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