17 bacterial strains belonging to all subdivisions, i.e., we identified alpha-, beta-, and gamma-proteobacteria, Cytophyaga/Flavobacteria (CF) and several Gram-positive bacteria . All except one Gram-positive strain were found in the guts of larvae fed with P . perfoliatus . Gram-positive bacteria and bacteria of the CF cluster were more sensitive to polyphenol-containing extracts of M . spicatum in an agar diffusion assay than strains of the alpha- or gamma-proteobacteria subdivision . Our results suggest an influence of food-derived tannins on gut microbiota in A . ephemerella.

J Gen Appl Microbiol, 2002 Jun, 48(3), 155 - 65
Phylogenetic structure of the genera Flexibacter, Flexithrix, and Microscilla deduced from 16S rRNA sequence analysis; Nakagawa Y et al.; The 16S rDNA sequences of 40 strains of 17 species in the genus Flexibacter, 5 strains of 4 species in the genus Microscilla, and 1 strain of Flexithrix dorotheae, including all type strains of approved and validated species in these genera, were determined to reveal their phylogenetic relationships . The 16S rRNA sequence analysis demonstrated the extreme heterogeneity of the genera Flexibacter and Microscilla . The strains examined diverged into 24 distinct lines of descent (1 group included both flexibacteria and flexithrix, and 1 group included both flexibacteria and microscilla) that were remote from each other at the genus level or higher . Flexibacter strains were scattered across the cytophaga-flavobacteria-bacteroides phylum and divided into 20 phylogenetic groups, and the genus Microscilla was separated into 5 groups . Flexibacter flexilis, the type species of the genus Flexibacter, and Microscilla marina, the type species of the genus Microscilla, were isolated from other organisms in their respective genera . This means that each genus should be restricted to only the type species . Flexithrix dorotheae, the type species of the genus Flexithrix, clustered with Flexibacter aggregans . The heterogeneity was found not only within genera but also within species . Flexibacter aggregans, Flexibacter aurantiacus, Flexibacter flexilis, Flexibacter roseolus, Flexibacter tractuosus, and "Microscilla sericea" each contained phylogenetically distant strains . The taxonomic concept of the genera Flexibacter, Flexithrix, and Microscilla should be reorganized in accordance with the natural relationships revealed in this study.

J Ind Microbiol Biotechnol, 2002 Nov, 29(5), 255 - 8
Isolation and characterization of a feather-degrading bacterium from the poultry processing industry; Riffel A et al.; A Flavobacterium sp . producing a high keratinolytic activity was isolated from a poultry industry after growth on selective feather meal agar . This bacterium grew on feather meal broth, producing keratinase, and was also capable of complete degradation of raw feathers . The proteolytic activity was assessed in the presence of specific protease inhibitors . The crude enzyme showed mainly metalloprotease character . This novel isolate would have potential biotechnological use in processes involving keratin hydrolysis.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3561 - 7
Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes; Mammeri H et al.; Myroides odoratus and Myroides odoratimimus (formerly designated in a single species as Flavobacterium odoratum) are gram-negative aerobes and sources of nosocomial infections in humans . They have variable susceptibility to beta-lactams and a decreased susceptibility to carbapenems . Using genomic DNAs of M . odoratus CIP 103105 and M . odoratimimus CIP 103073 reference strains, shotgun cloning of beta-lactamase genes was performed, followed by protein expression in Escherichia coli . The deduced amino acid sequences of these beta-lactamase genes revealed that TUS-1 and MUS-1 from M . odoratus CIP 103105 and M . odoratimimus CIP 103073, respectively, shared 73% amino acid identity . Mature proteins TUS-1 and MUS-1, with pI values of 7.8 and 5.2, respectively, had relative molecular masses of ca . 26 kDa . These beta-lactamases are members of the subclass B1 of metallo-beta-lactamases and are distantly related to other metalloenzymes, being most closely related to IND-1 from Chryseobacterium indologenes (42% amino acid identity) . However, phylogenic analysis showed that TUS-1 and MUS-1 belong to the same phylogenic lineage of subclass B1 enzymes that groups the subclass B1 beta-lactamases of Flavobacterium species . Kinetic parameters of purified beta-lactamases TUS-1 and MUS-1 detailed their hydrolysis spectra, which encompass most beta-lactams except aztreonam . beta-Lactamases TUS-1 and MUS-1 were classified in functional subgroup 3a of metalloenzymes . This work further characterizes chromosome-encoded metalloenzymes from Flavobacteriaceae species that explain at least part of their intrinsic resistance to beta-lactams.

Dis Aquat Organ, 2002 Aug 29, 51(2), 93 - 100
Diagnosis of flavobacteriosis by direct amplification of rRNA genes; Tiirola M et al.; A broad-range bacterial PCR method with universal 16S rDNA targeting primers and bacterial cultivation was used to identify the putative pathogen in flavobacterial outbreaks . Restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the partial 16S rDNA PCR products of 10 skin samples and 10 representative isolates derived from the same fish specimens revealed differences between direct molecular and cultivation-based analysis . Flavobacterium columnare-like sequences dominated in the direct molecular analysis in most cases, whereas most of the isolates belonged to a phylogenetically heterogeneous group of flavobacteria clustering with F . hibernum . F . columnare was isolated in only 1 outbreak . The possible explanations for the different results may be attributable to difficulties in the plate cultivation procedure of external flavobacterial samples . During plate cultivation, the dominating Flavobacterium species can be masked by saprophytic species of the same genus or other genera, or the growth of flavobacteria can be completely inhibited by antagonistic bacteria such as Pseudomonas . Direct analysis of the prevailing 16S rDNA sequences avoids the problems with cultivation and may thus be preferable for the diagnosis of flavobacterial diseases . When isolating flavobacteria from external samples, serial dilution of the sample before plating can improve the results.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1533 - 41
Aequorivita gen . nov., a member of the family Flavobacteriaceae isolated from terrestrial and marine Antarctic habitats; Bowman JP et al.; Several strains isolated from Antarctic winter sea water, sea-ice algal assemblages and quartz stone subliths were found to belong to a novel 16S rDNA sequence cluster within the family Flavobacteriaceae (Cytophaga-Flavobacterium-Bacteroides division) . The strains were gram-negative, non-motile, psychrotolerant, strictly aerobic, chemoheterotrophic rod-shaped cells that contained orange or yellow carotenoid pigments and required yeast extract when grown in defined mineral-salts media . The requirement for sodium ions varied between strains . Results of DNA-DNA hybridization analysis were used to divide the strains into four distinct genospecies, which were differentiated by physiological and nutritional characteristics . The DNA G+C content of the strains was 33-39 mol% . The fatty acid profiles of representative strains were very similar, with major constituents including i15:1omega10c, a15:1omega10c, i15:0, a15:0, i16:1omega6c, i17:1omega5c and 3-OH i16:0 . The novel genus Aequorivita gen . nov., which has widespread distribution in the Antarctic region, is proposed . The genus comprises four species: the type species Aequorivita antarctica sp . nov . (type strain SW49T = ACAM 640T = DSM 14231T), Aequorivita lipolytica sp . nov . (type strain Y10-2T = ACAM 641T = DSM 14236T), Aequorivita crocea sp . nov . (type strain Y12-2T = ACAM 642T = DSM 14239T) and Aequorivita sublithincola sp . nov . (type strain 9-3T= ACAM 643T = DSM 14238T).

Syst Appl Microbiol, 2002 Aug, 25(2), 259 - 66
Detection of the fish pathogen Flavobacterium psychrophilum in water from fish farms; Madetoja J et al.; Rainbow trout fry syndrome and cold-water disease are serious diseases in farmed salmonid fish . In the present study, three methods were compared, for the detection of the causative pathogen, Flavobacterium psychrophilum in water . The methods included traditional agar plate cultivation on tryptone yeast extract salts (TYES) agar, immunofluorescence antibody technique (IFAT) and nested PCR . The three methods were subsequently used for the detection of F . psychrophilum from fish farm environments . The nested PCR was the most sensitive method used for a detection of F . psychrophilum . As low as 3 CFU estimated by agar plate cultivation or 41 cells estimated by IFAT of F . psychrophilum per ml of non-sterile well water were needed for a detection using the nested PCR method . The obtained detection limits for the agar plate cultivation and the IFAT was 32 CFU/ml and 410 cells/ml, respectively . Using IFAT and nested PCR F . psychrophilum was detected most frequently in water samples from fish farms, but the pathogen was isolated from only a few samples using agar plate cultivation . In the present study IFAT and nested PCR proved to be rapid, specific and sensitive methods compared to traditional agar plate cultivation for the detection of F . psychrophilum from environmental samples . It is suggested that IFAT and nested PCR provide effective tools for the examination of F . psychrophilum in the environment.

Appl Environ Microbiol, 2002 Oct, 68(10), 5177 - 80
Simultaneous detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, three major fish pathogens, by multiplex PCR; Del Cerro A et al.; A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri . The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A . salmonicida, F . psychrophilum, and Y . ruckeri, respectively . The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks . Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.

Antimicrob Agents Chemother, 2002 Oct, 46(10), 3223 - 7
EBR-1, a novel Ambler subclass B1 beta-lactamase from Empedobacter brevis; Bellais S et al.; Empedobacter brevis (formerly designated Flavobacterium breve) is a gram-negative aerobe involved in nosocomial infections . The Ambler class B beta-lactamase gene bla(EBR-1) was cloned and expressed in Escherichia coli from E . brevis clinical strain ASS-1, which had reduced susceptibility to expanded-spectrum cephalosporins and carbapenems . Purified beta-lactamase EBR-1 hydrolyzed penicillins, cephalosporins, and carbapenems efficiently but not aztreonam . Kinetic parameters of EBR-1 were similar to those of class B enzymes such as BlaB, IND-2, and GOB-1 identified from other Flavobacteriaceae species, except for meropenem, which was more hydrolyzed by beta-lactamase GOB-1 . EBR-1, with a pI of 8.0 and a relative molecular mass of ca . 25 kDa, was classified in functional subgroup 3a, which includes most of the class B beta-lactamases . EBR-1, which belongs to molecular subclass B1 of metalloenzymes, shares 58, 57, and 42% amino acid identity with the most closely related beta-lactamases, IND-1/IND-2 from Chryseobacterium indologenes, CGB-1 from Chryseobacterium gleum, and BlaB from Chryseobacterium meningosepticum, respectively.

Anal Biochem, 2002 Sep 1, 308(1), 77 - 82
Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates; Rye CS et al.; Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates . We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode . Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined . The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases . The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.

Arch Microbiol, 2002 Oct, 178(4), 274 - 8 Epub 2002 Jul 24.
Formation of methylantimony species by an aerobic prokaryote: Flavobacterium sp; Jenkins RO et al.; Pure cultures of an aerobically grown Flavobacterium sp . were shown by hydride generation-cold trap-atomic absorption spectrometry to biomethylate inorganic antimony (III) supplied as potassium antimony tartrate . Growth inhibition of the Flavobacterium sp . by antimony (III) over the range 0-30 mg Sb l(-1) was assessed by optimising parameters within an extended logistic growth model . Antimony (III) concentrations over this range influenced both the extent of antimony biomethylation (up to 4.0 microg l(-1)) and the relative proportions of the involatile mono-, di, and trimethylantimony species formed . Provision of inorganic arsenic (III) alongside antimony (III) enhanced formation of the involatile methylantimony species up to eight-fold . The data are consistent with accumulation of involatile intermediates from an antimony or arsenic biomethylation pathway in culture supernatants . Low yields of methylantimony species (<0.03%) suggest that antimony biomethylation by the Flavobacterium sp . was a fortuitous rather than a primary resistance mechanism for this element . These findings demonstrate that anaerobiosis is not an obligate requirement for methylantimony formation in prokaryotes, thus broadening the range of habitats for potential formation of methylantimony species in nature.

Microb Ecol, 2002 Oct, 44(3), 252 - 9 Epub 2002 Sep 06.
Phylogenetic diversity of winter bacterioplankton of eutrophic siberian reservoirs as revealed by 16S rRNA gene sequence; Trusova MY et al.; Using 16S rRNA gene sequence analyses we investigated the bacterial diversity of winter bacterioplankton of two eutrophic Siberian reservoirs . These reservoirs show similarity in phytoplankton community composition in spring and autumn but tend to differ in summer in exhibiting cyanobacterial bloom . Forty-eight unique partial 16S RNA gene sequences retrieved from two libraries were mostly affiliated with the class Actinobacteria, b subdivision of the class Proteobacteria, and the phylum Cytophaga-Flavobacterium-Bacteroides . The clone library of the pond exhibiting summer cyanobacterial bloom showed more diversity in sequence composition . A significant number of bacterial 16S rRNA gene clones were closely related to freshwater bacteria previously found in different aquatic ecosystems . This finding confirms the assumption that some bacterial clades are globally distributed.

Biol Pharm Bull, 2002 Aug, 25(8), 1049 - 52
Prolyl endopeptidase inhibitors from the roots of Lindera strychnifolia F . Vill; Kobayashi W et al.; Prolyl endopeptidase (PEP, EC 3.4.21.26) has been proposed to play a role in degradation of proline-containing neuropeptides involved in the processes of learning and memory, e.g., vasopressin, substance P, and thyrotropin-releasing hormone (TRH) . In the course of our search for bioactive constituents in medicinal plants, we studied the PEP inhibitory constituents of the roots of Lindera strychnifolia F . VILL and isolated two known tannins, epicatechin (1) and aesculitannin B (2), and four known sesquiterpenes, linderene (3), linderene acetate (4), linderalactone (5) and isolinderalactone (6) as inhibitors . On the inhibitory activities of six compounds against PEP from Flavobacterium meningosepticum and that from rat brain supernatant, compounds 1, 2 and 4 inhibited the enzyme from Flavobacterium more strongly than that from rat brain supernatant . However, compounds 3, 5 and 6 inhibited the enzymes from both origins to the same extent and furthermore, compound 6 was the strongest natural inhibitor against PEP from rat brain supernatant . The kinetic study of these inhibitors indicated that compounds 1, 2 are noncompetitive inhibitors and compounds 3-6 are competitive inhibitors . This is the first example of non-phenolic constituents showing significant competitive inhibitory activity being isolated from natural medicines.

Antimicrob Agents Chemother, 2002 Sep, 46(9), 2791 - 6
Genetic and biochemical characterization of CGB-1, an Ambler class B carbapenem-hydrolyzing beta-lactamase from Chryseobacterium gleum; Bellais S et al.; Chryseobacterium gleum (previously included in the Flavobacterium IIb species) is a gram-negative aerobe that is a source of nosocomial infections . An Ambler class B beta-lactamase gene was cloned and expressed in Escherichia coli from reference strain C . gleum CIP 103039 that had reduced susceptibility to expanded-spectrum cephalosporins and carbapenems . The purified beta-lactamase, CGB-1, with a pI value of 8.6 and a determined relative molecular mass of ca . 26 kDa, hydrolyzed penicillins; narrow- and expanded-spectrum cephalosporins; and carbapenems . CGB-1 was a novel member of the molecular subclass B1 of metallo-enzymes . It had 83 and 42% amino acid identity with IND-1 from Chryseobacterium indologenes and BlaB from C . meningosepticum, respectively . Thus, in addition to the previously characterized clavulanic acid-inhibited extended-spectrum beta-lactamase CGA-1 of Ambler class A, C . gleum produces a very likely chromosome-borne class B beta-lactamase.

J Am Chem Soc, 2002 Aug 21, 124(33), 9756 - 67
Elucidation of the mechanism of polysaccharide cleavage by chondroitin AC lyase from Flavobacterium heparinum; Rye CS et al.; Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism resulting in disaccharides or oligosaccharides with delta4,5-unsaturated uronic acid residues at their nonreducing end . Mechanistic details concerning the ordering of the bond-breaking and -forming steps of this enzymatic reaction are nonexistent, mainly due to the inhomogeneous nature of the polymeric substrates . The creation of a new class of synthetic substrates for this enzyme has allowed the measurement of defined and reproducible k(cat) and K(m) values and has expanded the range of mechanistic studies that can be performed . The primary deuterium kinetic isotope effect upon k(cat)/K(m) for the abstraction of the proton alpha to the carboxylic acid was measured to be 1.67 +/- 0.07, showing that deprotonation occurs in a rate-limiting step . Using substrates with leaving groups of differing reactivity, a flat linear free energy relationship was produced, indicating that the C4-O4 bond is not broken in a rate-determining step . Taken together, these results strongly suggest a stepwise mechanism . Consistent with this was the measurement of a secondary deuterium kinetic isotope effect upon k(cat)/K(m) of 1.01 +/- 0.03 on a 4-{(2)H}-substrate, indicating that no sp2 character is developed at C4 during the rate-limiting step, thereby ruling out a concerted syn-elimination.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1374 - 7
Stabilization of flavobacterium meningosepticum glycerol kinase by introduction of a hydrogen bond; Sakasegawa S et al.; The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp {Protein Eng., 14, 663-667 (2001)} . Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme . If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329 . We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn . As we expected, S414N showed comparable thermostability to that of S329D . On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1325 - 9
Gelidibacter mesophilus sp . nov., a novel marine bacterium in the family Flavobacteriaceae; Macian MC et al.; Two Gram-negative, aerobic, heterotrophic, marine bacteria, isolated from Mediterranean sea water off the coast near Valencia (Spain), were the object of this study . These non-motile, yellow-pigmented, rod-shaped strains have been studied by means of DNA-DNA hybridization, 16S rRNA sequencing and cultural and physiological features . Phylogenetic analysis showed that both strains belong to the phylum Cytophaga-Flavobacterium-Bacteroides, and their closest neighbour is the psychrophilic bacterium Gelidibacter algens . The two strains differ from G . algens in their mesophilic behaviour, hydrolytic pattern and use of different carbon sources . There is 31% DNA-DNA hybridization between the proposed type strain and G . algens, and both isolates show 97.5% 16S rDNA similarity to G . algens . They represent a novel species of the genus Gelidibacter, for which the name Gelidibacter mesophilus sp . nov . is proposed, with strain 2SM29T (= CECT 5103T = DSM 14095T) as the type strain.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(5), 567 - 571
Cloning and Nucleotide Sequencing of Prolyl Endopeptidase Gene from Aeromonas punctata subsp . punctata; Shen GX et al.; Prolyl endopeptidase activity was found in Aeromonas punctata subsp . Punctata . The genomic DNA was partially digested with EcoRI and the recovered 8-16 kb DNA fragments were inserted into the EcoRI site of plasmid pUC19, and were transformated into Escherichia coli DH5alpha . The resulted clones were screened by using Benzyloxycarbonyl-Gly-Pro-beta-naphthylamide, the specific substrate of prolyl endopeptidase and a positive clone was obtained . The 12 kb insertion fragment of recombinant plasmid was digested with HincII and subcloned . The PEP gene was found in the 3.5 kb HincII/EcoRI fragment . Nucleotide sequence of the gene was completely sequenced by Auto Sequencer . The complete gene consisted of 2 073 bp corresponding to 690 amino acid residues with a calculated molecular weight of 76 467 Da . The amino acid sequence was 92.3% 53.2% 33.5% 33.2% and 20.5% homologous to those of Aeromonas hydrophila, Flavobacterium meningosepticum, porcine brain, human lymphocytes and Pyrococcus furiosus respectively . From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser(538) Asp(622) and His(657).

Diagn Microbiol Infect Dis, 2002 Jul, 43(3), 193 - 9
Investigation of an anaerobic microbial community associated with a corneal ulcer by denaturing gradient gel electrophoresis and 16S rDNA sequence analysis; Schabereiter-Gurtner C et al.; The bacterial community manifested in a corneal ulcer was investigated with culture-independent techniques . DNA was extracted from the eye swab, 200-bp fragments spanning the hypervariable V3 region of the 16S rRNA gene (16S rDNA) were amplified by broad-range PCR and genetic fingerprinting of the total bacterial community was performed by denaturing gradient gel electrophoresis (DGGE) . Additionally, 16S rDNA clone libraries containing 1500-bp fragments were constructed, clones were screened by DGGE and sequenced . Microorganisms were phylogenetically most closely related to the Cytophaga/Flavobacterium/Bacteroides phylum (eight clones), Fusobacteria (four clones), spirochetes (three clones) and to the low G+C Gram-positive bacteria (two clones) . Low sequence similarity values less than 93% to sequences of known bacteria indicated that some bacteria belonged to hitherto unknown genera . Bacteria which were detected in the healthy eye of the same patient, were phylogenetically related to the low G+C and high G+C Gram-positive bacteria (two clones) and to the Proteobacteria (one clone) . To our knowledge, this is the first time that such a complex and anaerobic bacterial community normally found in subgingival crevices is reported to play a role in corneal ulceration . Previous treatment of the ulcer with several topical antibiotics had shown no effect for six months . The followed culture-independent identification of spirochetes and Gram-negative, anaerobic bacilli facilitated the appropriate treatment with topical penicillin G, which stopped further destruction of the eye . Results demonstrated that 16S rDNA genotyping in combination with DGGE fingerprinting are appropriate molecular methods for the investigation of severe bacterial infections which might not be detected by conventional cultivation.

Lett Appl Microbiol, 2002, 35(2), 166 - 70
A proposed serotyping system for Flavobacterium psychrophilum; Mata M et al.; AIMS: To develop a common serological system for rapid and routine identification of the fish pathogen Flavobacterium psychrophilum . METHODS: Thirty-four isolates of Fl . psychrophilum from different fish species and different geographical areas were typed using a slide agglutination test and an enzyme-linked immunosorbent assay (ELISA) . RESULTS: Seven host-dependent serovars (1: salmon; 2: trout; 3: trout; 4: eel; 5: carp; 6: tench; 7: ayu) were found . Serovar 2 was divided into two antigenic subgroups (2a and 2b) . The results achieved by both slide agglutination and ELISA methods were totally consistent with each other . Although both techniques proved to be simple to carry out and useful, only the ELISA allowed identification of Fl . psychrophilum serovars using unabsorbed antiserum and whole-cells as antigens . SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proposes a harmonized scheme for serological identification of Fl . psychrophilum to be used for diagnostic and seroepidemiological studies of the diseases it causes.

Environ Sci Technol, 2002 Jun 15, 36(12), 2652 - 62
Phylogenetic characterization of microbial communities that reductively dechlorinate TCE based upon a combination of molecular techniques; Richardson RE et al.; An anaerobic microbial consortium (referred to as ANAS) that reductively dechlorinates trichloroethene (TCE) completely to ethene with the transient production of cisdichloroethene (cDCE) and vinyl chloride was enriched from contaminated soil obtained from Alameda Naval Air Station . ANAS uses lactate as its electron donor and has been functionally stable for over 2 years . Following a brief exposure to oxygen, a subculture (designated VCC) derived from ANAS could dechlorinate TCE only to vinyl chloride with lactate as its electron donor . Three molecular methods were used concurrently to characterize the community structure of ANAS and VCC: clone library construction/clone sequencing, terminal restriction fragment length polymorphism (T-RFLP) analysis, and fluorescent in situ hybridization (FISH) with rRNA probes . The community structure of ANAS did not change significantly over the course of a single feeding/dechlorination cycle, and only minor fluctuations occurred over many feeding cycles spanning the course of 1 year . Clone libraries and T-RFLP analyses suggested that ANAS was dominated by populations belonging to three phylogenetic groups: Dehalococcoides species, Desulfovibrio species, and members of the Clostridiaceae (within the low G + C Gram-positives) . FISH results suggest that members of the Cytophaga/Flavobacterium/Bacteroides (CFB) cluster and high G + C Gram-positives (HGCs) were numerically important in ANAS despite their under-representation in the clone libraries . Parallel analyses of VCC samples suggested that Dehalococcoides species and Clostridiaceae were only minor populations in this community . Instead, VCC had increased populations of organisms in the beta and gamma subclasses of the Proteobacteria as well as significant populations of organisms in the CFB cluster . It is possible that symbiotic interactions are occurring between some of ANAS's phylogenetic groups under the enrichment conditions, including interspecies hydrogen transfer from Desulfovibrio species to Dehalococcoides species . However, the nucleic acid-based analyses performed here would need to be supplemented with chemical species data in order to test any hypotheses about functional roles of various community members . Additionally, these results suggest that an organism outside the Dehalococcoides genus may be capable of dechlorinating cDCE to vinyl chloride.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1181 - 4
Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans; Yoshida E et al.; A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298 . The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000 . The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2 . Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.

J Org Chem, 2002 Jun 28, 67(13), 4505 - 12
Synthesis and evaluation of potential inhibitors of chondroitin AC lyase from Flavobacterium heparinum; Rye CS et al.; Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism, resulting in disaccharides or oligosaccharides with Delta4,5-unsaturated uronic acid residues at their nonreducing end . The syntheses and testing of two potential inhibitors of this lyase are described . Methyl O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1-->4)-alpha-L-threo-hex-4-enopyranoside, 1, has the trigonal geometry at C5 of the uronic acid moiety expected at the transition state, yet retains the "leaving group" sugar moiety . Surprisingly, compound 1 showed no inhibition of the enzyme . The novel 5-nitro sugar, phenyl (5S)-5-nitro-beta-D-xylopyranoside, 2, is a monosaccharide nitro analogue of the natural substrate, with C5 being a carbon acid of pK(a) 8.8 . The rate of reprotonation of the anion generated at this center is sufficiently low that the anion of 2 can be used directly in initial steady-state velocity measurements without significant interference from the conjugate carbon acid . The anion of compound 2 was found to be a competitive inhibitor with a K(i) value of 5 mM, whereas the conjugate acid had a K(i) value of 35 mM.

J Appl Microbiol, 2002, 93(1), 149 - 56
Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum; del Cerro A et al.; AIMS: This study developed a new diagnostic method for the bacterium Flavobacterium psychrophilum based on a TaqMan polymerase chain reaction (PCR) assay . METHODS AND RESULTS: Based on reported and newly designed PCR probes, a rapid procedure, that requires no post-PCR processing, was developed for the detection of F . psychrophilum by measuring the fluorescence produced during PCR amplification . Primers were designed to amplify a 971-bp fragment of the 16S rRNA as the target . When different F . psychrophilum strains and other bacterial species, that are taxonomically and ecologically related, were assayed the fluorogenic test was 100% specific in identifying all of the F . psychrophilum strains . The sensitivity of the assay was found to be 1.1 pg DNA and the assay was linear over a range of 0.1 pg-11.2 ng . With pure cultures of F . psychrophilum, the assay was linear over the range 0.4-4.7 x 104 cfu and was able to detect 4.7 cfu per reaction . The analysis was reproducible using either extracted DNA or pure culture . Results using artificially infected fish and diseased fry from natural fish farm outbreaks showed that the assay was useful for diagnosis . CONCLUSIONS: The data showed that the assay was as specific, sensitive, reproducible and rapid but less toxic than the PCR assays described and so very useful for the diagnosis of these micro-organisms . SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach permits a rapid, easy and safe routine laboratory diagnosis of F . psychrophilum.

J Biol Chem, 2002 Aug 23, 277(34), 31179 - 86 Epub 2002 Jun 12.
Biochemical characterization of the chondroitinase B active site; Pojasek K et al.; Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate . A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis . However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis . Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies . In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B . Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B . The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B . Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis . The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine . In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 1049 - 70
Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family; Bernardet JF et al.; In this paper minimal standards for the description of new genera and cultivable species in the family Flavobacteriaceae are proposed in accordance with Recommendation 30b of the Bacteriological Code (1990 Revision) . In addition to specified phenotypic characteristics, the description of new species should be based on DNA-DNA hybridization data, and the placement of new taxa should be consistent with phylogenetic data derived from 16S rRNA sequencing . An emended description of the family is also proposed as several new taxa have been described since 1996 . These proposals have been endorsed by the members of the Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes.

Biochim Biophys Acta, 2002 Jun 3, 1597(2), 260 - 70
Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum; Capila I et al.; Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues . Cleavage occurs through beta-elimination in a random endolytic action pattern . Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues . Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity . Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis . The current study demonstrates that the R292A mutation affords approximately 10-fold higher K(m) values but no significant change in V(max), consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues . Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic . Product studies using gel electrophoresis confirm the altered action pattern of this mutant . Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage.

Biochemistry, 2002 Jun 11, 41(23), 7424 - 34
Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity; Myette JR et al.; The soil bacterium Flavobacterium heparinum produces several enzymes that degrade heparan sulfate glycosaminoglycans (HSGAGs) in a sequence-specific manner . Among others, these enzymes include the heparinases and an unusual glycuronidase that hydrolyzes the unsaturated Delta4,5 uronic acid at the nonreducing end of oligosaccharides resulting from prior heparinase eliminative cleavage . We report here the molecular cloning of the Delta4,5 glycuronidase gene from the flavobacterial genome and its recombinant expression in Escherichia coli as a highly active enzyme . We also report the biochemical and kinetic characterization of this enzyme, including an analysis of its substrate specificity . We find that the Delta4,5 glycuronidase discriminates on the basis of both the glycosidic linkage and the sulfation pattern within its saccharide substrate . In particular, we find that the glycuronidase displays a strong preference for 1-->4 linkages, making this enzyme specific to heparin/heparan sulfate rather than 1-->3 linked glycosaminoglycans such as chondroitin/dermatan sulfate or hyaluronan . Finally, we demonstrate the utility of this enzyme in the sequencing of heparinase-derived HSGAG oligosaccharides.

Microb Ecol, 2002 May, 43(4), 455 - 66 Epub 2002 Apr 08.
Genetic diversity of attached bacteria in the hindgut of the deposit-feeding shrimp Neotrypaea (formerly Callianassa) californiensis (decapoda: thalassinidae); Lau WW et al.; Microbial colonization of marine invertebrate guts is widespread, but in general the roles that these bacteria play in the nutrition of their hosts are unknown . To examine the diversity and potential nutritional roles of hindgut microbiota in a deposit feeder, PCR-amplified 16S rRNA genes were cloned from the bacterial community attached to the hindguts of the thalassinid shrimp Neotrypaea californiensis exposed to different feeding treatments . Partial 16S rDNA sequences were analyzed for 30 clones for three shrimp per treatment for a total of 270 clones . No effects of host starvation or high-protein diets were apparent on hindgut bacterial community composition . Diversity analyses indicated high variability between bacterial communities in individual shrimp hindguts, but partial 16S rDNA sequences revealed remarkable species-level similarity (>98%) within clusters of sequences from the different shrimp hindguts, and many sequences from different shrimp hindguts were identical . Sequences belonged to three main groups of bacteria: Cytophaga-Flavobacteria-Bacteroides (CFB), proteobacteria, and gram-positives . Of the 270 sequences, 40% belonged to the alpha-proteobacteria, > or = 5% each to the gamma- and epsilon -proteobacteria, and > or =20% each to the gram-positive and CFB groups . All except one sequence are novel with < or = 95% sequence similarity to known genes . Despite weak similarity to known taxa,about 75% of the sequences were most closely related to known symbiotic and sedimentary bacteria . The bacteria in shrimp hindguts represent new species that have not yet been en-countered in other environments, and gut environments may be a rich source of the difficult-to-culture and novel components of marine bacterial diversity.

Microb Ecol, 2002 Apr, 43(3), 315 - 28 Epub 2002 Mar 13.
Phylogenetic diversity of numerically important Arctic sea-ice bacteria cultured at subzero temperature; Junge K et al.; Heterotrophic bacteria in sea ice play a key role in carbon cycling, but little is known about the predominant players at the phylogenetic level . In a study of both algal bands and clear ice habitats within summertime Arctic pack ice from the Chukchi Sea, we determined the abundance of total bacteria and actively respiring cells in melted ice samples using epifluorescence microscopy and the stains 4', 6'-diamidino-2-phenylindole 2HCl (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), respectively . Organic-rich and -poor culturing media were used to determine culturable members by plating (at 0 degrees C and 5 degrees C) and most-probable-number (MPN) analyses (at -1 degrees C) . Total bacterial counts ranged from 5.44 x 10(4) ml(-1) in clear ice to 2.41 x 10(6) ml(-1) in algal-band ice samples, with 2-27% metabolically active by CTC stain . Plating and MPN results revealed a high degree of culturability in both types of media, but greater success in oligotrophic media (to 62% of total abundance) and from clear ice samples . The bacterial enumeration anomaly, commonly held to mean <or= 0.01% cultured, was not demonstrated in any of our samples . Denaturing gradient gel electrophoresis was used to check the purity of 44 isolates and select representatives for subsequent sequencing . Phylogenetic analyses based on 16S rRNA sequences indicated close relationships exclusively to known marine psychrophiles within two bacterial divisions, Proteobacteria (in the genera Alteromonas, Colwellia, Glaciecola, Octadecabacter, Pseudoaltermonas and Shewanella) and Cytophaga-Flexibacter-Bacteroides (Cytophaga, Flavobacterium, Gelidibacter and Polaribacter) . All cultures from the clear ice sample with highest (62%) culturability were closely related to each other or to psychrophilic Cytophaga-Flexibacter-Bacteroides (94.9-99.6% sequence similarities) . Overall, these findings suggest limited, heterotrophic bacterial diversity at cold temperatures and may provide insight into the recent evolution of psychrophilic bacteria.

Dis Aquat Organ, 2002 Apr 5, 48(3), 175 - 85
Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes; Nilsson WB et al.; We report the application of a nucleic acid-based assay that enables direct detection and identification of bacterial pathogens in fish kidney tissue without the need for bacterial culture . The technique, known as terminal restriction fragment length polymorphism (T-RFLP), employs the polymerase chain reaction (PCR) using a primer pair that targets 2 highly conserved regions of the gene that encodes for the 16S small subunit of the bacterial ribosome . Each primer is 5' labeled with a different fluorescent dye, which results in each terminus of the resulting amplicon having a distinguishable fluorescent tag . The amplicon is then digested with a series of 6 restriction endonucleases, followed by size determination of the 2 labeled terminal fragments by capillary electrophoresis with laser-induced fluorescence detection . Comparison of the lengths of the full set of 12 terminal fragments with those predicted based on analyses of GenBank submissions of 16S sequences leads to presumptive identification of the pathogen to at least the genus, but more typically the species level . Results of T-RFLP analyses of genomic DNA from multiple strains of a number of fish bacterial pathogens are presented . The assay is further demonstrated on fish kidney tissue spiked with a known number of cells of Flavobacterium psychrophilum where a detection limit of ca . 30 CFU mg(-1) of tissue was estimated . A similar detection limit was observed for several other gram-negative pathogens . This procedure was also used to detect Aeromonas salmonicida and Renibacterium salmoninarum in the kidney tissue of 2 naturally infected salmonids.

J Bacteriol, 2002 Jun, 184(12), 3242 - 52
Expression system for high levels of GAG lyase gene expression and study of the hepA upstream region in Flavobacterium heparinum; Blain F et al.; A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described . hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains . The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F . heparinum grown in heparin-only medium . The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F . heparinum grown in chondroitin sulfate A-only medium . The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain . The transcriptional start sites were determined for hepA in both the wild-type F . heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain . The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

Microb Ecol, 2001 Aug, 42(2), 136 - 149
Phylogenetic Analysis of Microbial Diversity in the Rhizoplane of Oilseed Rape (Brassica napus cv . Westar) Employing Cultivation-Dependent and Cultivation-Independent Approaches; Kaiser O et al.; The structure of the microbial rhizoplane community of the important crop plant oilseed rape was studied by using a culture-dependent as well as a culture-independent approach based on 16S rDNA amplification . After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica napus cv . Westar), the collected suspension was divided into two parts . One part was used for cultivation of bacteria onto three different growth media to establish a culture collection . From the other part of the rhizoplane suspension, genomic DNA was isolated and purified . Thereafter, 16S rDNA was amplified by PCR and cloned to obtain a library of 16S rDNA genes representative for the bacterial communities of this habitat . Phylogenetic 16S rDNA sequence analysis of 103 clones of this library revealed considerable differences from the corresponding nucleotide sequences of 111 cultured bacteria . Whereas the 16S rDNA clone library was dominated by a-Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups . More than 64% of the cultivated isolates were allocated to the b- and g-subclasses of the Proteobacteria, which were present in the clone library at about 14% . Most of the clones of the a-Proteobacteria of the library showed highest similarity to Bradyrhizobium sp . No such bacteria were found in the culture collection . Similarly, the second dominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate . The phylogenetic analysis of isolates of the culture collection clearly emphasized the need to use different growth media for recovery of rhizoplane bacteria . Whereas most of the a-Proteobacteria were recovered on complex medium, most of the b-Proteobacteria were isolated onto minimal media . Our results demonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.

Microb Ecol, 2001 Oct, 42(3), 359 - 371
Changes in the Epilimnetic Bacterial Community Composition, Production, and Protist-Induced Mortality along the Longitudinal Axis of a Highly Eutrophic Reservoir; Simek K et al.; We studied changes in the epilimnetic bacterial community composition (BCC), bacterial biomass and production, and protistan succession and bacterivory along the longitudinal axis of the canyon-shaped, highly eutrophic Sau Reservoir (NE Spain) during two sampling campaigns, in April and July 1997 . Longitudinal changes in BCC from the river inflow to the dam area of the reservoir were detected by using oligonucleotide probes targeted to the kingdom Bacteria, to the alpha, beta, and gamma subclasses (ALFA, BETA, and GAMA) of the class Proteobacteria, and to the Cytophaga/Flavobacterium (CF) cluster . In general, the inflow of the organically loaded Ter river, with highly abundant allochthonous bacterial populations, induced a clearly distinguishable longitudinal succession of the structure of the microbial food web . The most dynamic changes in microbial parameters occurred at the plunge point, the mixing area of river water and the reservoir epilimnion . Changes within members of BETA and CF were the most important in determining changes in BCC, bacterial abundance and biomass . Much less relevant changes occurred within the less abundant ALFA and GAMA bacteria . From the plunge point downstream, we described a significant shift in BCC in the form of decreased proportions of BETA and CF . This shift spatially coincided with the highest values of heterotrophic nanoflagellate bacterivory (roughly doubled the bacterial production) . CF numerically dominated throughout the reservoir without any marked longitudinal changes in their mean cell volume . In contrast, very large cells affiliated to BETA clearly dominated in the allochthonous bacterial biomass brought by the river . BETA showed a marked downstream trend of decreasing mean cell volume . We conclude that the observed BCC shift and the longitudinal shift in food web structure (bacteria-heterotrophic nanoflagellates-ciliates) resulted from highly complex interactions brought about by several major factors: varying hydrology, the high localized allochthonous input of organic matter brought by the river, downstream changing substrate availability, and selective protistan bacterivory.

Microb Ecol, 2001 Oct, 42(3), 274 - 285
Community Dynamics of Free-living and Particle-associated Bacterial Assemblages during a Freshwater Phytoplankton Bloom; Riemann L et al.; Bacterial community dynamics were followed in a 19-day period during an induced diatom bloom in two freshwater mesocosms . The main goal was to compare diversity and succession among free-living (<10 mm) and particle-associated (>10 mm) bacteria . Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rDNA showed the highest number of bands among free-living bacteria, but with a significant phylogenetic overlap in the two size fractions indicating that free-living bacteria were also important members of the particle-associated bacterial assemblage . Whereas the number of bands in the free-living fraction decreased during the course of the bloom, several phylotypes unique to particles appeared towards the end of the experiment . Besides the primer set targeting Bacteria, a primer set targeting most members of the Cytophaga-Flavobacterium (CF)-cluster of the Cytophaga-Flavobacterium-Bacteroides group and a primer set mainly targeting a-Proteobacteria were applied . PCR-DGGE analyses revealed that a number of phylotypes targeted by those primer sets were found solely on particles . Almost all sequenced bands from the bacterial DGGE gel were related to phylogenetic groups commonly found in freshwater: a-Proteobacteria, CF, and Firmicutes . Despite the use of primers intended to be specific mainly for a-Proteobacteria most bands sequenced from the a-proteobacterial DGGE gel formed a cluster within the Verrucomicrobiales subdivision of the Verrucomicrobia division and were not related to a-Proteobacteria . Bands sequenced from the CF DGGE gel were related to members of the CF cluster . From the present study, we suggest that free-living and particle-associated bacterial communities should not be perceived as separate entities, but rather as interacting assemblages, where the extent of phylogenetic overlap is dependent on the nature of the particulate matter.

Environ Microbiol, 2002 Apr, 4(4), 204 - 15
Wide bacterial diversity associated with tubes of the vent worm Riftia pachyptila; Lopez-Garcia P et al.; We carried out a 16S rDNA-based molecular survey of the prokaryotic diversity associated with the chitin tubes of the giant vent tubeworm Riftia pachyptila (collected at the East Pacific Rise, 9 degrees N and 13 degrees N) . Scanning electron microscopy showed dense microbial populations, particularly on the external surface of the middle and upper tube regions, which included very diverse prokaryotic morphotypes . We used archaeal- and bacterial-specific primers for polymerase chain reaction (PCR) amplification, but only bacterial amplicons were obtained . We analysed a total of 87 clones . Most belonged to the epsilon-Proteobacteria, but also to the delta-, alpha- and gamma-Proteobacteria . A broad diversity of phylotypes belonging to other bacterial divisions was detected, including Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides group and the candidate division OP8 . We also retrieved a sequence, R76-B150, of uncertain phylogenetic affiliation, which could represent a novel candidate division . The sequence of the R . pachyptilagamma-proteobacterial endosymbiont was not detected . The bacterial diversity found suggests that complex metabolic interactions, particularly based on sulphur chemistry, may be occurring in different microniches of the R . pachyptila tubes.

Folia Microbiol (Praha), 2002, 47(1), 51 - 5
Antifungal efficacy of bacteria isolated from marine sedentary organisms; Mohapatra BR et al.; The antibiotic-producing ability of 57 bacteria isolated from 8 marine sedentary organisms, 6 sponges (Spirastrella sp., Phyllospongia sp., Ircinia sp., Aaptos sp., Azorica sp., Axinella sp.), 1 soft coral (Lobophytum sp.) and 1 alga (Sargassum sp.), was evaluated against 6 phytopathogenic fungi (Helminthosporium oryzae, Rhizoctonium solani, Pyricularia oryzae, Fusarium oxysporum, Aspergillus oryzae and A . fumigatus) . Bacteria of the genus Bacillus (20%), Pseudomonas (33%) and Flavobacterium (40%) were predominant among the heterotrophic bacteria isolated from the marine sponges, soft coral and alga, respectively . Bioassay results revealed that 36 (63%) bacterial isolates displayed antifungal activity against at least one fungus, the alga (Sargassum sp.) being the source of highest number (80%) of producer strains . Twelve bacterial isolates inhibited all fungi . The MIC of the organic extracts of 12 bacteria ranged from 0.3 to 22.8 mg/L.

Biochem J, 2002 Aug 1, 365(Pt 3), 857 - 63
Identification of the two essential groups in the family 3 beta-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis; Chir J et al.; beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) catalyses the hydrolysis of beta-1,4-glucosidic bonds via a two-step double-displacement mechanism in which two amino acid residues act as nucleophile and acid/base catalyst . Definitive identification of these two residues is provided by the two active-site-directed inactivators, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-d-glucoside (2FDNPG) and N-bromoacetyl-beta-d-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbgl, respectively . Pseudo-first-order inactivation rate constants (k(i)) of 0.25+/-0.01 and 0.05+/-0.01 min(-1) and dissociation constants (K(i)) of 90+/-15 and 4.4+/-0.2 mM are determined for 2FDNPG and NBGN, respectively . Proteolytic digestion of the labelled proteins, followed by peptide mapping and tandem MS analysis identify Asp-247 and Glu-473 as the catalytic nucleophile and acid/base residues, respectively, of Fbgl . This study confirms that the catalytic nucleophile of family 3 glycohydrolase is conserved across sub-families . However, different sub-families may have unique general acid/base catalysts.

J Bacteriol, 2002 May, 184(9), 2370 - 8
Mutations in Flavobacterium johnsoniae gldF and gldG disrupt gliding motility and interfere with membrane localization of GldA; Hunnicutt DW et al.; Flavobacterium johnsoniae moves rapidly over surfaces by a process known as gliding motility . The mechanism of this form of motility is not known . Four genes that are required for F . johnsoniae gliding motility, gldA, gldB, gldD, and ftsX, have recently been described . GldA is similar to the ATP-hydrolyzing components of ATP binding cassette (ABC) transporters . Tn4351 mutagenesis was used to identify two additional genes, gldF and gldG, that are required for cell movement . gldF and gldG appear to constitute an operon, and a Tn4351 insertion in gldF was polar on gldG . pMK314, which carries the entire gldFG region, restored motility to each of the gldF and gldG mutants . pMK321, which expresses GldG but not GldF, restored motility to each of the gldG mutants but did not complement the gldF mutant . GldF has six putative membrane-spanning segments and is similar in sequence to channel-forming components of ABC transporters . GldG is similar to putative accessory proteins of ABC transporters . It has two apparent membrane-spanning helices, one near the amino terminus and one near the carboxy terminus, and a large intervening loop that is predicted to reside in the periplasm . GldF and GldG are involved in membrane localization of GldA, suggesting that GldA, GldF, and GldG may interact to form a transporter . F . johnsoniae gldA is not closely linked to gldFG, but the gldA, gldF, and gldG homologs of the distantly related gliding bacterium Cytophaga hutchinsonii are arranged in what appears to be an operon . The exact roles of F . johnsoniae GldA, GldF, and GldG in gliding are not known . Sequence similarities of GldA to components of other ABC transporters suggest that the Gld transporter may be involved in export of some material to the periplasm, outer membrane, or beyond.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 599 - 605
Obligate bacterial endosymbionts of Acanthamoeba spp . related to the beta-Proteobacteria: proposal of 'Candidatus Procabacter acanthamoebae' gen . nov., sp . nov; Horn M et al.; All obligate bacterial endosymbionts of free-living amoebae currently described are affiliated with the alpha-Proteobacteria, the Chlamydiales or the phylum Cytophaga-Flavobacterium-Bacteroides . Here, six rod-shaped gram-negative obligate bacterial endosymbionts of clinical and environmental isolates of Acanthamoeba spp . from the USA and Malaysia are reported . Comparative 16S rDNA sequence analysis demonstrated that these endosymbionts form a novel, monophyletic lineage within the beta-Proteobacteria, showing less than 90% sequence similarity to all other recognized members of this subclass . 23S rDNA sequence analysis of two symbionts confirmed this affiliation and revealed the presence of uncommon putative intervening sequences of 146 bp within helix-25 that shared no sequence homology to any other bacterial rDNA . In addition, the 23S rRNA of these endosymbionts displayed one polymorphism at the target site of oligonucleotide probe BET42a that is conserved in all other sequenced beta-Proteobacteria . Intra-cytoplasmatic localization of the endosymbionts within the amoebal host cells was confirmed by electron microscopy and fluorescence in situ hybridization with a specific 16S rRNA-targeted oligonucleotide probe . Based on these findings, the provisional name 'Candidatus Procabacter acanthamoebae' is proposed for classification of a representative of the six endosymbionts of Acanthamoeba spp . studied in this report . Comparative 18S rDNA sequence analysis of the Acanthamoeba host cells revealed their membership with either Acanthamoeba 18S rDNA sequence type T5 (Acanthamoeba lenticulata) or sequence type T4, which comprises the majority of all Acanthamoeba isolates.

Invest Ophthalmol Vis Sci, 2002 Apr, 43(4), 1210 - 21
Effect of dietary zeaxanthin on tissue distribution of zeaxanthin and lutein in quail; Toyoda Y et al.; PURPOSE: The xanthophyll carotenoids (lutein and zeaxanthin) are hypothesized to delay progression of age-related macular degeneration . The quail has a cone-dominant retina that accumulates carotenoids . The purpose of these experiments was to characterize the carotenoid composition of retina, serum, liver, and fat in quail and to determine whether dietary enrichment with zeaxanthin alters zeaxanthin or lutein concentrations in these tissues . METHODS: Quail were fed for 6 months with a commercial turkey diet (T group; n = 8), carotenoid-deficient diet (C- group; n = 8), or a carotenoid-deficient diet supplemented with 35 mg 3R,3'R-zeaxanthin per kilogram of food, (Z+ group; n = 8) . Zeaxanthin was derived from Sphingobacterium multivorum (basonym Flavobacterium) . Carotenoids in serum, retina, liver, and fat were analyzed by HPLC . RESULTS: As in the primate fovea, the retina accumulated zeaxanthin, lutein, and cryptoxanthin, and preferentially absorbed zeaxanthin (P < 0.005) . In contrast, lutein was preferentially absorbed by liver (P < 0.01) and fat (P < 0.0001) . In supplemented females, zeaxanthin increased approximately 4-fold in retina, and 74-, 63- and 22-fold in serum, liver, and fat, respectively . In males, zeaxanthin was elevated approximately 3-fold in retina, and 42-, 17-, and 12-fold in serum, liver, and fat, respectively . Birds fed the Z+ diet absorbed a higher fraction of dietary lutein into serum, but lutein was reduced in the retina (P < 0.05) . CONCLUSIONS: Xanthophyll profiles in quail mimic those in primates . Dietary supplements of zeaxanthin effectively increased zeaxanthin concentrations in serum, retina, liver, and fat . The robust response to zeaxanthin supplementation identifies the quail as an animal model for exploration of factors regulating delivery of dietary carotenoids to the retina.

Appl Environ Microbiol, 2002 Apr, 68(4), 1706 - 14
Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations; Casamayor EO et al.; Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting . Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times . DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members . The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups . Thus, the sequences of two Synechococcus spp . (both had a similarity of 97% to Synechococcus sp . strain PCC6307 in 16S rRNA), two Thiocystis spp . (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp . (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp . of 93%, respectively) were obtained . The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar . The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time . Finally, two of the Cytophaga spp . showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter . Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained . Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.

Appl Environ Microbiol, 2002 Apr, 68(4), 1674 - 83
Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds; Abed RM et al.; We studied the microbial diversity of benthic cyanobacterial mats inhabiting a heavily polluted site in a coastal stream (Wadi Gaza) and monitored the microbial community response induced by exposure to and degradation of four model petroleum compounds in the laboratory . Phormidium- and Oscillatoria-like cyanobacterial morphotypes were dominant in the field . Bacteria belonging to different groups, mainly the Cytophaga-Flavobacterium-Bacteriodes group, the gamma and beta subclasses of the class Proteobacteria, and the green nonsulfur bacteria, were also detected . In slurry experiments, these communities efficiently degraded phenanthrene and dibenzothiophene completely in 7 days both in the light and in the dark . n-Octadecane and pristane were degraded to 25 and 34% of their original levels, respectively, within 7 days, but there was no further degradation until 40 days . Both cyanobacterial and bacterial communities exhibited noticeable changes concomitant with degradation of the compounds . The populations enriched by exposure to petroleum compounds included a cyanobacterium affiliated phylogenetically with Halomicronema . Bacteria enriched both in the light and in the dark, but not bacteria enriched in any of the controls, belonged to the newly described Holophaga-Geothrix-Acidobacterium phylum . In addition, another bacterial population, found to be a member of green nonsulfur bacteria, was detected only in the bacteria treated in the light . All or some of the populations may play a significant role in metabolizing the petroleum compounds . We concluded that the microbial mats from Wadi Gaza are rich in microorganisms with high biodegradative potential.

Fish Shellfish Immunol, 2002 Feb, 12(2), 169 - 79
The outer membrane fraction of Flavobacterium psychrophilum induces protective immunity in rainbow trout and ayu; Rahman H et al.; Flavobacterium psychrophilum is the causative agent of coldwater disease, which is responsible for serious losses in fish aquaculture in several parts of the world . No commercial vaccines are currently available for the prevention of coldwater disease . The present study sought to assess the efficacy of a F . psychrophilum vaccine based on the antigenic outer membrane fraction (OMF) . This fraction induced significantly higher protection against coldwater disease in both rainbow trout (Oncorhynchus mykiss) and ayu (Plecoglossus altivelis) compared to inactivated whole cell F . psychrophilum bacterin . Coincident with higher protection, sera of fish immunised with the OMF vaccine had higher agglutination titres than those of fish immunised with inactivated whole cell F . psychrophilum.

Fish Shellfish Immunol, 2002 Feb, 12(2), 141 - 53
Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro; Wiklund T et al.; Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro . A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells . After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C . In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either . The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum . In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed . The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C . In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h . The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F . psychrophilum . However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.

Microbes Infect, 2002 Mar, 4(3), 279 - 83
Adherence of Flavobacterium psychrophilum on the body surface of the ayu Plecoglossus altivelis; Kondo M et al.; Bacterial cold water disease in the ayu Plecoglossus altivelis caused by Flavobacterium psychrophilum is a serious problem in the Japanese freshwater culture industry . The distribution and activity of this bacterium on the body surface of the ayu in the infection process was investigated . The survival of F . psychrophilum in tap water showed that this bacterium might sustain its infectivity for 24 h . In an experimental infection, juvenile ayu were immersed in water containing 10(8.9) CFU/ml F . psychrophilum, and the progressing infection was followed by scanning electron microscopy during a 24-h period . This bacterium was observed in the ayu for 24 h adhering to the lower jaw and caudal peduncle, where the epidermis tissue was collapsed . This study showed that bacterial suspension in water sustains the activity of this bacterium . F . psychrophilum attaches especially to the jaw and caudal peduncle, growing at these sites, collapsing the dermal structure and invading the tissues.

J Immunol, 2002 Mar 15, 168(6), 2939 - 43
Toll-like receptor 4-MD-2 complex mediates the signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum; Gomi K et al.; Flavolipin, an amino acid-containing lipid isolated from Flavobacterium meningosepticum, induces many immune responses . It has been shown that flavolipin does not induce an immune response of macrophages derived from C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4) . To determine whether TLR4 or the molecular complex of TLR4 and TLR4 association molecule MD-2 mediates the flavolipin signal, flavolipin responsiveness was examined by measuring NF-kappaB activation in Ba/F3 cells and Ba/F3 transfectants expressing TLR4 or both TLR4 and MD-2 . Flavolipin-induced NF-kappaB activation was detected in the cells expressing both TLR4 and MD-2, but not in the other cells . Expression of CD14 in the transfectant expressing both TLR4 and MD-2 increased the sensitivity to flavolipin . Furthermore, flavolipin stereoisomers were chemically synthesized, and their abilities to induce NF-kappaB activation were examined . (R)-Flavolipin, in which the configuration of the lipid moiety is R, induced NF-kappaB activation via the TLR4-MD-2 complex, but (S)-flavolipin did not . In this study, we demonstrated the involvement of TLR4-MD-2 and CD14 in flavolipin signaling and the importance of the (R)-configuration of the flavolipin lipid moiety for the induction of an immune response via TLR4-MD-2.

Proc Natl Acad Sci U S A, 2002 Apr 2, 99(7), 4662 - 7 Epub 2002 Mar 05.
A mitochondrial-like aconitase in the bacterium Bacteroides fragilis: implications for the evolution of the mitochondrial Krebs cycle; Baughn AD et al.; Aconitase and isocitrate dehydrogenase (IDH) enzyme activities were detected in anaerobically prepared cell extracts of the obligate anaerobe Bacteroides fragilis . The aconitase gene was located upstream of the genes encoding the other two components of the oxidative branch of the Krebs cycle, IDH and citrate synthase . Mutational analysis indicates that these genes are cotranscribed . A nonpolar in-frame deletion of the acnA gene that encodes the aconitase prevented growth in glucose minimal medium unless heme or succinate was added to the medium . These results imply that B . fragilis has two pathways for alpha-ketoglutarate biosynthesis-one from isocitrate and the other from succinate . Homology searches indicated that the B . fragilis aconitase is most closely related to aconitases of two other Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria, Cytophaga hutchinsonii and Fibrobacter succinogenes . Phylogenetic analysis indicates that the CFB group aconitases are most closely related to mitochondrial aconitases . In addition, the IDH of C . hutchinsonii was found to be most closely related to the mitochondrial/cytosolic IDH-2 group of eukaryotic organisms . These data suggest a common origin for these Krebs cycle enzymes in mitochondria and CFB group bacteria.

J Appl Microbiol, 2002, 92(3), 510 - 6
Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum; Baliarda A et al.; AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples . METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples . Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain) . This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only . The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid . Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl . psychrophilum . The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O . kisutch) samples were contaminated . CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl . psychrophilum in the ovarian fluid . SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl . psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.

J Org Chem, 2002 Feb 8, 67(3), 871 - 5
Biosynthesis of zeaxanthin via mevalonate in Paracoccus species strain PTA-3335 . A product-based retrobiosynthetic study; Eisenreich W et al.; Cultures of the zeaxanthin-producing bacterium Paracoccus species strain PTA-3335 (formerly Flavobacterium) were grown with supplements of (13)C-labeled glucose . Zeaxanthin was isolated and analyzed by (13)C NMR spectroscopy . The data showed that the isoprenoid precursors of zeaxanthin were biosynthesized via the mevalonate pathway . The microorganism was found to utilize glucose mainly via the Entner-Doudoroff pathway.

Biochemistry, 2002 Feb 26, 41(8), 2751 - 9
Identification of the general acid/base catalyst of a family 3 beta-glucosidase from Flavobacterium meningosepticum; Li YK et al.; beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) (also known as Chryseobacterium meningosepticum) has been classified as a member of the family 3 glycohydrolases . It is a retaining enzyme involving a two-step, double-displacement mechanism . D247 was shown to function as the nucleophile of the enzymatic reaction {Li, Y.-K., Chir, J., and Chen, F.-Y . (2001) Biochem . J . 355, 835-840} . However, the general acid/base catalyst of this enzyme and of all other family 3 glycohydrolases has not yet been identified . On the basis of amino acid sequence alignment of 15 family 3 enzymes, 11 residues (D71, R129, E132, E136, D137, K168, H169, E177, D247, D458, and E473) are highly conserved . All of these residues are studied by site-directed mutagenesis and kinetic investigation . Analyzing the catalytic power of all mutants reveals E473 residue is the best candidate of the acid/base catalyst . Detailed studies supporting this suggestion are summarized as follows . (1) The k(cat) and K(m) values for the hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside (2,4-DNPG) by E473G are reduced 3300- and 900-fold, respectively, compared with those of the wild type (WT) . (2) The k(cat) values of E473G-catalyzed hydrolysis are virtually invariant with pH over the range of 5.0-9.0 . (3) The activity of E473G with 2,4-DNPG is enhanced by the addition of azide, and beta-glucosyl azide is formed . (4) The k(cat) of the reaction of 2-carboxyphenyl beta-glucoside catalyzed by E473G is comparable to that for hydrolysis by wild-type Fbgl and is 100- and 320-fold better than the k(cat) values for the E473G-catalyzed hydrolysis of 4-carboxyphenyl beta-glucoside and the corresponding methyl ester, respectively . (5) The accumulated glucosyl-enzyme intermediate was directly observed by mass analysis in the reaction of 2,4-DNPG with E473G . All of these results confirm that E473 is the general acid/base catalyst of Fbgl.

Microbiol Immunol, 2001, 45(12), 813 - 8
Changes in the cell structure of Flavobacterium psychrophilum with length of culture; Kondo M et al.; Flavobacterium psychrophilum, the pathogen of bacterial cold-water disease, causes serious problems in ayu Plecoglossus altivelis culture . This study investigated the effect of the culture period of F . psychrophilum and on the structure of its cells . From the SDS-PAGE of total proteins of cellular components, much difference was found between the 36 hr culture and the 48 and 72 hr cultures . A SEM observation of the cells showed many fragments, especially on the cell surface of the 36 hr culture . These fragments consisted of an outer membrane, seen by TEM observation, and may contain substances causing the virulence . Specific proteins observed by the SDS-PAGE and fragments in the 36 hr culture may be related to the virulence of F . psychrophilum.

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 173 - 8
Anaerophaga thermohalophila gen . nov., sp . nov., a moderately thermohalophilic, strictly anaerobic fermentative bacterium; Denger K et al.; The strictly anaerobic gram-negative bacterium strain Fru22T grows at 50 degrees C in media containing up to 75 g NaCl l(-1) . Hexoses and pentoses are fermented to equal molar amounts of acetate, propionate and succinate, and no CO2 is formed . An orange-red pigment similar to flexirrubin is produced during stationary phase upon exposure to light for several days . Cells also produce a surface-active extracellular compound which lowers the surface tension of the medium . This tenside is heat-tolerant up to 70 degrees C and is destroyed by treatment with proteinase K or trypsin, but not by lipase . Comparative 16S rDNA sequence analysis confirmed a phylogenetic affiliation of strain Fru22T to the phylum Bacteroides (Cytophaga/Flavobacterium/Bacteroides), moderately related to the genus Marinilabilia . Therefore, on the basis of phylogenetic, phenotypic and physiological evidence, a new genus, Anaerophaga, is proposed to harbour strain Fru22T (DSM 12881T, OCM 798T) which is described as the type strain of a new species, Anaerophaga thermohalophila gen . nov., sp . nov.

Protein Expr Purif, 2002 Feb, 24(1), 90 - 8
Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F; Loo T et al.; PNGase F is a widely used deglycosidase, secreted in small amounts by the gram-negative bacterium Flavobacterium meningosepticum . We have designed a T7 promoter-based Escherichia coli expression system to provide a high-yield source of recombinant enzyme . When expressed intracellularly, the enzyme was produced in a largely insoluble state . However, when expressed as a fusion with the leader sequence from the ompA gene, hexahistidine-tagged PNGase F was efficiently processed and exported to the E . coli periplasm . Single-step purification using immobilized metal affinity chromatography yielded 8 mg of pure enzyme per liter of culture, which is fully active on a range of protein and peptide substrates .

Zhonghua Jie He He Hu Xi Za Zhi, 2001 Jun, 24(6), 345 - 7
{Analysis of the risk factors and drug resistance of lower respiratory tract infection by Flavobacteria}; Feng J et al.; OBJECTIVE: To investigate risk factors of 26 cases with lower respiratory tract infection caused by Flavobacteria and its antimicrobial susceptibility in vitro . METHODS: Retrospective study of the clinical materials of 26 cases of lower respiratory tract infection caused by Flavobacteria . Antimicrobial susceptibility (MIC) was determined with the method of agar dilution . RESULTS: All of 26 cases suffered from underlying diseases, among them malignant tumors were most common (31%) illness . 50% of the cases were mixed infections . Risk factors of infections were application of multiple antibiotics (81%), host immune suppression (77%), invasive manasements (31%), and prolonged hospitalization . No specific clinical manifestations and chest X-ray appearance revealed . Isolates were highly resistant to most of antimicrobial agents . Ciprofloxacin, trimethoprim-sulfamethoxazole and piperacillin are the most sensitive agents . CONCLUSIONS: Lower respiratory tract infection caused by Flavobacteria developed most commonly in patients who suffered from various underlying diseases, immunodeficiency, and longtime antibiotic overuse . Clinical isolates were highly resistant to most kinds of antimicrobial agents.

FEMS Microbiol Lett, 2002 Jan 2, 206(1), 51 - 5
Isolation of a Pseudomonas monteilli strain with a novel phosphotriesterase; Horne I et al.; A Pseudomonas monteilli strain (designated C11) that uses the phosphotriester coroxon as its sole phosphorus source has been isolated . Native PAGE and activity staining identified a single isozyme with significant phosphotriesterase activity in the soluble fraction of the cell . This phosphotriesterase could hydrolyse both coumaphos and coroxon . The hydrolysis product of coroxon, diethylphosphate, and the thion analogue, coumaphos, could not serve as phosphorus sources when added to the growth medium . The majority of the phosphotriesterase and phosphatase activity was contained in the soluble fraction of the cell . Phosphatase activity was inhibited by vanadate as well as by dialysis against the metal chelator, EDTA . Phosphotriesterase activity was not affected by either vanadate or dialysis with EDTA or 1,10-phenanthroline . Phosphotriesterase activity was regulated by the amounts of both phosphate and coroxon in the medium, whereas total phosphatase activity was regulated by phosphate but not coroxon . A lack of hybridisation using a probe against the opd (organophosphate degradation) gene encoding a phosphotriesterase from Flavobacterium sp . ATCC27551 against bulk DNA from P . monteilli C11 suggested that this strain does not contain opd . The work presented here indicates the presence of a novel phosphotriesterase in P . monteilli C11.

Appl Environ Microbiol, 2002 Jan, 68(1), 379 - 88
Phylogeny of culturable estuarine bacteria catabolizing riverine organic matter in the northern Baltic Sea; Kisand V et al.; The objective of our study was to isolate and determine the phylogenetic affiliation of culturable estuarine bacteria capable of catabolizing riverine dissolved organic matter (RDOM) under laboratory conditions . Additions of RDOM consistently promoted the growth of estuarine bacteria in carbon-limited dilution cultures, with seasonal variation in growth rates and yields . At least 42 different taxa were culturable on solid agar media and, according to quantitative DNA-DNA hybridizations, constituted 32 to 89% of the total bacterial number in the enriched treatments . Five species in the Cytophaga-Flexibacter-Bacteroides group and one in the gamma-proteobacteria phylogenetic group (Marinomonas sp.) were numerically dominant during the stationary phase of the RDOM-enriched dilution cultures but not in the control cultures . Four of the isolates in Cytophaga-Flexibacter-Bacteroides group were putatively affiliated with the genus FLAVOBACTERIUM: All dominating isolates were determined to be new species based on comparison to the current databases . The same group of species dominated independently of the season investigated, suggesting a low diversity of bacteria catabolizing RDOM in the estuary . It also suggested a broad tolerance of the dominating species to seasonal variation in hydrography, chemistry, and competition with other species . Taken together, our results suggest that a limited group of bacteria, mainly in the Flavobacterium genus, played an important role in introducing new energy and carbon to the marine system in the northern Baltic Sea.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 1997 - 2006
Muricauda ruestringensis gen . nov., sp . nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment; Bruns A et al.; A gram-negative, facultatively anaerobic bacterium with appendages was isolated from continuous cultures with a seawater-sediment suspension containing hexadecane as the sole carbon source . Although this organism was isolated from a hexadecane-degrading bacterial community, it was not able to degrade hexadecane . However, this bacterium was able to use different sugars and amino acids for growth, indicating that it probably profits from the lysis or from products like surfactants of other cells in the community . 16S rDNA analysis demonstrated that the isolated strain is phylogenetically related to the family Flavobacteriaceae of the phylum 'Cytophaga-Flavobacterium-Bacteroides' . Evidence based on phenotypic characteristics and 16S rDNA analysis supports the conclusion that this bacterium is distinct from its nearest relative, Zobellia uliginosa (90.72% similarity in 16S rRNA gene sequence), and from the other genera of the Flavobacteriaceae . It is therefore proposed that the isolated marine bacterium represents a novel taxon, designated Muricauda ruestringensis gen . nov., sp . nov . The type strain is strain B1T (= DSM 13258T = LMG 19739T).

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 1987 - 95
Arenibacter gen . nov., new genus of the family flavobacteriaceae and description of a new species, Arenibacter latericius sp . nov; Ivanova EP et al.; Five dark-orange-pigmented, Gram-negative, rod-shaped, non-motile, aerobic bacterial strains were isolated from sandy sediment samples collected in the South China Sea in the Indian Ocean, from a holothurian, Apostichopus japonicus, in the Sea of Japan and from a brown alga, Chorda filum, from the Sea of Okhotsk in the Pacific Ocean . Phenotypic data were collected, demonstrating that the bacteria are chemo-organotrophic and require seawater-based media for growth . Polar lipids were analysed and 27% of the total extract comprised phosphatidylethanolamine as the major component . The predominant cellular fatty acids were branched-chain saturated and unsaturated {i-C15:0, i-C15:1, a-C15:0, C15:0, C16:1(n-7)} . The DNA base composition was 37.5-38.2 mol % G+C . The level of DNA homology of the five isolates was 83-94%, indicating that these isolates belong to the same species . A 16S rDNA sequence of the type strain KMM 426T was determined and phylogenetic analysis, based on neighbour-joining and Fitch-Margoliash methods, revealed that the type strain formed a distinct phyletic line in a clade corresponding to the family Flavobacteriaceae and represented a new genus . From the results of this polyphasic taxonomic analysis, it is proposed that the bacterial strains be classified in a new genus, Arenibacter gen . nov., and species, Arenibacter latericius sp . nov . The type strain is KMM 426T (VKM B 2137DT = LMG 19694T = CIP 106861T).

J Microbiol Methods, 2002 Jan, 48(1), 53 - 68
Automated image analysis and in situ hybridization as tools to study bacterial populations in food resources, gut and cast of Lumbricus terrestris L; Schonholzer F et al.; An image analysis procedure was developed for bacterial cells after staining with the DNA-intercalating dye 4'-6-diamidino-2-phenylindole (DAPI), and after in situ hybridization with Cy3-labeled, rRNA-targeted oligonucleotide probes . DAPI- and Cy3-images were captured separately from the same scenery with a cooled digital video camera with three CCD chips for the basic colors red (R), green (G) and blue (B) . Using the appropriate filter sets, images of DAPI-stained cells were captured with the red channel shut down, while Cy3-stained cells were captured with the green and blue channels shut down . DAPI images and Cy3 images were subsequently merged to produce virtual color (RGB)-images . Processing of all color channels allowed to specifically enumerate DAPI-stained and hybridized bacteria, to measure their cell sizes, to subsequently calculate their biovolumes and to estimate their biomass . Using this procedure, significant differences were detected in bacterial populations in food resources, digestive tract and cast of the earthworm L . terrestris L . In leaves, bacteria were on average ten times more abundant and two times larger than in soil . In the digestive tract of L . terrestris, however, numbers and volumes of bacteria were comparable to those in soil indicating the disruption of cells originating from leaves before arriving in the foregut . Passage through the digestive tract of L . terrestris significantly reduced bacterial populations belonging to the alpha-, beta- and gamma-subdivisions of Proteobacteria . While these populations did not recover during incubation of cast, populations of the delta-subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster of the CFB phylum increased in cast . These results suggest a large impact of passage through the digestive tract of L . terrestris on bacterial community structure and demonstrate the usefulness of our image analysis procedure for the determination of cell sizes and biovolumes and thus biomass of specific bacterial populations in different terrestrial habitats.

Ceylon Med J, 2001 Jun, 46(2), 45 - 7
Microbiological aspects of peritonitis in patients undergoing chronic peritoneal dialysis at the dialysis unit of Sri Jayawardenapura General Hospital; Perera S et al.; BACKGROUND: Peritoneal dialysis (PD) is an established form of therapy in the management of end stage renal disease . Peritonitis is the main complication of PD . OBJECTIVES: To study the incidence and microbial aetiology of peritonitis in patients undergoing chronic PD at the dialysis unit of Sri Jayewardenapura General Hospital (SJGH); to assess the diagnostic value of the Gram's stain; and to study the relationship of the total white cell count of effluent to peritonitis . DESIGN: A prospective study over three months . SETTING: Dialysis unit of SJGH . PATIENT POPULATION: The study involved 18 patients undergoing manual intermittent peritoneal dialysis (IPD), 4 patients undergoing chronic ambulatory peritoneal dialysis (CAPD), and 1 patient undergoing nocturnal intermittent peritoneal dialysis (NIPD) . MEASUREMENTS: Clinical presentation of patients with peritonitis; total and differential white blood cell counts of effluent samples; Gram stain and culture of the centrifuged deposit to determine microbial aetiology; incidence of peritonitis in different categories of dialysis . RESULTS: 32 samples were examined from patients on IPD, and 17 from patients on CAPD . In IPD most episodes were due to Gram negative organisms whereas in CAPD most episodes were due to Gram positive organisms . Sensitivity of Gram's stain in relation to culture was 32.4% . 98% of effluent samples had white blood cell counts of > 100/ml and none showed neutrophil counts of < 49% . CONCLUSIONS: The incidence of IPD associated peritonitis was 11.1 episodes per patient year, and the incidence of CAPD associated peritonitis was 14 episodes per patient year . Flavobacterium spp . were the predominant organisms in IPD associated peritonitis, whereas CAPD associated peritonitis was commonly caused by coagulase negative staphylococci . Gram's stain was not useful in the initial identification of the causative agent, but the white cell and neutrophil counts were found to be sensitive indicators of peritonitis.

Appl Environ Microbiol, 2001 Dec, 67(12), 5392 - 402
Microbial communities in the chemocline of a hypersaline deep-sea basin (Urania basin, Mediterranean Sea); Sass AM et al.; The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea . Since this basin is located at a depth of approximately 3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon . In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated . The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface . Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated . Employing 11 cultivation methods, we isolated a total of 70 bacterial strains . The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods . These strains were identified as flavobacteria, Alteromonas macleodii, and Halomonas aquamarina . All 70 strains could grow chemoorganoheterotrophically under oxic conditions . Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates . Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically . While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unusually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source . Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.

Protein Eng, 2001 Sep, 14(9), 663 - 7
Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region; Sakasegawa S et al.; The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was investigated . A single Escherichia coli transformant, which produced a more thermostable glycerol kinase than the parent enzyme, was obtained . The nucleotide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys . Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostability and Km for ATP had changed . The thermostability of FGK2615 was apparently increased; the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation of FGK2615 was determined to be 72.1 degrees C which was 3.1 degrees C higher than that of the parent FGK . Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermostability and Km for substrates were evaluated . The effect of the substitution of Ser329 to Asp is discussed.

Comp Biochem Physiol B Biochem Mol Biol, 2001 Dec, 130(4), 513 - 9
Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica; Jeong J et al.; Acharan sulfate is a glycosaminoglycan (GAG), having the structure -->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose(1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->, isolated from the body of the giant African snail Achatina fulica . This GAG represents 3-5% of the dry weight of this snail's soft body tissues . Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate . Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules . A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation . Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography . Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate . These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.

Environ Microbiol, 2001 Sep, 3(9), 570 - 7
Isolation of bacteria and 16S rDNAs from Lake Vostok accretion ice; Christner BC et al.; Lake Vostok, the largest subglacial lake in Antarctica, is separated from the surface by approximately 4 km of glacial ice . It has been isolated from direct surface input for at least 420 000 years, and the possibility of a novel environment and ecosystem therefore exists . Lake Vostok water has not been sampled, but an ice core has been recovered that extends into the ice accreted below glacial ice by freezing of Lake Vostok water . Here, we report the recovery of bacterial isolates belonging to the Brachybacteria, Methylobacterium, Paenibacillus and Sphingomonas lineages from a sample of melt water from this accretion ice that originated 3593 m below the surface . We have also amplified small-subunit ribosomal RNA-encoding DNA molecules (16S rDNAs) directly from this melt water that originated from alpha- and beta-proteobacteria, low- and high-G+C Gram-positive bacteria and a member of the Cytophaga/Flavobacterium/Bacteroides lineage.

Appl Environ Microbiol, 2001 Nov, 67(11), 5210 - 8
Comparison of cellular and biomass specific activities of dominant bacterioplankton groups in stratified waters of the Celtic Sea; Zubkov MV et al.; A flow-sorting technique was developed to determine unperturbed metabolic activities of phylogenetically characterized bacterioplankton groups with incorporation rates of {(35)S}methionine tracer . According to fluorescence in situ hybridization with rRNA targeted oligonucleotide probes, a clade of alpha-proteobacteria, related to Roseobacter spp., and a Cytophaga-Flavobacterium cluster dominated the different groups . Cytometric characterization revealed both these groups to have high DNA (HNA) content, while the alpha-proteobacteria exhibited high light scatter (hs) and the Cytophaga-Flavobacterium cluster exhibited low light scatter (ls) . A third abundant group with low DNA (LNA) content contained cells from a SAR86 cluster of gamma-proteobacteria . Cellular specific activities of the HNA-hs group were 4- and 1.7-fold higher than the activities in the HNA-ls and LNA groups, respectively . However, the higher cellular protein synthesis by the HNA-hs could simply be explained by their maintenance of a larger cellular protein biomass . Similar biomass specific activities of the different groups strongly support the main assumption that underlies the determination of bacterial production: different bacteria in a complex community incorporate amino acids at a rate proportional to their protein synthesis . The fact that the highest growth-specific rates were determined for the smallest cells of the LNA group can explain the dominance of this group in nutrient-limited waters . The metabolic activities of the three groups accounted for almost the total bacterioplankton activity, indicating their key biogeochemical role in the planktonic ecosystem of the Celtic Sea.

Appl Environ Microbiol, 2001 Nov, 67(11), 5134 - 42
Isolation of novel pelagic bacteria from the German bight and their seasonal contributions to surface picoplankton; Eilers H et al.; We tested new strategies for the isolation of abundant bacteria from coastal North Sea surface waters, which included reducing by several orders of magnitude the concentrations of inorganic N and P compounds in a synthetic seawater medium . Agar plates were resampled over 37 days, and slowly growing colonies were allowed to develop by repeatedly removing all newly formed colonies . A fivefold increase of colonies was observed on plates with reduced nutrient levels, and the phylogenetic composition of the culture collection changed over time, towards members of the Roseobacter lineage and other alpha-proteobacteria . Novel gamma-proteobacteria from a previously uncultured but cosmopolitan lineage (NOR5) formed colonies only after 12 days of plate incubation . A time series of German Bight surface waters (January to December 1998) was screened by fluorescence in situ hybridization (FISH) with isolate-specific and general probes . During spring and early summer, a prominent fraction of FISH-detectable bacteria (mean, 51%) were affiliated with the Cytophaga-Flavobacterium group (CF) of the Bacteroidetes . One Cytophaga sp . lineage with cultured representatives formed almost 20% of the CF group . Members of the Roseobacter cluster constituted approximately 50% of alpha-proteobacteria, but none of the Roseobacter-related isolates formed populations of >1% in the environment . Thus, the readily culturable members of this clade are probably not representative of Roseobacter species that are common in the water column . In contrast, members of NOR5 were found at high abundances (>10(5) cells ml(-1)) in the summer plankton . Some abundant pelagic bacteria are apparently able to form colonies on solid media, but appropriate isolation techniques for different species need to be developed.

J Food Prot, 2001 Oct, 64(10), 1515 - 20
Effect of sampling method on the representative recovery of microorganisms from the surfaces of aquacultured finfish; Nedoluha PC et al.; The objective of this study was to determine if a gentle rinse procedure was equivalent to the combination of excision and homogenization with a stomacher for the relative removal of various microorganisms from finfish fillets . Fillets of hybrid striped bass and rainbow trout were obtained from local markets and sampled using three methods: rinse (R), excision followed by homogenization in a stomacher (S), and homogenization of fillets following a rinse (RS) . Microorganisms were enumerated on selective and nonselective media, and randomly selected colonies from aerobic plate counts were identified using MIDI Sherlock and BIOLOG microbial identification systems . Enrichments and selective media were used for the isolation of Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica . This study confirms previous reports that stomaching is superior to rinsing for enumerating total microbial populations from fish fillets . Rinsing was more effective for rainbow trout than for striped bass . Sampling method did not affect the relative magnitude of plate counts on media selective for aeromonads, pseudomonads, Shewanella, lactic acid bacteria, enterics, and gram-positive cocci . In the compositional analysis of random isolates, R recovered significantly lower fractions of aeromonads than did S or RS, but sampling method did not affect the percent recovery of lactic acid bacteria, pseudomonads, Shewanella, Moraxellaceae, or Cytophaga/Flavobacterium . However, observations suggest that with increased replication, differences among Moraxellaceae, Pseudomonas, and gram positives might be significant . Only one L . monocytogenes colony was isolated, and no Salmonella or Y . enterocolitica, so the effect of sampling method could not be determined for these organisms . Differences in predominant bacterial populations were seen between fish species.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1639 - 52
Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria: proposal for Tenacibaculum gen . nov . with Tenacibaculum maritimum comb . nov . and Tenacibaculum ovolyticum comb . nov., and description of Tenacibaculum mesophilum sp . nov . and Tenacibaculum amylolyticum sp . nov; Suzuki M et al.; Bacterial strains were isolated from sponge and green algae which were collected on the coast of Japan and Palau . The phylogenetic relationships of these isolates among marine species of the Cytophaga-Flavobacterium-Bacteroides complex were analysed by using their gyrB nucleotide sequences and translated peptide sequences (GyrB) in addition to 16S rDNA sequences . These isolates were closely related to the previously characterized marine Flexibacter species, {Flexibacter} maritimus and {Flexibacter} ovolyticus . These Flexibacter species are distantly related to Flexibacter flexilis, the type species of the genus Flexibacter, and phylogenetically belong to the family Flavobacteriaceae (according to analysis using both 16S rDNA and GyrB sequences) . Their phylogenetic, chemotaxonomic and phenotypic characteristics prompted the proposal that these two species should be transferred to the new genus Tenacibaculum, as Tenacibaculum maritimum and Tenacibaculum ovolyticum, respectively . Two additional new species of the genus Tenacibaculum, Tenacibaculum mesophilum gen . nov., sp . nov . (= MBIC 1140T = IFO 16307T) and Tenacibaculum amylolyticum gen . nov., sp . nov . (= MBIC 4355T = IFO 16310T), which were isolated from sponges and macroalgae, are also reported . For taxonomic considerations at the species level, the resolution of gyrB sequences was superior to that of 16S rDNA sequences, and the grouping based on the gyrB phylogram was consistent with DNA-DNA hybridization results.

Appl Environ Microbiol, 2001 Oct, 67(10), 4414 - 25
Changes in populations of rhizosphere bacteria associated with take-all disease of wheat; McSpadden Gardener BB et al.; Take-all, caused by Gaeumannomyces graminis var . tritici, is one of the most important fungal diseases of wheat worldwide . Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen . Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a Pseudomonas-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA) . Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from Pseudomonas to Chryseobacterium species in the rhizosphere of diseased plants . Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria . The functional significance of some of these populations was investigated in vitro . Of those isolated, only a small subset of the most abundant Pseudomonas spp . and a phlD(+) Pseudomonas sp . showed any significant ability to inhibit G . graminis var . tritici directly . When cultured strains were mixed with the inhibitory phlD(+) Pseudomonas strain, the Chryseobacterium isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in Chryseobacterium populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producing phlD(+) pseudomonads.

Mikrobiologiia, 2001 Jul-Aug, 70(4), 567 - 73
{Microbiota of the Orchid rhizoplane}; Tsavkelova EA et al.; Six bacterial strains isolated from the underground roots of the terrestrial orchid Calanthe vestita var . rubrooculata were found to belong to the genera Arthrobacter, Bacillus, Mycobacterium, and Pseudomonas . Strains isolated from the aerial roots of the epiphytic orchid Dendrobium moschatum were classified into the genera Bacillus, Curtobacterium, Flavobacterium, Nocardia, Pseudomonas, Rhodococcus, and Xanthomonas . The rhizoplane of the terrestrial orchid was also populated by cyanobacteria of the genera Nostoc and Oscillatoria, whereas that of the epiphytic orchid was populated by one genus, Nostoc . In orchids occupying different econiches the spectra of the bacterial genera revealed differed . The microbial complex of the terrestrial orchid rhizoplane differed from that of the surrounding soil.

Lett Appl Microbiol, 2001 Sep, 33(3), 178 - 82
Adhesion of the fish pathogen Flavobacterium psychrophilum to unfertilized eggs of rainbow trout (Oncorhynchus mykiss) and n-hexadecane; Vatsos IN et al.; AIMS: The ability of Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS) in fish, to attach to unfertilized rainbow trout (Oncorhynchus mykiss) eggs and to hydrocarbon n-hexadecane was examined in the present study . METHODS AND RESULTS: Five different isolates of Fl . psychrophilum obtained from a variety of origins were compared . The effect of the age of the bacterium and conditions of starvation on the ability of the bacterium to adhere, were also evaluated . CONCLUSION: The different isolates were found to exhibit a similar ability to attach to both substrates . Increased surface hydrophobicity and a greater ability to attach to the surface of the eggs were observed with bacteria aged for one month, compared to bacteria cultured in Cytophaga agar for only three days . SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide useful information regarding the pathogenicity of RTFS, especially during the initial steps of infection.

Environ Microbiol, 2001 Jul, 3(7), 440 - 9
Members of the Cytophaga-Flavobacterium-Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'; Horn M et al.; Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp . isolates were analysed . Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity) . One endocytobiont could neither be cultivated outside its original Acanthamoeba host (Acanthamoeba sp . TUMSJ-321) nor transferred into other amoebae . Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane . According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity) . Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe . Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as 'Candidatus Amoebophilus asiaticus' . Comparative 18S rRNA sequence analysis of the host of 'Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.

Microbios, 2001, 106 Suppl 2, 105 - 16
Polyamine distribution profiles in newly validated genera and species within the Flavobacterium-Flexibacter-Cytophaga-Sphingobacterium complex; Hamana K et al.; Cellular polyamines of 58 strains belonging to the Flavobacterium-Flexibacter-Cytophaga-Sphingobacterium complex were analysed by HPLC . Homospermidine was found in all species of Flavobacterium, Chryseobacterium, Empedobacter, Myroides, Cellulophaga, Salegentibacter, Psychroserpens and Gelidibacter of the family Flavobacteriaceae . Flavobacterium ferrugineum located outside of this family also contained homospermidine . Cytophaga fermentans and C . xylanolytica belonging to the family Bacteroidaceae contained spermidine . Cytophaga marinoflava and C . latercula belonging to Flavobacteriaceae contained homospermidine . The Cytophaga hutchinsonii/C . aurantiaca group contained homospermidine which was the major polyamine in Flexibacter maritimus/ F . ovolyticus of the family Flavobacteriaceae . The Flexibacter sancti/F filiformis/ Cytophaga arvensicola group, F . elegans, F . ruber, F . canadensis, F . flexilis and F . tractuosus, were located separately in different six clusters, and contained homospermidine . The Flexibacter litoralis/F . polymorphus/F . aggregans group contained spermidine, which was detected in Flexibacter roseolus belonging to a divergent cluster . Sphingobacterium and Pedobacter species of the family Sphingobacteriaceae contained homospermidine . Polyamine profiles serve, as a phenotypic chemotaxonomic marker, for the classification of this complex.

Annu Rev Microbiol, 2001, 55, 49 - 75
Bacterial gliding motility: multiple mechanisms for cell movement over surfaces; McBride MJ; The mechanisms responsible for bacterial gliding motility have been a mystery for almost 200 years . Gliding bacteria move actively over surfaces by a process that does not involve flagella . Gliding bacteria are phylogenetically diverse and are abundant in many environments . Recent results indicate that more than one mechanism is needed to explain all forms of bacterial gliding motility . Myxococcus xanthus "social gliding motility" and Synechocystis gliding are similar to bacterial "twitching motility" and rely on type IV pilus extension and retraction for cell movement . In contrast, gliding of filamentous cyanobacteria, mycoplasmas, members of the Cytophaga-Flavobacterium group, and "adventurous gliding" of M . xanthus do not appear to involve pili . The mechanisms of movement employed by these bacteria are still a matter of speculation . Genetic, biochemical, ultrastructural, and behavioral studies are providing insight into the machineries employed by these diverse bacteria that enable them to glide over surfaces.

Syst Appl Microbiol, 1992 Dec, 15(4), 513 - 21
A partial phylogenetic analysis of the "flavobacter-bacteroides" phylum: basis for taxonomic restructuring; Gherna R et al.; On the basis of small subunit rRNA sequence analyses five major subgroups within the flavobacteria-bacteroides phylum have been defined . These are tentatively designated the cytophaga subgroup (comprising largely Cytophaga species), the flavobacter subgroup (comprising the true flavobacteria and the polyphyletic genus Weeksella), the bacteroides subgroup (comprising the bacteroides and certain cytophaga-like bacteria), the sphingobacter subgroup (which contains the known sphingolipid-producing members of the phylum), and the saprospira subgroup (comprising particular species of Flexibacter, Flavobacterium, Haliscomenobacter, and, of course, the genus Saprospira) . These groupings are given not only by evolutionary distance analysis, but can be defined and distinguished on the basis of a simple small subunit rRNA signatures.

Planet Space Sci, 1995 Jan-Feb, 43(1-2), 139 - 47
Meteorite organics in planetary environments: hydrothermal release, surface activity, and microbial utilization; Mautner MN et al.; Up to 50% of the organics in the Murchison meteorite, possibly including some of the polymer, is released in high temperature and pressure aqueous environments, to 350 degrees C and 250 bar, that simulate submarine volcanic, hydrothermal or impact-induced conditions . Meteorite organics of prebiotic significance, such as nonanoic acid, glycine, and pyrene survive the hydrothermal conditions . The released material is surface active with surface pressures up to 19.8 x 10(-3) N m-1, and exhibits an extended surface tension isotherm which suggests a mixture of amphiphilic components . One component, nonanoic acid, is shown to form vesicles . The materials extracted under mild conditions, at 120 degrees C, are nutrients for the humic acid bacterium Pseudomonas maltophilia and efficient nutrients for the oligotroph Flavobacterium oryzihabitans, demonstrating the capability of microorganisms to metabolize extraterrestrial organics.

Microbiology, 2001 Sep, 147(Pt 9), 2611 - 22
The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the Cytophaga-Flavobacterium-Bacteroides division; Griffiths E et al.; The phylogenetic placement of the rumen bacterium Fibrobacter succinogenes was determined using a signature sequence approach that allows determination of the relative branching order of the major divisions among Bacteria {Gupta, R . S . (2000) FEMS Microbiol Rev 24, 367-402} . For this purpose, segments of the Hsp60 (groEL), Hsp70 (dnaK), CTP synthase and alanyl-tRNA synthetase genes, which are known to contain signature sequences that are useful for phylogenetic deterministic purposes, were cloned . Using degenerate oligonucleotide primers for highly conserved regions in these proteins, 1.4 kb, 0.75 kb, 401 bp and 171 bp fragments of the Hsp70, Hsp60, CTP synthase and alanyl-tRNA synthetase genes respectively were amplified by PCR, and these fragments were cloned and sequenced . These primers, because of their high degree of conservation, could also be used for cloning these genes from other bacterial species . The Hsp70 homologues from different Gram-negative bacteria contain a 21-23 aa insert that is not found in any Gram-positive bacteria . The presence of this insert in the F . succinogenes Hsp70 supports its placement within the Gram-negative group of bacteria . A conserved insert in F . succinogenes Hsp60 that is commonly present in all bacterial species, except various Gram-positive bacteria, Deinococcus-Thermus groups and green non-sulphur bacteria, provides evidence that F . succinogenes does not belong to these taxa . A particularly useful signature consisting of a 4 aa insert is found in Ala-tRNA synthetase . This insert is present in all proteobacterial homologues as well as in homologues from species belonging to the Chlamydia and Cytophaga-Flavobacterium- Bacteroides (CFB) groups, but it is not found in homologues from any other groups of bacteria . The presence of this insert in F . succinogenes Ala-tRNA synthetase provides evidence that this species is related to these groups . However, two other signatures in CTP synthase and Hsp70 proteins, that are distinctive of the proteobacterial species, are not present in the F . succinogenes homologues . These results provide evidence that F . succinogenes does not belong to the proteobacterial division and thus should be placed in a similar position as the Chlamydia and CFB groups of species.

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1542 - 8
Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp . endo-beta-N-acetylglucosaminidase; Fujita K et al.; The gene encoding the endo-beta-N-acetylglucosaminidase from Flavobacterium sp . (Endo-Fsp) was sequenced . The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea . The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme . Endo-Fsp had 60% sequence identity with the endo-beta-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved . Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H . The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated . Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity . Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127 . Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.

Biochem Biophys Res Commun, 2001 Aug 17, 286(2), 343 - 51
Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum; Pojasek K et al.; Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation . Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs . Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum . In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time . Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step . The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique . In addition, the Michaelis-Menten parameters were determined for these enzymes . With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1) . This is the first report of kinetic parameters for chondroitinase AC with this substrate . With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1) . Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate . Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS .

Microbios, 2001, 106(413), 7 - 17
Polyamine distribution profiles in the eighteen genera phylogenetically located within the Flavobacterium-Flexibacter-Cytophaga complex; Hamana K et al.; Cellular polyamines of eighteen genera belonging to the Flavobacterium-Flexibacter-Cytophaga complex were analysed by ion exchange liquid chromatography . Homospermidine was the major polyamine in the genera Bergeyella, Riemerella, Ornithobacterium, Weeksella, Capnocytophaga, Polaribacter and Psychroflexus belonging to the family Flavobacteriaceae . In the family Spirosomaceae, Runella, Spirosoma and Flectobacillus species contained spermidine whereas Cyclobacterium species contained homospermidine . Within a divergent cluster, Haliscomenobacter and Lewinella species contained spermidine whereas Saprospira grandis contained agmatine alone . The major polyamine of Chitinophaga and Sporocytophaga species was homospermidine . Flexithrix dorotheae contained spermidine . Microscilla marina, the type species of the genus Microscilla, contained spermidine and cadaverine . However, 'Microscilla sericea' contained homospermidine, 'Microscilla furvescens' contained spermidine, and 'Microscilla arenaria' lacked all polyamines . Polyamine profiles serve as a phenotypic chemotaxonomic marker for the reclassification of the genera belonging to the complex.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1235 - 43
Flavobacterium frigidarium sp . nov., an aerobic, psychrophilic, xylanolytic and laminarinolytic bacterium from Antarctica; Humphry DR et al.; A psychrophilic, aerobic bacterium designated A2iT was isolated from marine sediment recovered from shallow waters surrounding Adelaide Island, Antarctica (67 degrees 34' S, 68 degrees 07' W) . The organism exhibited xylanolytic and laminarinolytic activity and was halotolerant . Basic characterization showed that it was gram-negative, non-motile, yellow-pigmented (beta,beta-carotene-3,3'-diol) and positive for oxidase and catalase synthesis . Analysis of the 16S rDNA sequence suggests that the organism belongs to the Flexibacter-Cytophaga-Bacteroides phylum . On the basis of its 16S rDNA sequence, the bacterium is 96.8% similar to Flavobacterium columnare ATCC 43622--its closest relation . The genomic DNA G+C content was 35 mol% . Growth on xylan occurs optimally at 15 degrees C, though growth also occurs at 0 degrees C, and the doubling times are 9.6 and 34.8 h, respectively . The maximum growth temperature on xylan is at 24 degrees C . The bacterium is a neutrophile, growing across the pH range 5.6-8.4 and having an optimum at pH 7.5 . Analysis of the 16S rDNA sequence, together with phenotypic characterization, suggests that the organism is a member of the genus Flavobacterium . DNA-DNA hybridization experiments have shown that it is a novel species; it is proposed, therefore, that the organism be designated as the type strain of Flavobacterium frigidarium sp . nov . (= ATCC 700810T = NCIMB 13737T).

Mar Environ Res, 2001 Mar, 51(2), 95 - 112
Strong ligands for thorium complexation in marine bacteria; Hirose K et al.; The interaction between thorium and marine organisms (cultured heterotrophic bacteria) was experimentally examined by using chemical equilibrium techniques . Thorium (Th) quantitatively reacts with a binding site on bacteria (Alteromonas, Vibrio, Pseudomonas and Flavobacterium) in 0.1 M HCl solution . According to mass balance analysis of adsorption experiment data, Th forms a 1:1 complex with a binding site similar in reactivity among bacteria used in this study, whose conditional stability constants are in the range from 10(6.63) to 10(7.07) M-1 under the experimental conditions of a 0.1 M HCl solution . The mole ratio of the strong ligand to organic carbon in bacteria ranged from 2.3 to 4.3 mmol/mol C . The strong ligand/carbon ratios in bacteria were more than one order of magnitude greater than in phytoplankton, zooplankton or other organic ligands in surface waters . The results suggest that the strong organic ligand reacting with Th is one of the functional groups commonly existing in oceanic microorganisms . The conditional stability constants of the Th complexes with the binding site in marine microorganisms are in the same order of magnitude as that with the strong ligand found in particulate and dissolved organic matter . These findings strongly suggest that the strong ligand in particulate and dissolved organic matter, reacting with trace metals under the conditions of seawater, originates from marine organisms.

Eur J Emerg Med, 2001 Jun, 8(2), 93 - 7
The yield of blood cultures in a department of emergency medicine; Stalnikowicz R et al.; This study sought to determine the yield of blood cultures drawn in the department of emergency medicine . The results of 730 blood cultures taken from 718 patients were retrospectively analysed . The total percentage of positive cultures was 9.7% . Only 3.4% of the blood cultures were classified as true bacteraemia and the rest as contaminants . The commonest type of isolate was coagulase-negative staphylococci (49%), which were considered contaminants in all cases . Other contaminants represented 13.2% of all the positive blood cultures . The following bacteria comprised the group of true bacteraemia: Escherichia coli (12.6%), Streptococcus pneumoniae (9.8%), viridans streptococci (7%), Staphylococcus aureus (2.8%), Bacteroides fragilis (2.8%), Moraxella species (1.4%) and Flavobacterium species (1.4%) . Blood cultures were positive in 3.6% of patients with pneumonia and in 10% of patients with urinary tract infections . In patients with fever of unclear source blood cultures were positive in 3.1% of children between 0-36 months of age and in 1.1% of patients older than 16 years . As a whole, patients with positive blood cultures were clinically sicker, a higher percentage of them required admission to the hospital and had higher temperatures or rapidly fatal disease, compared with the group of patients with negative blood cultures . In order to improve the yield of blood cultures in febrile patients, first, better a priori identification of those subjects at high risk for bacteraemia will reduce the number of unnecessary blood cultures and second, sterile venipuncture techniques should be improved in order to reduce the number of contaminants.

Prep Biochem Biotechnol, 2001 May, 31(2), 113 - 24
Enzymatic preparation of heparin disaccharides as building blocks in glycosaminoglycan synthesis; Kim YS et al.; Pharmaceutical heparin and heparan sulfate, isolated from a side-stream of a commercial heparin manufacturing process, have been enzymatically depolymerzed with heparin lyases obtained from Flavobacterium heparinun . Heparin afforded a trisulfated disaccharide product that was recovered from the reaction mixture using gel permeation chromatography . Heparan sulfate afforded unsulfated disaccharide that was conveniently recovered from the product mixture by ion exchange chromatography . Both disaccharides were obtained in gram amounts at 90% or higher purity . Both enzymatically prepared disaccharides were chemically protected to prepare building blocks required for the future chemical synthesis of therapeutically valuable heparin oligosaccharides.

J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 320 - 325
Phylogeny of Sphingomonas species that degrade pentachlorophenol; Crawford RL et al.; Four pentachlorophenol (PCP)-degrading bacteria isolated from geographically diverse areas have been examined in detail as regards their physiology and phylogeny . According to traditional biochemical methods, these strains had been classified as members of the genera Arthrobacter, Flavobacterium, Pseudomonas, and Sphingomonas . The PCP degradation pathway has been studied extensively in Sphingomonas (Flavobacterium) sp strain ATCC 39723 and the first three degradation steps catalyzed by a PCP-4-monooxygenase (PcpB) and a reductive dehalogenase (PcpC) that functions twice are well established . A fourth step appears to involve ring-fission of the aromatic nucleus (PcpA) . Molecular analyses revealed that the PCP degradation pathway in these four strains was rather conserved, leading to a phylogenetic analysis using 16S rDNA . The results revealed a much closer phylogenetic relationship between these organisms than traditional classification indicated, placing them into the more recently established genus Sphingomonas where they may even represent a single species . With 16S rDNA analysis, many bacterial isolates involved in degradation of xenobiotic compounds that were previously classified into diverse genera have been reclassified into the genus Sphingomonas.

J Bacteriol, 2001 Jul, 183(14), 4167 - 75
Cloning and characterization of the Flavobacterium johnsoniae gliding motility genes gldD and gldE; Hunnicutt DW et al.; Cells of Flavobacterium johnsoniae move over surfaces by a process known as gliding motility . The mechanism of this form of motility is not known . Cells of F . johnsoniae propel latex spheres along their surfaces, which is thought to be a manifestation of the motility machinery . Three of the genes that are required for F . johnsoniae gliding motility, gldA, gldB, and ftsX, have recently been described . Tn4351 mutagenesis was used to identify another gene, gldD, that is needed for gliding . Tn4351-induced gldD mutants formed nonspreading colonies, and cells failed to glide . They also lacked the ability to propel latex spheres and were resistant to bacteriophages that infect wild-type cells . Introduction of wild-type gldD into the mutants restored motility, ability to propel latex spheres, and sensitivity to bacteriophage infection . gldD codes for a cytoplasmic membrane protein that does not exhibit strong sequence similarity to proteins of known function . gldE, which lies immediately upstream of gldD, encodes another cytoplasmic membrane protein that may be involved in gliding motility . Overexpression of gldE partially suppressed the motility defects of a gldB point mutant, suggesting that GldB and GldE may interact . GldE exhibits sequence similarity to Borrelia burgdorferi TlyC and Salmonella enterica serovar Typhimurium CorC.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 985 - 97
Zobellia galactanovorans gen . nov., sp . nov., a marine species of Flavobacteriaceae isolated from a red alga, and classification of; Barbeyron T et al.; A mesophilic, aerobic, non-flagellated, gliding bacterium, forming yellow colonies and designated DsijT, was isolated from a red alga on the sea-shore of Roscoff, Brittany, France . DsijT was selected for its ability to actively degrade both agars and carrageenans . The Gram-negative cells occurred singly or in pairs as long rods . The temperature range for growth was 13-45 degrees C, with an optimum at 35 degrees C . The pH range for growth at 35 degrees C was from 6.0 to 8.5, with an optimum around pH 7.0 . The NaCl concentrations required for growth at 35 degrees C and pH 7.0 ranged from 5 to 60 g l(-1), with an optimum around 25 g l(-1) . The G+C content of the genomic DNA was 42-43 mol% . Phylogenetic analysis of 16S rRNA gene sequences indicated that strain DsijT is closely related to {Cytophaga} uliginosa DSM 2061T . Phenotypic features, however, allowed DsijT and {Cytophaga} uliginosa strains to be distinguished on the basis of ten traits (spreading behaviour, assimilation of eight compounds and amylase production) . Their total protein profiles were also different and DNA-DNA hybridization experiments confirmed that DsijT constitutes a new species, distinct from {Cytophaga} uliginosa . Based on the phenotypic features and the phylogenetic relationships of the Flavobacteriaceae, a new genus designated Zobellia gen . nov . is proposed to include Zobellia galactanovorans gen . nov., sp . nov., while {Cytophaga} uliginosa becomes Zobellia uliginosa comb . nov . The type strain of Zobellia galactanovorans is DsijT (= DSM 12802T = CIP 106680T).

J Cataract Refract Surg, 2001 Jun, 27(6), 917 - 23
Diffuse lamellar keratitis: isolation of endotoxin and demonstration of the inflammatory potential in a rabbit laser in situ keratomileusis model; Peters NT et al.; PURPOSE: To systematically examine sources of endotoxin contamination in eye centers as a potential cause of diffuse lamellar keratitis (DLK) and to demonstrate the inflammatory potential of endotoxin in a rabbit model of laser in situ keratomileusis (LASIK) surgery . SETTING: University of Calgary, Calgary, Alberta, Canada . METHODS: In this prospective study, all water sources that routinely come in contact with LASIK instruments, including sterilizer reservoirs, eyedrops, microkeratome blades, and cleaning solutions, were examined for endotoxins at 5 eye centers . Bacterial cultures were performed on water samples from 5 sterilizer reservoirs . A LASIK flap was created in 8 rabbit eyes using an Automated Corneal Shaper microkeratome (Bausch & Lomb) . The flaps were reflected, and a dose of endotoxin at various concentrations was placed on the interface . After 1 minute, the flap was irrigated and repositioned . The rabbit eyes were examined daily with a slitlamp biomicroscope for 3 days for the development of DLK, which was classified on a scale from grade 1 to 4 (mild to severe) . The rabbits were killed at the conclusion of the study, and the interfaces were stained to rule out infectious etiologies . RESULTS: Endotoxin was detected in significant concentrations in tap water, filtered and distilled water, instrument washbasins, and sterilizer reservoirs at all 5 centers . The cultures of the water samples taken from the sterilizer reservoirs ranged from no growth to the presence of >100 colony-forming units of Flavobacterium and Pseudomonas aeruginosa . Endotoxins caused DLK-like interface inflammation in all eyes tested . Examination of stained scrapings showed no microorganisms in the interface of the rabbit eyes . CONCLUSION: Endotoxin contamination was detected in water sources that routinely come in contact with LASIK instruments . Endotoxins were capable of inducing interface inflammation in a rabbit model and may therefore be a significant factor in epidemic DLK.

Plasmid, 2001 May, 45(3), 227 - 32
Sequence analysis of the plasmid pRRI2 from the rumen bacterium Prevotella ruminicola 223/M2/7 and the use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors; Mercer DK et al.; pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli . Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames . Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively . ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp . plasmid pFL1 and 30% amino acid sequence identity with BmpH from B . fragilis Tn5520 . Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp . hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate . pRRI2 shows no significant similarity with the only other P . ruminicola plasmid to have been studied previously, pRAM4 .

Syst Appl Microbiol, 2001 Apr, 24(1), 98 - 107
Characterization of facultative oligotrophic bacteria from polar seas by analysis of their fatty acids and 16S rDNA sequences; Mergaert J et al.; One hundred and seventy three bacterial strains, isolated previously after enrichment under oligotrophic, psychrophylic conditions from Arctic (98 strains) and Antarctic seawater (75 strains), were characterized by gas-liquid chromatographic analysis of their fatty acid compositions . By numerical analysis, 8 clusters, containing 2 to 59 strains, could be delineated, and 8 strains formed separate branches . Five clusters contained strains from both poles, two minor clusters were confined to Arctic isolates, and one cluster consisted of Antarctic isolates only . The 16S rRNA genes from 23 strains, representing the different fatty acid profile clusters and including the unclustered strains, were sequenced . The sequences grouped with the alpha and gamma Proteobacteria, the high percent G+C gram positives, and the Cytophaga-Flavobacterium-Bacteroides branch . The sequences of strains from 4 clusters and of 7 unclustered strains were closely related (sequence similarities above 97%) to reference sequences of Sulfitobacter mediterraneus, Halomonas variabilis, Alteromonas macleodii, Pseudoalteromonas species, Shewanella frigidimarina, and Rhodococcus fascians . Strains from the other four clusters and an unclustered strain showed sequence similarities below 97% with nearest named neighbours, including Rhizobium, Glaciecola, Pseudomonas, Alteromonas macleodii and Cytophaga marinoflava, indicating that the clusters which they represent form as yet unnamed taxa.

Arch Microbiol, 2001 Apr, 175(4), 259 - 62
Isolation and characterization of a Chryseobacterium strain from the gut of the American cockroach, Periplaneta americana; Dugas JE et al.; A 16S rDNA sequence cloned directly from whole-gut microbiota of the American cockroach, Periplaneta americana, indicated the presence of a member of the Bacteroides/Flavobacterium group most closely related to the genus Flavobacterium . In an attempt to confirm this finding, we isolated a yellow-pigmented bacterium (strain FR2) from the hindgut of this insect . Strain FR2 was phylogentically and phenotypically most similar to species of Flavobacterium and related bacteria, namely Chryseobacterium indologenes . Fifty-four other yellow-pigmented bacteria isolated during a 1-year study shared the salient phenotypic characteristics of Chryseobacterium spp., and thus were considered the same phenotype . This phenotype's abundance was related to the fiber content of the insect diet, being consistently detected only in cockroaches fed a high-fiber diet (30% crude fiber by weight) . The highest population density was in the hindgut, ranging from 2 x 10(6) to 1.2 x 10(7) colony forming units ml(-1) during a 1-year period . The nature of the symbiosis between the FR2 phenotype and P . americana is discussed.

Appl Environ Microbiol, 2001 Jun, 67(6), 2723 - 33
Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir; Simek K et al.; Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-microm treatment) or enhance (<5-microm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags . Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells . Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes . Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates . In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant . In the "grazer-free" (0.8-microm-filtered) treatment, subject only to a relatively low mortality rate (approximately 17% day(-1)) from viral lysis, bacteria increased markedly in concentration . While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria . In the grazing-enhanced treatment (5-microm filtrate), grazing mortality was approximately 200% and viral lysis resulted in mortality of 30% of daily production . Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment . Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage . Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs . Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.

Appl Environ Microbiol, 2001 Jun, 67(6), 2436 - 44
Purification and characterization of a psychrophilic, calcium-induced, growth-phase-dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum; Secades P et al.; Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids . This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa . This enzyme, designated Fpp1 (F . psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography . On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride . Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability . In addition to calcium, strontium and barium can activate the protein . The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40 degrees C, and its activity decreased rapidly at 45 degrees C . Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II . Fpp1 also degraded actin and myosin, basic elements of the fish muscular system . The presence of this enzyme in culture media was specifically dependent on the calcium concentration . Fpp1 production started early in the exponential growth phase and reached a maximum during this period . Addition of calcium during the stationary phase did not induce Fpp1 production at all . Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1 . In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature . The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F . psychrophilum . Consequently, we suggest that environmental host factors govern Fpp1 production.

Environ Microbiol, 2001 Apr, 3(4), 246 - 55
Molecular detection of marine bacterial populations on beaches contaminated by the Nakhodka tanker oil-spill accident; Kasai Y et al.; In January 1997, the tanker Nakhodka sank in the Japan Sea, and more than 5000 tons of heavy oil leaked . The released oil contaminated more than 500 km of the coastline, and some still remained even by June 1999 . To investigate the long-term influence of the Nakhodka oil spill on marine bacterial populations, sea water and residual oil were sampled from the oil-contaminated zones 10, 18, 22 and 29 months after the accident, and the bacterial populations in these samples were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments . The dominant DGGE bands were sequenced, and the sequences were compared with those in DNA sequence libraries . Most of the bacteria in the sea water samples were classified as the Cytophaga-Flavobacterium-Bacteroides phylum, alpha-Proteobacteria or cyanobacteria . The bacteria detected in the oil paste samples were different from those detected in the sea water samples; they were types related to hydrocarbon degraders, exemplified by strains closely related to Sphingomonas subarctica and Alcanivorax borkumensis . The sizes of the major bacterial populations in the oil paste samples ranged from 3.4 x 10(5) to 1.6 x 10(6) bacteria per gram of oil paste, these low numbers explaining the slow rate of natural attenuation.

Jpn J Ophthalmol . 2001 Jan;45(1):115.
Bacterial Infection in the Conjunctiva of Patients with Adenoviral Conjunctivitis; Watanabe Y et al.; Purpose: We evaluate the microbiological features of mixed infection in adenovirus-infected conjunctiva.Subjects: Isolation of bacteria was performed in 82 samples of adenoviral conjunctivitis at six eye clinics in Japan.Methods: For microbiological diagnosis, we performed immunochromatography (IC) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for detection and serotyping of adenovirus, and PCR for detection of herpes simplex virus (HSV) and Chlamydia trachomatis antigens out of 82 samples with adenoviral conjunctivitis.Results: Pathogenic bacteria were isolated in 6 samples out of 82 . Out of these 6 cases, 5 samples were gram-negative rods and most of them were Flavobacterium meningosepticum (4 samples) . Adenovirus type 8 was isolated from all these mixed infection cases . However, HSV-1 and Chlamydia trachomatis were not found.Conclusions: From these results, it is suggested that gram-negative rods, especially F . meningosepticum, are the most common bacteria causing mixed bacterial infection in adenoviral conjunctivitis.

Int Microbiol, 2000 Dec, 3(4), 235 - 8
Rapid and low-level toxic PCR-based method for routine identification of Flavobacterium psychrophilum; Cepeda C et al.; We describe a rapid, low-toxicity and simple method for the detection of the bacterial fish pathogen Flavobacterium psychrophilum . The method, based on the polymerase chain reaction (PCR), combined the electrophoresis of PCR products in a vertical agarose gel and a modified methylene blue stain . DNA was amplified directly either from bacterial suspensions or from tissues experimentally infected with F . psychrophilum, using different non-toxic commercial DNA extraction kits . The protocol allowed to detect 15 to 150 cells of the pathogen in bacterial suspension, without prior DNA extraction, and 7500 to 75,000 cells in seeded spleen tissue and ovarian fluid using Dynabeads DNA DIRECT extraction system . This method, which has the advantage of not using hazardous products, is proposed as a fast tool for routine identification of F . psychrophilum.

Clin Diagn Lab Immunol, 2001 May, 8(3), 522 - 7
Biological properties of lipid A isolated from Flavobacterium meningosepticum; Tanamoto K et al.; The biological properties of the lipid A from Flavobacterium meningosepticum, which we recently isolated and whose complete chemical structure has been determined (H . Kato, T . Iida, Y . Haishima, A . Tanaka, and K . Tanamoto . J . Bacteriol . 180:3891--3899, 1998), were studied . The lipid A exhibited generally moderate activity compared to Salmonella enterica subsp . enterica serovar abortus equi lipopolysaccharide (LPS) used as a control in the assay systems tested; lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells, induction of tumor necrosis factor alpha (TNF-alpha) release from mouse peritoneal macrophages and J774-1 mouse macrophage-like and human THP-1 line cells, nitric oxide induction activity from J774-1 cells, and Limulus gelation activity . The moderate activity of the F . meningosepticum lipid A may be explained by its unique fatty acid composition and the lack of a phosphate group in position 4' . It is noteworthy that the lipid A apparently induced TNF-alpha release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice and that the activation was suppressed by the LPS-specific antagonist, succinylated lipid A precursor . Significant splenocyte mitogenicity in C3H/HeJ mice was also observed with the lipid A . Taken together with the previous results concerning Porphyromonas gingivalis lipid A, which has a high level of structural similarity to the lipid A of F . meningosepticum, and the induction of TNF-alpha release in macrophages from C3H/HeJ mice, the lipid A of F . meningosepticum, which has novel fatty acids, may possibly play an role for the activation of C3H/HeJ macrophages.

Biochemistry, 2001 Feb 27, 40(8), 2359 - 72
Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis; Huang W et al.; The crystal structures of Flavobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide (HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively . The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution . For each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and HA(tetra)) ordered sugars are visible in electron density maps . The lyase AC DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel . We suggest that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area . The putative catalytic site is located between the substrate recognition area and the product release area, carrying out catalysis at the +1 subsite . Four residues near the catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic tetrad . The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg292Ala mutant . Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis . Candidates for the general base, removing the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or His225 . Tyrosine 234 is a candidate to protonate the leaving group . Arginine 288 likely contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis.

Eur J Biochem, 2001 May, 268(9), 2710 - 6
The structure of the lipopolysaccharide O-antigen produced by Flavobacterium psychrophilum (259-93); MacLean LL et al.; Flavobacterium psychrophilum, a Gram-negative bacterium, is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic infections in reared salmonids . In humans Flavobacterium spp . have been associated with neonatal meningitis and septicemia, catheter-associated bacteremia, and pneumonia . Recently, several F . psychrophilum surface molecules, including lipopolysaccharide (LPS), have been implicated in its pathogenesis and identified as potential vaccine and diagnostic candidate macromolecules . Studies on the LPS produced by the bacterium are reported herein . The structure of the antigenic O-polysaccharide contained in the LPS of F . psychrophilum was deduced by the application of analytical NMR spectroscopy, mass spectrometry, glycose and methylation analysis, and partial hydrolysis degradations, and was found to be an unbranched polymer of trisaccharide repeating units composed of L-rhamnose (L-Rhap), 2-acetamido-2-deoxy-L-fucose (L-FucpNAc) and 2-acetamido-4-((3S,5S)-3,5-dihydroxyhexanamido)-2,4-dideoxy-D-quinovose (D-Quip2NAc4NR, 2-N-acetyl-4-N-((3S,5S)-3,5-dihydroxyhexanoyl)-D-bacillosamine) (1 : 1 : 1) and having the structure: -->4)-alpha-L-FucpNAc-(1-->3)-alpha-D-Quip2NAc4NR-(1-->2)- alpha-L-Rhap-(1--> where R is (3S,5S)-CH3CH(OH)CH2CH(OH)CH2CO-.

Environ Microbiol, 2001 Feb, 3(2), 110 - 22
In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process; Liu WT et al.; Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods . Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the alpha- (9.5% of total cells), beta- (41.3%) and gamma- (6.8%) subclasses of the class Proteobacteria, Flexibacter-Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%) . With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the beta-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel gamma-proteobacterial group were the predominant populations . Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na . As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups . Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA . In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA . No cellular inclusions were detected in most members of the alpha-Proteobacteria and the Cytophaga-Flavobacterium group . The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.

FEMS Microbiol Ecol, 2001 May, 35(3), 267 - 275
A molecular phylogenetic survey of sea-ice microbial communities (SIMCO); Brown MV et al.; 16S rDNA clone library analysis was used to identify bacterial biodiversity in a variety of sea-ice microbial communities (SIMCO) . DNA was extracted from seven Antarctic sea-ice samples and one Arctic sea-ice sample and 16S rDNA PCR-amplified using universal and Archaea-specific primers . Recombinant 16S rDNA clones were obtained and dereplicated using restriction fragment length polymorphism analysis (RFLP) . After RFLP analysis, 100 distinct phylotypes (a unique clone or group of clones with sequence similarity of >0.98) were defined . From the clone libraries 16S rDNA sequences of bacterial and eukaryotic origin were detected, however Archaea were not detected either with universal or Archaea-specific 16S rDNA primer sets . Bacterial phylotypes grouped within the alpha and gamma proteobacteria, the Cytophaga-Flavobacterium-Bacteroides division, the Gram-Positive bacteria and the orders Chlamydiales and Verrucomicrobiales . The majority of bacterial phylotypes were affiliated with heterotrophic taxa and many grouped closely with cultivated genera and species . Eukaryotic clones were affiliated with a variety of autotrophic and heterotrophic nanoplankton and included a large number of chloroplast 16S rDNA genes . The findings of this investigation corroborated culture data indicating bacterial biodiversity increased in SIMCO displaying high levels of primary production, however the bacterial communities within SIMCO were highly heterogeneous at the genus/species-level between different samples . A comparison of Antarctic and Arctic SIMCO revealed certain sea-ice dwelling bacterial genera are common at both poles.

FEMS Microbiol Ecol, 2001 May, 35(3), 249 - 258
The microbial diversity in picoplankton enrichment cultures: a molecular screening of marine isolates; Uphoff HU et al.; Picoplankton bacteria from a North Sea water sample were cultured under a variety of different conditions (nutrients, temperature, light, agitation, adhesion) . Fluorescent in situ hybridization (FISH) analysis of the enrichments showed complex communities which were dominated by gamma-Proteobacteria or beta-Proteobacteria, followed by alpha-Proteobacteria and bacteria from the Cytophaga/Flavobacterium/Bacteroides (CFB) cluster . Among 410 isolates, a high degree of diversity was found, both with respect to colony color and morphology and with respect to genetic diversity . Isolated bacteria were classified into the main taxa by a special PCR approach, termed signature PCR (SIG-PCR) . It was based on an oligo primer mixture targeting 16S rDNA which yielded PCR products of taxon-specific lengths . Again, gamma-Proteobacteria dominated (48%), followed by alpha-Proteobacteria (20%) . beta-Proteobacteria were rarely isolated (eight strains of 410) . The CFB cluster comprised the second largest phylum (14%), and 7.5% of all isolates belonged to the high-GC Gram-positives . Thus, isolated bacteria were representative of enrichment communities with the exception of the beta-Proteobacteria, which were detected in high abundance in certain enrichments by FISH but not isolated, and the high-GC Gram-positives, which were cultivated but not detected by FISH . A genomic fingerprinting technique, randomly amplified polymorphic DNA, showed that among 58 CFB isolates only 18 identical genotypes were found, and among the 84 alpha-Proteobacteria only eight identical genotypes were present . The data show the enormous diversity of cultivated bacteria from picoplankton enrichment cultures of one North Sea water sample, which is only a small fraction of the total picoplankton community.

Biochem J, 2001 May 1, 355(Pt 3), 835 - 40
Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247; Li YK et al.; A family 3 beta-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed . The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies . The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction . Based on the k(cat) values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed . This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl-enzyme intermediate . The large Bronsted constant (beta=-0.85) for the leaving-group-dependent portion (pK(a) of leaving phenols >7) indicates substantial bond cleavage at the transition state . Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta-D-glucopyanoside, o-nitrophenyl beta-D-glucopyanoside and p-cyanophenyl beta-D-glucopyanoside as substrates were 1.17+/-0.02, 1.19+/-0.02 and 1.04+/-0.02 respectively . These results support an S(N)1-like mechanism for the deglucosylation step and an S(N)2-like mechanism for the glucosylation step . Site-directed mutagenesis was also performed to study essential amino acid residues . The activities (k(cat)/K(m)) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity . These results indicate that Asp-247 is an essential amino acid . It is likely that this residue functions as a nucleophile in the reaction . This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

Chem Pharm Bull (Tokyo), 2001 Apr, 49(4), 396 - 401
Prolyl endopeptidase inhibitors from the underground part of Rhodiola sachalinensis; Fan W et al.; The methanolic extract of the underground part of Rhodiola sachalinensis was found to show inhibitory activity on prolyl endopeptidase (PEP, EC . 3.4.21.26), an enzyme that plays a role in the metabolism of proline-containing neuropeptidase which is recognized to be involved in learning and memory . From the MeOH extract, five new monoterpenoids named sachalinols A (24), B (25) and C (26) and sachalinosides A (23) and B (27) were isolated, together with twenty-two known compounds, gallic acid (1), trans-p-hydroxycinnamic acid (2), p-tyrosol (3), salidroside (4), 6n-O-galloylsalidroside (5), benzyl beta-D-glucopyranoside (6), 2-phenylethyl beta-D-glucopyranoside (7), trans-cinnamyl beta-D-glucopyranoside (8), rosarin (9), rhodiocyanoside A (10), lotaustralin (11), octyl beta-D-glucopyranoside (12), 1,2,3,6-tetra-O-galloyl-beta-D-glucose (13), 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (14), kaempferol (15), kaempferol 3-O-beta-D-xylofuranosyl(1-->2)-beta-D-glucopyranoside (16), kaempferol 3-O-beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranoside (17), rhodionin (18), rhodiosin (19), (-)-epigallocatechin (20), 3-O-galloylepigallocatechin-(4-->8)-epigallocatechin 3-O-gallate (21) and rosiridin (22) . Among these, nineteen compounds other than 3, 4 and 9 have been isolated for the first time from R . sachalinensis, and six (6, 8, 13, 16, 17, 20) are isolated from Rhodiola plants for the first time . Among them, six compounds (13, 14, 18, 19, 21, 22) showed noncompetitive inhibition against Flavobacterium PEP, with an IC50 of 0.025, 0.17, 22, 41, 0.44 and 84 microM, respectively.

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