Flavobacterium

J Food Prot, 2001 Feb, 64(2), 268 - 71
Aflatoxin B1 degradation by flavobacterium aurantiacum in the presence of reducing conditions and seryl and sulfhydryl group inhibitors; D'Souza DH et al.; This study was undertaken to determine the effects of reducing conditions (L-cysteine) and seryl (phenylmethylsulfonyl fluoride) and sulfhydryl (divalent cadmium) group inhibitors on aflatoxin B1 (AFB1) degradation by Flavobacterium aurantiacum . High-performance liquid chromatography was used to determine AFB1 concentrations in 72-h cultures of F . aurantiacum . The addition of 0.1, 1, or 10 mM L-cysteine did not have any significant effect on AFB1 degradation by these cultures after incubation for 4, 24, or 48 h (P > 0.05) . The addition of 0.1 mM phenylmethylsulfonyl fluoride did not significantly decrease AFB1 degradation (P > 0.05), but 1 mM phenylmethylsulfonyl fluoride significantly decreased AFB1 degradation after 4, 24, and 48 h of incubation (P < or = 0.05) . No significant difference in AFB1 degradation was obtained with 0.1 mM Cd2+ after 4, 24, or 48 h of incubation (P > 0.05) . The addition of 1 and 10 mM Cd2+ significantly decreased AFB1 degradation compared with the cells containing AFB1 alone after 4 and 24 h (P < or = 0.05) . The addition of chelators, 1 mM EDTA and 1 mM o-phenanthroline, did not result in removal of inhibition of AFB1 degradation observed with 1 and 10 mM Cd2+ . Higher concentration of chelators (>1 mM) are necessary to overcome the inhibitory effect . Further work on the cellular fractions and/or crude enzyme preparations is necessary to determine if indeed sulfhydryl and seryl groups of the enzymes are involved in AFB1 degradation (by maintaining either the structure or function of the enzyme).

J Hosp Infect, 2001 Mar, 47(3), 188 - 92
Chryseobacterium (Flavobacterium) meningosepticum outbreak associated with colonization of water taps in a neonatal intensive care unit; Hoque SN et al.; From September 1994 to May 1996, a strain of multi-resistant Chryseobacterium (Flavobacterium) meningosepticum was isolated from eight neonates on a neonatal intensive care unit . The strain was resistant to ampicillin, ceftazidime, imipenem, gentamicin, ciprofloxacin and trimethoprim-sulphamethoxazole, susceptible to piperacillin and amikacin, and had variable susceptibility to rifampicin and vancomycin . Two neonates were infected (one had pneumonia and one septicaemia and meningitis); the remaining six neonates were colonized in the respiratory secretions . Two cases occurred that could not be explained by cross-infection during the outbreak . Environmental screening recovered C . meningosepticum from sink taps . Pulsed-field gel electrophoresis of chromosomal macrorestriction digests of patient and environmental isolates showed them to be representatives of a single strain . The outbreak was controlled after staff were required to use an alcoholic handrub after washing hands, and toiletting of babies was done with sterile water instead of tap-water . Repair and chlorination of the water-tanks and changing the sink-taps resolves the outbreak .

Microbiology, 2001 Mar, 147(Pt 3), 581 - 9
Development of a genetic system for the transfer of DNA into Flavobacterium heparinum; Su H et al.; Flavobacterium heparinum (now Pedobacter heparinus) is a Gram-negative soil bacterium which can produce yellow pigments . It synthesizes five enzymes that degrade glycosoaminoglycan molecules . The study of this unique bacterium has been limited by the absence of a genetic manipulation system . In this paper, the construction of a conjugation/integration plasmid system and a broad-host-range plasmid, both of which contain a F . heparinum functional selective marker created by placing the trimethoprim resistance gene, dhfrII, under the control of the hepA regulatory region is described . Both plasmids were introduced into F . heparinum by conjugation and/or electroporation, and trimethoprim resistant colonies were obtained . Fifty electroporants were obtained per microgram covalently closed circular plasmid DNA . The existence of integrated plasmid DNA was confirmed by Southern hybridization and PCR . The existence of a derivative of the broad-host-range plasmid pBBR1 in F . heparinum was demonstrated by plasmid digestion and Southern hybridization, and by transformation of Escherichia coli.

Burns, 2001 Mar, 27(2), 179 - 82
Chryseobacterium in burn wounds; Kienzle N et al.; Chryseobacteria are gram negative organisms, formerly known as Flavobacteria, which rarely cause infections of burn wounds . This article documents three cases of Chryseobacterium infection in burn wounds and adds to the other two cases that have been reported in English literature . Two patients died, with one of the deaths linked to a Chryseobacteria bacteraemia . In two patients, there was an associated history of first aid treatment with untreated water . Patients whose burn wounds are suspected to be infected with Chryseobacterium require wound excision and coverage in combination with antibiotic therapy such as ciprofloxacin, vancomycin and rifampicin.

Environ Microbiol, 2000 Apr, 2(2), 191 - 201
Changes in community composition during dilution cultures of marine bacterioplankton as assessed by flow cytometric and molecular biological techniques; Fuchs BM et al.; Dilution cultures are a common technique for measuring the growth of bacterioplankton communities . In this study, the taxonomic composition of marine bacterioplankton dilution cultures was followed in water samples from Plymouth Sound and the English Channel (UK) . Bacterial abundances as well as protein and DNA content were closely monitored by flow cytometry . Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments and fluorescence in situ hybridization (FISH) were applied directly to the water samples and to cells sorted from the dilution cultures based on their protein and DNA content . As expected, a rapid activation of bacteria occurred . However, molecular techniques showed that the community developed in the dilution culture within 1 day was significantly different from that in the original water samples . Whereas in the original samples, cells detectable by FISH were dominated by members of the Cytophagal Flavobacterium (CF) cluster, in dilution cultures, gamma-proteobacteria accounted for the majority of cells detected, followed by alpha-proteobacteria . An actively growing and an apparently non-growing population with average cellular protein contents of 24 and 4.5 fg respectively, were sorted by flow cytometry . FISH indicated mostly gamma- (64%) and alpha-proteobacteria (33%) in the first active fraction and 78% members of the CF cluster in the second fraction . Sequencing of DGGE bands confirmed the FISH assignments of the latter two groups . The data presented clearly show that even relatively short-term dilution experiments do not measure in situ growth, but rather growth patterns of an enrichment . Furthermore, it was demonstrated that the combination of flow cytometric analysis and sorting combined with FISH and DGGE analysis presented a fairly rapid method of analysing the taxonomic composition of marine bacterioplankton.

Clin Appl Thromb Hemost, 2001 Jan, 7(1), 44 - 52
Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I; Demir M et al.; Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa . Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs . A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis) . In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest, were used . Amidolytic anti-Xa and anti-IIa activities were also measured . The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated . The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg . SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa . Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions . However, the anti-Xa activities were only partially neutralized . Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH . SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate . These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.

J Microbiol Methods, 2001 Mar 1, 44(2), 173 - 82
PCR primers and functional probes for amplification and detection of bacterial genes for extracellular peptidases in single strains and in soil; Bach HJ et al.; A set of primers and functional probes was developed for the detection of peptidase gene fragments of proteolytic bacteria . Based on DNA sequence data, degenerate PCR primers and internal DIG-labeled probes specific for genes encoding alkaline metallopeptidases (apr) (E.3.4.24), neutral metallopeptidases (npr) (E.3.4.24) and serine peptidases (sub) (E.3.4.21) were derived by multiple sequence alignments.Type strains with known peptidase genes and proteolytic bacteria from a grassland rhizosphere soil, a garden soil and an arable field were investigated for their genotypic proteolytic potential . For 52 out of 53 proteolytic bacterial isolates, at least one of the three peptidase classes could be identified by this approach . The amplified gene fragments were of the expected sizes with each of the three primer sets . The functional probes APR, NPR and SUB have been shown to hybridize specifically to the corresponding gene fragments . sub and npr genes were mainly found in Bacillus species . apr genes were only found in the Pseudomonas fluorescens biotypes and in two morphologically identical Flavobacterium-Cytophaga strains from two different sites . In most of the Bacillus spp., both sub and the npr and in the Flavobacterium-Cytophaga strains even all the three genes could be detected . PCR with DNA isolated from soil led to one main product of the expected size with each primer pair whose identity was additionally confirmed by Southern blot hybridization with the corresponding probes.

Appl Environ Microbiol, 2001 Feb, 67(2), 750 - 9
Antigenic characterization of the fish pathogen Flavobacterium psychrophilum; Crump EM et al.; Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens . The psychrotroph Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids . Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically . A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates . F . psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens . Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo-N-hydoxysuccinimide-biotin and glycosyl groups with biotin hydrazide . The predominant approximately 16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants . Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant approximately 20-kDa protein . This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F . psychrophilum strains.

Thromb Res, 2000 Dec 15, 100(6), 549 - 56
Heparinase I acts on a synthetic heparin pentasaccharide corresponding to the antithrombin III binding site; Yu G et al.; A synthetic pentasaccharide, containing an intact antithrombin III (ATIII) binding site that is in clinical studies a specific antifactor Xa agent, serves as a substrate for a heparin lyase (heparinase I, EC 4.2.2.7) from Flavobacterium heparinum . Heparinase I, currently being assessed as a heparin reversal agent, also reverses the antifactor Xa activity of this synthetic pentasaccharide by breaking it down to inactive disaccharide and trisaccharide products.

FEMS Microbiol Ecol, 2001 Jan, 34(3), 243 - 253
Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations; Schafer H et al.; A mesocosm experiment was performed to study the influence of nutrients on activity and diversity of bacterial assemblages from the Mediterranean Sea . Changes in the diversity of the predominant bacterial populations were monitored by DGGE fingerprinting of PCR products derived from 16S rRNA encoding genes . Fluctuations in the diversity of the most active populations was inferred by performing the DGGE fingerprinting on the basis of the cellular rRNA after reverse transcription and PCR amplification . DNA-derived DGGE patterns obtained from duplicate control and nutrient-enriched mesocosms showed differences in the development of the bacterial communities between control and nutrient-enriched experimental mesocosms . Multidimensional scaling analysis of the DNA-derived DGGE fingerprints indicated that duplicate treatments were reproducible . DNA- and RNA-derived DGGE fingerprints of bacterial assemblages changed over time, showing that the composition of the bacterial assemblages, as well as the most active bacterial populations changed during different phases of the incubation . Sequences of predominant DGGE bands in RNA-derived patterns were similar to 16S rRNA gene sequences of members of the alpha-, gamma- and delta-Proteobacteria and of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB) . Bands corresponding to Ruegeria-like bacteria and members of the CFB became especially dominant during the course of incubation, suggesting that these populations were important contributors to bacterial production and activity in the post-grazing phase of the experiment.

Appl Environ Microbiol, 2001 Jan, 67(1), 434 - 44
Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile; Webster NS et al.; Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile . The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments . Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis . The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives . FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions . The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R . odorabile . High microbial diversity was inferred from low duplication of clones in a library with 70 representatives . Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture . Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.

Appl Environ Microbiol, 2001 Jan, 67(1), 387 - 95
Quantitative molecular analysis of the microbial community in marine arctic sediments (Svalbard); Ravenschlag K et al.; Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard) . FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment . Up to 65.4% +/- 7.5% of total DAPI (4',6'-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% +/- 1.5% hybridized to the archaeal probe ARCH915 . Besides delta-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA . Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts . In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments . Members of the gamma-proteobacteria also constituted a significant fraction in this sediment (6.1% +/- 2.5% of total cell counts, 14.4% +/- 3.6% of prokaryotic rRNA) . A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed . A significant number of cells was detected by this probe (2.1% +/- 0.7% of total DAPI cell counts, 13.2% +/- 4 . 6% of prokaryotic rRNA), showing no clear zonation along the vertical profile . Gram-positive bacteria and the beta-proteobacteria were near the detection limit in all sediments.

Appl Microbiol Biotechnol, 2000 Nov, 54(5), 652 - 8
Microbial and cytoplasmic membrane-based potentiometric biosensors for direct determination of organophosphorus insecticides; Gaberlein S et al.; Potentiometric biosensors for the determination of organophosphorus (OP) insecticides were developed by applying either immobilized whole cells or cytoplasmic membrane fractions of wild-type Flavobacterium sp . on the surface of a glass pH electrode . The ability of Flavobacterium sp . to degrade OP compounds as sole carbon source was demonstrated for parathion with a degradation rate of almost 100% after 30 min and for chlorpyrifos of 33% after 48 h incubation . The products of hydrolysis of these compounds, p-nitrophenol and 3,5,6-trichloro-2-pyridinol, were accumulated in the medium and not used as substrates for growth by Flavobacterium sp . In the course of hydrolysis, which is catalyzed by organophosphorus hydrolase, two protons are released for each substrate molecule hydrolyzed . This stoichiometry forms the electrochemical basis of the potentiometric biosensors . Direct determination without previous extraction of OP was carried out in a stirred measuring cell with a pH electrode as transducer . Poly(carbamoyl sulfonate) (PCS) prepolymer, a hydrogel with good adhesive properties, was used for immobilization of whole cells and membrane-associated organophosphorus hydrolase . The sensor with cytoplasmic membrane fractions was superior to the one with whole cells and showed a linear range for paraoxon from 0.01 to 0.47 mM and 3 weeks' working stability.

Dis Aquat Organ, 2000 Oct 25, 43(1), 27 - 38
Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss; Madetoja J et al.; The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector . Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures . The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F . psychrophilum into the affected fish in bath and cohabitation challenges . Disruption of the skin is discussed as an important invasion route for F . psychrophilum into the fish . The shedding rate of F . psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish . High numbers of F . psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish . The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms . In immunohistochemical examinations of organs and tissues of orally infected fish, F . psychrophilum cells were detected in only 1 fish out of 31 studied . Mortality of the orally challenged fish was not observed in the experiment.

Indian J Pathol Microbiol, 1999 Oct, 42(4), 491 - 2
An unusual case of Flavobacterium meningosepticum pneumonia in an immunocompromised patient; Shivananda PG; An unusual case of Flavobacterium meningosepticum pneumonia in an immunocompromised patient is reported . The necessity to recognize this opportunistic pathogen and its implication is discussed.

J Nat Toxins, 2000 Nov, 9(4), 409 - 17
Isolation of bacteria from toxic dinoflagellate Alexandrium minutum and their effects on algae toxicity; Lu YH et al.; Attempts were made to isolate the bacteria from toxic dinoflagellate Alexandrium minutum T1 and to study the effect of these bacteria on the growth and toxicity of A . minutum T1 . It was found that intracellular bacterial species including Pasteurella haemolytica, Pseudomonas vesicularis, and Sphingomonas sp., and extracellular bacterial species including Pasteurolla pneumotropica, Morganella wisconsensis, Flavobacterium oryzihabitans, Pseudomonas pseudomallei, and Sphingomonas sp . All of them were cultured and determined to have non-PSP-producing ability . The maximum cell number of A . minutum cultured without isolated bacteria was higher than that cultured with isolated bacteria . The total toxicity of A . minutum cultured with bacteria was similar to that of A . minutum T1 cultured without bacteria from lag phase to stationary phase, but it was lower after stationary phase . The growth of A . minutum T1 cultured without antibiotics was also better than that cultured with antibiotics . The total toxicity of A . minutum cultured without antibiotics was higher than that of A . minutum cultured with antibiotics . However, the cell toxicity of A . minutum did not decrease even if the culture medium was added with antibiotics.

Dis Aquat Organ, 2000 Sep 28, 42(3), 191 - 7
Standardization of experimental infection with Flavobacterium psychrophilum, the agent of rainbow trout Oncorhynchus mykiss fry syndrome; Garcia C et al.; Rainbow trout fry syndrome (RTFS) is a septicaemic infection of young rainbow trout Oncorhynchus mykiss occurring at low temperatures and responsible for severe economic losses in European fish farming . The causative agent, Flavobacterium psychrophilum, is a gliding bacterium, and difficulties in culturing it have long been an impediment to investigations on pathogenesis and immunity . Successful attempts at experimentally inducing the disease have been reported, but no experimental model resulting in well-controlled and quantitatively reproducible effects has been described . Recent improvements in F . psychrophilum cultivation made it possible to produce bacterial suspensions with nearly constant viability and to complete challenge injections in rainbow trout fingerlings, using accurately adjusted infective doses . Parenteral injection resulted in significant mortality, which was higher when administered intramuscularly (IM) than intraperitoneally (IP) . Lethal doses 50 % lower than 10(3) colony forming units were consistently obtained in trout weighing 3 to 5 g, and the regular shape of the cumulative mortality curves appeared to lend itself to quantitative analyses . Bath experiments produced milder effects, although mortality ranging between 45 and 60 % was obtained in 6 g trout when skin lesions or stressors were induced along with bacterial exposure . Temperature, salinity and the process of preserving isolates (at least over short periods of time) did not seem to be associated with the severity of infection . Nevertheless, infection trials performed at 2 different locations differing both in water quality and in the system of fish maintenance resulted in different mortalities . These findings notwithstanding, the proposed IM model appears easy to apply under standardized experimental conditions and should contribute to effective advances in the study of the disease.

J Biochem (Tokyo), 2000 Dec, 128(6), 975 - 82
Cloning and characterization of pcd encoding delta'-piperideine-6-carboxylate dehydrogenase from flavobacterium lutescens IFO3084; Fujii T et al.; The pcd gene from Flavobacterium lutescens IFO3084 encoding Delta'-piperideine-6-carboxylate dehydrogenase (PCD) was cloned, sequenced, and expressed in Escherichia coli . The deduced amino acid sequence of PCD from F . lutescens IFO3084 showed strong similarity to that from Streptomyces clavuligerus . The molecular mass of the recombinant PCD was estimated to be approximately 58,000 Da by SDS-PAGE and native PAGE, which indicated that the enzyme molecule is a monomer . The in vitro analysis of L-alpha-aminoadipic acid (L-AAA) production showed that L-AAA is synthesized from L-lysine in two steps catalyzed by L-lysine 6-aminotransferase (LAT) and PCD from F . lutescens IFO3084.

Appl Microbiol Biotechnol, 2000 Oct, 54(4), 461 - 6
Biodegradation of nylon oligomers; Negoro S; This mini-review is a compendium of the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium strains . The results are summarized as follows: 1 . Three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endotype 6-aminohexanoate-oligomer hydrolase (EIII) were responsible for degradation of the oligomers . 2 . The genes coding these enzymes were located on pOAD2, one of three plasmids harbored in Flavobacterium sp . KI72, which comprised 45,519 bp . 3 . The gene coding the EII' protein (a protein having 88% homology with EII) and five IS6100 elements were identified on pOAD2 . 4 . The specific activity of EII was 200-fold higher than that of EII' . However, altering two amino acid residues in the EII' enzyme enhanced the activity of EII' to the same level as that of the EII enzyme . 5 . The deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (encoding oligopeptide permease), ftsX (filamentation temperature sensitivity), penDE (isopenicillin N-acyltransferase) and rep (plasmid replication) . 6 . The EI and EII genes of Pseudomonas sp . NK87 (another nylon oligomer-degrading bacterium) were also located on plasmids . 7 . Through selective cultivation using nylon oligomers as a sole source of carbon and nitrogen, two strains which initially had no metabolic activity for nylon oligomers, Flavobacterium sp . KI725 and Pseudomonas aeruginosa PAO1, were given the ability to degrade xenobiotic compounds . A molecular basis for the adaptation of microorganisms toward xenobiotic compounds was described.

Microbiol Res, 2000 Sep, 155(3), 149 - 56
Microflora of technogenous wastes characterised by fatty acid profiling; Kozdroj J; Fatty acid methyl ester (FAME) profiles obtained directly in situ have been used to estimate microbial community structure in different technogenous wastes . The effect of nutrients added, simulating the effect of plant-derived exudates on the indigenous microflora in the heaps during the reclamation process, was also studied in microcosms . The wastes such as coal-mine spoil, non-ferrous metallurgical slag and coal fly-ash were characterised by a poorly developed microflora as compared to a typical sandy loam soil . However, the most similar to the soil was the community structure in the coalmine spoil . The high content of 18: 2omega6,9 found in the metallurgical slag indicated the domination of fungi in this waste . In contrast, representatives of the Cytophaga-Flavobacterium group dominated the coal fly-ash, for which 16:1omega5c was used as the marker acid . The waste amendment resulted in changes of FAME profiles obtained . However, the changes were site-specific, indicating response of particular microbial groups to the added nutrients.

Appl Environ Microbiol, 2000 Nov, 66(11), 5053 - 65
Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria; Glockner FO et al.; In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenkollesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia) . The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin . Six of the clusters were affiliated with the alpha, four were affiliated with the beta, and one was affiliated with the gamma subclass of the Proteobacteria; four were affiliated with the Cytophaga-Flavobacterium-Bacteroides group; and one was affiliated with the class Actinobacteria (formerly known as the high-G+C gram-positive bacteria) . The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments . Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 x 10(5) cells ml(-1) in Lake Gossenkollesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated . Cell size measurements revealed that Actinobacteria in Lake Gossenkollesee can account for up to 63% of the bacterioplankton biomass . A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class Actinobacteria in freshwater ecosystems.

Appl Environ Microbiol, 2000 Nov, 66(11), 4908 - 15
Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four danish rainbow trout farms; Schmidt AS et al.; Surveillance of bacterial susceptibility to five antimicrobial agents was performed during a 1-year period in and around four freshwater fish farms situated along a stream in western Denmark . Besides assessing the levels of antibiotic resistance among the culturable fraction of microorganisms in fish, water, and sediment samples, two major fish pathogens (88 Flavobacterium psychrophilum isolates and 134 Yersinia ruckeri isolates) and 313 motile Aeromonas isolates, representing a group of ubiquitous aquatic bacteria, were isolated from the same samples . MICs were obtained applying a standardized agar dilution method . A markedly decreased susceptibility of F . psychrophilum isolates to most antimicrobial agents presently available for use in Danish aquaculture was detected, while the collected Y . ruckeri isolates remained largely sensitive to all therapeutic substances . Comparing the inlet and outlet samples, the increase of the antibiotic-resistant proportions observed among the culturable microflora was more pronounced and statistically significant among the motile aeromonads . High levels of individual and multiple antimicrobial resistances were demonstrated within the collected flavobacteria and aeromonads, thus indicating a substantial impact of fish farming on several groups of bacteria associated with aquacultural environments.

Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1505 - 7
Crystallization and preliminary crystallographic studies of a new L-asparaginase encoded by the Escherichia coli genome; Borek D et al.; A new Escherichia coli L-asparaginase belonging to the class of Ntn amidohydrolases has been crystallized using the vapour-diffusion method and PEG 4000 as the precipitant . The crystals belong to the orthorhombic space group P2(1)2(1)2(1) (unit-cell parameters a = 50 . 3, b = 77.6, c = 148.2 A) and diffract to 1.65 A resolution . The structure has been solved by molecular replacement using aspartylglucosaminidase from Flavobacterium meningosepticum as the search model . The asymmetric unit contains four protein chains composed into a dimer of alphabeta heterodimers, where the subunits alpha and beta are the product of autoproteolytic cleavage of the immature protein.

Antimicrob Agents Chemother, 2000 Nov, 44(11), 3028 - 34
Genetic diversity of carbapenem-hydrolyzing metallo-beta-lactamases from Chryseobacterium (Flavobacterium) indologenes; Bellais S et al.; The class B carbapenem-hydrolyzing beta-lactamase IND-1 has been characterized for Chryseobacterium indologenes strain 001 . With internal primers for the bla gene for IND-1 (bla(IND-1)) and an internal bla(IND-1) probe, PCR amplifications failed, while hybridization results were positive when DNA from another C . indologenes isolate, strain CIP101026, was used as a template . Thus, a bla(IND)-related gene was cloned from this C . indologenes reference strain . Sequencing of the insert of a recombinant plasmid conferring resistance to carbapenems revealed an open reading frame with a G + C content of 39.9% and coding for a 243-amino-acid preprotein named IND-2 . IND-2 shared 80% amino acid identity with IND-1 and had a similar broad-spectrum resistance profile, including resistance to carbapenems . It was classified in functional subgroup 3a of class B carbapenem-hydrolyzing beta-lactamases . IND-1 and IND-2, despite their genetic diversity, possessed similar kinetic parameters, except that ceftazidime was hydrolyzed less by IND-2 . To obtain the entire bla(IND)-related gene sequences of eight other C . indologenes isolates, PCR was performed using internal and external primers, followed by inverse PCR techniques . The likely chromosome-mediated metallo-beta-lactamases of the 10 C . indologenes isolates were divided into several groups and subgroups . IND-1, IND-2, IND-2a, IND-3, and IND-4 shared 77 to 99% amino acid identity.

Res Vet Sci, 2000 Oct, 69(2), 165 - 9
Difficulties in experimental infection studies with Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) using immersion, oral and anal challenges; Decostere A et al.; This study was carried out in order to try to establish an efficacious and reliable experimental infection model for Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome, using contact, oral and anal challenges . Ten F psychrophilum strains of different origin were included . The influence of water temperature, scarification, water quality, stress and growth conditions of the pathogen on the experimental infection was assessed . For each challenge protocol, all strains failed to reproduce disease signs or mortality in rainbow trout (Oncorhynchus mykiss L.) fry . Histological and bacteriological examination of the skin, gills and internal organs of the fish 3 weeks following inoculation were found to be negative . Different hypotheses to explain the inability of the challenge models to reproduce the disease experimentally are discussed .

Dis Aquat Organ, 2000 Aug 10, 42(1), 53 - 9
Ultraviolet irradiation inactivates the waterborne infective stages of Myxobolus cerebralis: a treatment for hatchery water supplies; Hedrick RP et al.; The effects of ultraviolet (UV) irradiation on the viability of the waterborne triactinomyxon stages of Myxobolus cerebralis were evaluated by vital staining and the infectivity for juvenile rainbow trout Oncorhynchus mykiss . A dose of 1300 mWs cm-2 was required to inactivate 100% of the triactinomyxons held under a static collimated beam of UV as determined by vital staining . Juvenile rainbow trout were protected from infections with M . cerebralis when exposed to 14,000 or 1400 triactinomyxon spores per fish that had been treated with the collimating beam apparatus (1300 mWs cm-2) . Among all fish receiving UV-treated triactinomyxons, none had clinical signs of whirling disease, or evidence of microscopic lesions or spores of M . cerebralis after 5 mo at water temperatures of 15 degrees C . In contrast, 100% of the fish receiving the higher dose of untreated triactinomyxons developed clinical signs of whirling disease and both microscopic signs of infection and spores were detected in all of the high and low dose trout receiving untreated triactinomyxon exposures . Two additional trials evaluated the Cryptosporidium Inactivation Device (CID) for its ability to treat flow-through 15 degrees C well water to which triactinomyxons were added over a 2 wk period . CID treatments of a cumulative dose exceeding 64,000 triactinomyxons per fish protected juvenile rainbow from infections with M . cerebralis . Rainbow trout controls receiving the same number of untreated triactinomyxons developed both microscopic lesions and cranial spore concentrations up to 10(4.6) per 1/2 head, although no signs of clinical whirling disease were observed . UV (126 mWs cm-2, collimated beam apparatus) was also effective in killing Flavobacterium psychrophilum, the agent causing salmonid bacterial coldwater disease, as demonstrated by the inability of bacterial cells to grow on artificial media following UV treatment.

Proc Natl Acad Sci U S A, 2000 Sep 12, 97(19), 10365 - 70
Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin; Shriver Z et al.; Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known . Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III) . Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects . The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it . The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II . Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site . Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage . Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site . We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.

Nippon Ganka Gakkai Zasshi, 2000 Aug, 104(8), 555 - 8
{Bacterial infection in the conjunctiva of patients with adenoviral conjunctivitis}; Watanabe Y et al.; PURPOSE: We evaluate the microbiological features of mixed infection in adenovirus-infected conjunctiva . SUBJECTS: Isolation of bacteria was performed in 82 samples of adenoviral conjunctivitis at six eye clinics in Japan . METHODS: For microbiological diagnosis, we performed immunochromatography (IC) and polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis for detection and serotyping of adenovirus, and PCR for detection of herpes simplex virus (HSV) and Chlamydia trachomatis antigens out of 82 samples with adenoviral conjunctivitis . RESULTS: Pathogenic bacteria were isolated in 6 samples out of 82 . Out of these 6 cases, 5 samples were gram-negative rods and most of them were Flavobacterium meningosepticum (4 samples) . Adenovirus type 8 was isolated from all these mixed infection cases . However, HSV-1 and Chlamydia trachomatis were not found . CONCLUSIONS: From these results, it is suggested that gram-negative rods, especially F . meningosepticum, are the most common bacteria causing mixed bacterial infection in adenoviral conjunctivitis.

Acta Paediatr, 2000 Aug, 89(8), 942 - 6
Etiologic spectrum and pattern of antimicrobial drug susceptibility in bacterial meningitis in Sokoto, Nigeria; Emele FE; Etiologic agents of meningitis were prospectively investigated among patients admitted to Usman Danfodio University Teaching Hospital, Sokoto . Of 1097 cerebrospinal fluid (CSF) samples submitted to the microbiology laboratory from various wards of the hospital, 289 (26%) were microscopically, culturally and/or serologically proven to be bacterial meningitis . The etiologic spectrum was as follows: Neisseria meningitidis (61%), Streptococcus pneumoniae (18%), Haemophilus influenzae (10%), Staphylococcus aureus (6%), Coliform bacilli (3%), Escherichia coli (0.7%), Mycobacterium tuberculosis (0.7%), Listeria monocytogenes (0.4%), Flavobacterium meningosepticum (0.4%) and Pseudomonas putrifasciens (0.4%) . Bacterial meningitis was most prevalent (195 or 68%) among children aged 1-9 y, while adults and neonates were least affected . Coliform bacilli caused five of eight neonatal cases . Males were more frequently affected than females (chi2 = 12.50; p < 0.05) . Culture and microscopy were comparatively less efficient than the search for bacterial antigens, especially in the diagnosis of Haemophilus meningitis . Antimicrobial susceptibility of N . meningitidis to ampicillin and benzyl penicillin reduced progressively over the years (F = 406.98; p < 0.001) . Nineteen (11%) of the isolates (5 Meningococci, 7 Staph . aureus, 1 Haem . influenza and 6 others) showed simultaneous resistance to chloramphenicol, ampicillin and benzyl penicillin.

FEMS Microbiol Ecol, 2000 Aug 1, 33(2), 157 - 170
Characterization of the microbial community of lotic organic aggregates ('river snow') in the Elbe River of Germany by cultivation and molecular methods; Bockelmanna U et al.; Aerobic and anaerobic cultivation techniques, 16S rDNA-based phylogeny, and fluorescent in situ hybridization were used to describe the phylogenetic diversity and physiological versatility of lotic microbial aggregates ('river snow') obtained from the river Elbe . In the course of the year the 'river snow' community changed . It was characterized by a great bacterial diversity in spring, the predominant occurrence of algae in summer and reduction of the total bacterial cell count in autumn and winter . In all 'river snow' samples, more than 70% of the bacteria counted with the general DNA stain DAPI also hybridized with the Bacteria-specific probe EUB338 . In situ analysis of the bacterial 'river snow' community with a comprehensive suite of specific rRNA-targeted probes revealed population dynamics to be governed by seasonal factors . During all seasons, beta-Proteobacteria constituted the numerically most important bacterial group forming up to 54% of the total cell counts . In contrast to this, the relative abundance of other major bacterial lineages ranged from 2% for the order Planctomycetales to 36% for Cytophaga-Flavobacteria . Cultivation of 'river snow' under aerobic and anaerobic conditions with a variety of different media resulted in the isolation of 40 new bacterial strains . Phenotypic and phylogenetic analyses revealed these new strains to be mostly unknown organisms affiliated to different bacterial phyla . Application of newly developed specific oligonucleotide probes proved the cultivated bacteria, including clostridia and the numerically abundant beta-Proteobacteria, as relevant in situ members of the 'river snow' community.

J Biochem (Tokyo), 2000 Sep, 128(3), 441 - 7
Directed evolution to improve the thermostability of prolyl endopeptidase; Uchiyama H et al.; Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues . Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin . Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme . First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum . Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters . Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated . The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme . The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.

J Biochem (Tokyo), 2000 Sep, 128(3), 391 - 7
Characterization of L-lysine 6-aminotransferase and its structural gene from Flavobacterium lutescens IFO3084; Fujii T et al.; L-Lysine 6-aminotransferase (LAT) is an enzyme involved in L-lysine catabolism in a wide range of living organisms . LAT from Flavobacterium lutescens IFO3084 was purified, and its structural gene (lat) was cloned, sequenced and expressed in Escherichia coli . Native PAGE analysis of purified LAT gave a single band corresponding to a molecular weight of about 110,000 . lat encoded a protein of 493 amino acids with a deduced molecular weight of 53,200, which is very close to that of purified LAT determined on SDS-PAGE . Expression of lat in E . coli revealed that lat encodes a single subunit protein leading to LAT activity . These data suggested that LAT from F . lutescens IFO3084, like most other aminotransferases, is derived from a single ORF and is active as a homodimer.

Dis Aquat Organ, 2000 Jul 14, 41(3), 173 - 9
Effect of Flavobacterium psychrophilum strains and their metabolites on the oxidative activity of rainbow trout oncorhynchus mykiss phagocytes; Lammens M et al.; The oxidative activity of rainbow trout phagocytes was studied using a chemiluminescence technique using 12 different Flavobacterium psychrophilum strains and their metabolites . Phagocytes were obtained from the head kidney of rainbow trout Oncorhynchus mykiss . The addition of viable F . psychrophilum or their metabolites to the phagocytes resulted in an immediate chemiluminescence response . The stimulating effects of both the F . psychrophilum and their metabolites on the phagocytes were found to be heat stable . No significant differences in stimulation capacity were found between the strains tested . To investigate the nature of the stimulating agent, both the bacteria and the supernatant were treated with either sodium metaperiodate or polymyxin B . Adding polymyxin B to the bacterial cells and supernatant did not change the chemiluminescence pattern, suggesting that the capacity of F . psychrophilum to stimulate the phagocytes probably is not due to lipopolysaccharides (LPS) . However, following incubation of the bacteria and their metabolites with sodium metaperiodate, the capacity to stimulate phagocytes was significantly impaired . This suggests that a carbohydrate component most likely plays an important role in the ability of F . psychrophilum to stimulate phagocytes . Opsonisation of the bacteria with native trout serum or with rabbit anti-F . psychrophilum serum resulted in an additional chemiluminescence peak which was significantly higher than the first peak . This extra peak disappeared following heat treatment of the trout serum and the rabbit anti-F . psychrophilum serum, pointing towards the involvement of heat labile complement in opsonisation.

Mol Biotechnol, 1999 Nov, 13(1), 1 - 15
The FokI methyltransferase from Flavobacterium okeanokoites . Purification and characterization of the enzyme and its truncated derivatives; Kaczorowski T et al.; The gene encoding the FokI methyltransferase from Flavobacterium okeanokoites was cloned into an Escherichia coli vector . The transcriptional start sites were mapped as well as putative -10 and -35 regions of the fokIM promoter . Enzyme overproduction was ensured by cloning the fokIM gene under the phi 10 promoter of phase T7 . M.FokI was purified using a two-step chromatography procedure . M.FokI is a monomeric protein with a M(r) = 76,000 +/- 1,500 under denaturing conditions . It contains 21 Arg residues, and at least one of which is required for activity as shown by inhibition using 2,3-butanedione . Deletion mutants in the N- and C-terminus of M.FokI were isolated and characterized . The N-terminal derivative (M.FokIN) methylates the adenine residue within the sequence 5'-GGATG-3', whereas the C-terminal derivative (M.FokIC) modifies the adenine residue within the sequence 5'-CATCC-3' . Substrate-protection studies, utilizing chemical modification combined with data on the effect of divalent cations and pH on methylation activity, proved the existence of two catalytic centers within the FokI methyltransferase molecule . M.FokI and its truncated derivatives require S-adenosyl-L-methionine as the methyl-group donor, and they are strongly inhibited by divalent cations (Mg2+, Ca2+, Ba2+, Mn2+, and Zn2+) and S-adenosyl-L-homocysteine . The Km values for the methyl donor, S-adenosyl-L-methionine are 0.6 microM (M.FokI), 0.4 microM (M.FokIN), and 0.9 microM (M.FokIC) while the Km values for substrate lambda DNA are 1.2 nM (M.FokI), 1.4 nM (M.FokIN), and 1.3 nM (M.FokIC).

Appl Environ Microbiol, 2000 Aug, 66(8), 3125 - 33
Diversity of thiosulfate-oxidizing bacteria from marine sediments and hydrothermal vents; Teske A et al.; Species diversity, phylogenetic affiliations, and environmental occurrence patterns of thiosulfate-oxidizing marine bacteria were investigated by using new isolates from serially diluted continental slope and deep-sea abyssal plain sediments collected off the coast of New England and strains cultured previously from Galapagos hydrothermal vent samples . The most frequently obtained new isolates, mostly from 10(3)- and 10(4)-fold dilutions of the continental slope sediment, oxidized thiosulfate to sulfate and fell into a distinct phylogenetic cluster of marine alpha-Proteobacteria . Phylogenetically and physiologically, these sediment strains resembled the sulfate-producing thiosulfate oxidizers from the Galapagos hydrothermal vents while showing habitat-related differences in growth temperature, rate and extent of thiosulfate utilization, and carbon substrate patterns . The abyssal deep-sea sediments yielded predominantly base-producing thiosulfate-oxidizing isolates related to Antarctic marine Psychroflexus species and other cold-water marine strains of the Cytophaga-Flavobacterium-Bacteroides phylum, in addition to gamma-proteobacterial isolates of the genera Pseudoalteromonas and Halomonas-Deleya . Bacterial thiosulfate oxidation is found in a wide phylogenetic spectrum of Flavobacteria and Proteobacteria.

J Biochem (Tokyo), 2000 Aug, 128(2), 323 - 8
Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15; Kim BT et al.; A novel type of heparinase (heparin lyase, no EC number) has been purified from Bacteroides stercoris HJ-15, isolated from human intestine, which produces three kinds of heparinases . The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, CM-Sephadex C-50, hydroxyapatite, and HiTrap SP chromatographies with a final specific activity of 19.5 mmol/min/mg . It showed optimal activity at pH 7.2 and 45 degrees C and the presence of 300 mM KCl greatly enhanced its activity . The purified enzyme activity was inhibited by Cu(2+), Pb(2+), and some agents that modify histidine and cysteine residues, and activated by reducing agents such as dithiothreitol and 2-mercaptoethanol . This purified Bacteroides heparinase is an eliminase that shows its greatest activity on bovine intestinal heparan sulfate, and to a lesser extent on porcine intestinal heparan sulfate and heparin . This enzyme does not act on acharan sulfate but de-O-sulfated acharan sulfate and N-sulfoacharan sulfate were found to be poor substrates . The substrate specificity of this enzyme is similar to that of Flavobacterial heparinase II . However, an internal amino acid sequence of the purified Bacteroides heparinase shows significant (73%) homology to Flavobacterial heparinase III and only 43% homology to Flavobacterial heparinase II . These findings suggest that the Bacteroidal heparinase is a novel enzyme degrading GAGs.

Syst Appl Microbiol, 2000 Apr, 23(1), 107 - 14
16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides; Weller R et al.; Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities . A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats . For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum . Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates . Probe CFB563 detects marine as well as animal-associated isolates . Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates . All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 16S rRNA sequences from the CFB-phylum . We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach . Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human-associated species, was not further characterized . The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum . It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.

Appl Environ Microbiol, 2000 Jul, 66(7), 3102 - 9
Identification of 16S ribosomal DNA-defined bacterial populations at a shallow submarine hydrothermal vent near Milos Island (Greece); Sievert SM et al.; In a recent publication (S . M . Sievert, T . Brinkhoff, G . Muyzer, W . Ziebis, and J . Kuever, Appl . Environ . Microbiol . 65:3834-3842, 1999) we described spatiotemporal changes in the bacterial community structure at a shallow-water hydrothermal vent in the Aegean Sea near the isle of Milos (Greece) . Here we describe identification and phylogenetic analysis of the predominant bacterial populations at the vent site and their distribution at the vent site as determined by sequencing of DNA molecules (bands) excised from denaturing gradient gels . A total of 36 bands could be sequenced, and there were representatives of eight major lineages of the domain Bacteria . Cytophaga-Flavobacterium and Acidobacterium were the most frequently retrieved bacterial groups . Less than 33% of the sequences exhibited 90% or more identity with cultivated organisms . The predominance of putative heterotrophic populations in the sequences retrieved is explained by the input of allochthonous organic matter at the vent site.

Appl Environ Microbiol, 2000 Jul, 66(7), 3044 - 51
Culturability and In situ abundance of pelagic bacteria from the North Sea; Eilers H et al.; The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques . We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH) . A culture collection of 145 strains was established by plating on oligotrophic medium . Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs . The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio . Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community . They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria . Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all . Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved . The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter . The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library . Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria . This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.

FEMS Immunol Med Microbiol, 2000 Jul, 28(3), 205 - 9
Modulation of chemotaxis, O(2)(-) production and myeloperoxidase release from human polymorphonuclear leukocytes by the ornithine-containing lipid and the serineglycine-containing lipid of Flavobacterium; Kawai Y et al.; The ornithine-containing lipid (OL) and the serineglycine-containing lipid (SGL) of Flavobacterium activated and modulated the functions of human polymorphonuclear leukocytes (PMNs) . The OL and the SGL strongly activated fMet-Leu-Phe- and interleukin-8-induced chemotaxis of PMNs at the concentration of 0.1 microg ml(-1), and a synthetic OL also activated the function of PMNs . Further, the OL strongly activated O(2)(-) production from PMNs . Although the OL and the SGL slightly modulated myeloperoxidase release from PMNs, inhibition effects of their component fatty acid analogues were observed . O(2)(-) production-inducing activity is a common biological activity between the OL and bacterial lipopolysaccharides, but OL and SGL, unlike lipopolysaccharide, are potent activators of PMN chemotaxis.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 311 - 7
Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase; Tsugawa W et al.; The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp . can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters . Ethyl 4-nitrophenyl phenylphosphonothioate (EPN) is a chiral organophosphate . Optical isomers of EPN show differences in their toxicity . R-EPN is known to be more toxic to hens and houseflies than S-EPN . We determined the Ki value of each enantiomer toward electric eel acetylcholinesterase . R-EPN (Ki = 6 microM) inhibited acetylcholinesterase much more effectively than S-EPN (Ki = 52 microM) did in vitro . Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively . When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration . At 20% DMSO, the maximum activity was observed . The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed . The Km value for R-EPN decreased from 0.24 to 0.03 mM, and the Vmax value increased from 0.25 to 0.60 U/mg of protein . Vmax/Km values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1055 - 63
Taxonomy of Antarctic Flavobacterium species: description of Flavobacterium gillisiae sp . nov., Flavobacterium tegetincola sp . nov., and Flavobacterium xanthum sp . nov., nom . rev . and reclassification of {Flavobacterium} salegens as Salegentibacter salegens gen . nov., comb . nov; McCammon SA et al.; 16S rRNA phylogenetic analysis of a number of yellow- and orange-pigmented strains isolated from a variety of Antarctic habitats including sea ice, lakewater and cyanobacterial mats indicated a close relationship to the genus Flavobacterium but distinct from known Flavobacterium species . Phenotypic properties, DNA G+C content and whole-cell fatty acid profiles of the Antarctic strains were consistent with those of the genus Flavobacterium . DNA-DNA hybridization analysis indicated the presence of two distinct and novel genospecies each isolated from a different Antarctic habitat . From polyphasic taxonomic data it is proposed that the two groups represent new species with the following proposed names: Flavobacterium gillisiae (ACAM 601T) and Flavobacterium tegetincola (ACAM 602T) . In addition polyphasic analysis of the species '{Cytophaga} xantha' (Inoue and Komagata 1976), isolated from Antarctic mud, indicated it was a distinct member of the genus Flavobacterium and was thus revived as Flavobacterium xanthum . Phylogenetic and fatty acid analyses also indicate that the species {Flavobacterium} salegens (Dobson et al . 1993), from Organic Lake, Antarctica, is misclassified at the genus level . It is proposed that this species belongs to a new genus, Salegentibacter salegens gen . nov., comb . nov.

Appl Environ Microbiol, 2000 Jun, 66(6), 2400 - 7
Bacterial community structure and physiological state within an industrial phenol bioremediation system; Whiteley AS et al.; The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater . Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor . Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the alpha, beta and gamma subclasses of the class Proteobacteria {alpha-, beta-, and gamma-Proteobacteria, respectively}, the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting . At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the gamma-Proteobacteria . The alpha subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the beta subclass of the Proteobacteria exhibited local dominance in several of the processing compartments . Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA . For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content . The gamma subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate . While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters . The potential importance of the gamma-Proteobacteria and the Cytophaga-Flavobacteria during this bioremediation process was highlighted.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 145 - 8
Bacteroides acidifaciens sp . nov., isolated from the caecum of mice; Miyamoto Y et al.; During studies of the bacterial flora in the intestines of mice, we isolated characteristic strains which lowered the pH of peptone-yeast broth containing Fildes' digest . Based on 16S rRNA sequence comparison, these isolates were considered to belong to the Bacteroides cluster in the bacteroides subgroup of the Cytophaga-Flavobacterium-Bacteroides phylum, and were divided into two groups . Their phenotypic characteristics, i.e . growth in 20% bile, aesculin hydrolysis, and glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) activity, were the same as those of the 'Bacteroides fragilis group' . The low level of DNA-DNA hybridization with type strains in the Bacteroides cluster confirmed the novel species status of these isolates . It is proposed that these isolates be named Bacteroides acidifaciens, the type strain of which is A40T (= JCM 10556T).

Antimicrob Agents Chemother, 2000 Jun, 44(6), 1448 - 52
Carbapenemases of Chryseobacterium (Flavobacterium) meningosepticum: distribution of blaB and characterization of a novel metallo-beta-lactamase gene, blaB3, in the type strain, NCTC 10016; Woodford N et al.; Genes encoding carbapenemases in 15 reference strains of Chryseobacterium (Flavobacterium) meningosepticum from the United Kingdom National Collection of Type Cultures and in one recent clinical isolate were investigated . All the strains hydrolyzed imipenem, but their levels of resistance to carbapenems varied, with imipenem and meropenem MICs ranging from 2 to >32 microg/ml . The blaB gene, which encodes a molecular-class B carbapenemase, was detected in only six reference strains and in clinical isolate 97/P/5448 . The gene from 97/P/5448 had 98% nucleotide identity with the published sequence of blaB (from strain NCTC 10585) and was designated blaB2 . A distinct carbapenemase gene, designated blaB3, was cloned from the type strain of C . meningosepticum, NCTC 10016 . blaB3 had an open reading frame of 750 bp with 82% nucleotide identity to blaB and blaB2 and encoded a beta-lactamase of 249 amino acids, including the putative signal peptide . This beta-lactamase showed 87.6 and 86.7% amino acid homology with BlaB and BlaB2, respectively . blaB3 was detected in one other reference strain besides NCTC 10016, but the genetic basis of the carbapenemase activity detected in the other seven reference strains was not defined . Thus, neither blaB nor blaB3 was ubiquitous in the strains of C . meningosepticum studied, indicating that the reference strains may represent more than one bacterial species, each with its own intrinsic metallo-beta-lactamase . Further taxonomic studies of C . meningosepticum are necessary to resolve this topic . Chryseobacterium spp . are environmental organisms and occasional opportunist pathogens . They apparently represent a reservoir of diverse metallo-beta-lactamases, which potentially spread to gram-negative bacteria of greater clinical significance.

Mikrobiologiia, 2000 Mar-Apr, 69(2), 248 - 56
{Cell division in the volume of Flavobacterium sp.22 colonies}; Puzyr' AP et al.; Six-day-old colonies of Flavobacterium sp . 22 were studied by electron microscopy . Direct evidence was obtained of bacterial cell division across the entire colony volume, indicating that the colony growth of Flavobacterium sp . 22 is not purely peripheral . It is argued that the colony shape is determined not only by peripheral growth but also by physical forces acting upon a droplet of liquid on the surface . For bacterial colonies developing on solid nutrient media, the intercellular matrix plays the role of such a liquid.

Southeast Asian J Trop Med Public Health, 1998 Dec, 29(4), 872 - 7
Serotyping of Flavobacterium meningosepticum by co-agglutination method; Salasawati H et al.; The purpose of this investigation was to evaluate the usefulness of a co-agglutination procedure for the typing of Flavobacterium meningosepticum . The sensitivity and specificity of the co-agglutination test was compared to the slide agglutination test using reference strains of the bacterial species . Antisera were characterized by both technics to determine their titer and working dilution . The specificity of the sera was assessed by performing tests which include strains of other species and serotypes . A collection of 47 strains of F . meningosepticum isolated from clinical specimens were typed by both co-agglutination and slide agglutination methods . Co-agglutination proved to be markedly more specific than the slide procedure although both methods were similar in sensitivity . It was concluded that co-agglutination proved to be an excellent method for the serotyping of F . meningosepticum.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 731 - 4
Phylogenetic characterization of a novel radiation-resistant bacterium from irradiated pork: description of Hymenobacter actinosclerus sp . nov; Collins MD et al.; A phylogenetic analysis was performed on a red-pigmented, radiation-resistant, Gram-negative, rod-shaped organism originating from irradiated pork . Comparative 16S rRNA gene sequencing showed the bacterium was a member of the Cytophaga-Flavobacterium-Bacteroides line of descent and represents a new subline within the genus Hymenobacter . A new species, Hymenobacter actinosclerus, is described for this novel radiation-resistant bacterium . The type strain of Hymenobacter actinosclerus is CCUG 39621T.

Biochemistry, 2000 Apr 11, 39(14), 4012 - 9
Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III; Pojasek K et al.; The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically . Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate . Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III . Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)) . The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified . As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme . Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity.

Biosci Biotechnol Biochem, 2000 Feb, 64(2), 333 - 40
Purification and some properties of a beta-glucosidase from Flavobacterium johnsonae; Okamoto K et al.; Flavobacterium johnsonae was isolated as a microorganism that produced a beta-glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides . The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl . The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE . An isoelectric point of pI 8.8 was estimated by isoelectric focusing . The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0 . The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C . The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside . The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta-glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose . The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%) . The pNPG hydrolysis was also inactivated to almost the same degrees . Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.

J Appl Microbiol, 2000 Feb, 88(2), 299 - 307
Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification; Wiklund T et al.; Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids . Some of the presently available techniques for the detection of Fl . psychrophilum are either time consuming or lack sufficient sensitivity . In the present investigation, the possible detection of Fl . psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes . The DNA was extracted using Chelex(R) 100 chelating resin . The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl . psychrophilum . The obtained detection limit of Fl . psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg-1 brain tissue . The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl . psychrophilum . In non-sterile fresh water seeded with Fl . psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml-1 water . The PCR-assay detected Fl . psychrophilum in water samples taken from a rainbow trout farm, but Fl . psychrophilum could not be isolated using inoculation on selective agar . The method presented here has the potential to detect low levels of Fl . psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl . psychrophilum.

Antimicrob Agents Chemother, 2000 Apr, 44(4), 997 - 1003
TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli; Silva J et al.; Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem . This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0 . Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E . coli R170 to E . coli J53-2 . The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length . The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E . coli DH5alpha . Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG . The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E . coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis . The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime . The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid . TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.

J Food Prot, 2000 Mar, 63(3), 415 - 8
Preliminary evidence that degradation of aflatoxin B1 by Flavobacterium aurantiacum is enzymatic; Smiley RD et al.; The ability of crude protein extracts from Flavobacterium aurantiacum to degrade aflatoxin B1 (AB1) in aqueous solution was evaluated . Crude protein extracts (800 microg of total protein per ml) degraded 74.5% of AB1 in solution . An average of 94.5% of AB1 was recovered after incubation with heat-treated crude protein extracts (800 microg of total protein per ml) . DNase I-treated crude protein extracts degraded 80.5% of AB1 in solution, suggesting that removal of aflatoxin by F . aurantiacum is not due to nonspecific binding with the bacterium's genomic DNA . Proteinase K-treated crude protein extracts degraded 34.5% of AB1, providing evidence that degradation of aflatoxin is linked to a protein that is possibly an enzyme . Solution pH affected the amount of AB1 degraded by crude protein extracts after 24 h . Maximum degradation was observed at pH 7 (pH levels tested: 5, 6, 7, and 8), with some AB1 degradation occurring at pH levels as low as 5 and as high as 8 . Acidic pH levels were more detrimental to the ability of crude protein extracts to degrade AB1 than was basic pH . The results of this work indicate that the degradation of AB1 by F . aurantiacum may be enzymatic.

Int J Biol Macromol, 2000 Mar 16, 27(1), 49 - 57
A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana; Chavante SF et al.; With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates . These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues . The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks . Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A . franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin).

J Bacteriol, 2000 Mar, 182(6), 1671 - 9
Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division; Kempf MJ et al.; Flavobacterium johnsoniae is a gram-negative bacterium that exhibits gliding motility . To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn4351 mutagenesis . Seventeen of these mutants exhibited filamentous cell morphology . The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced . The transposon was inserted in a gene that was similar to Escherichia coli ftsX . Two of the remaining 16 filamentous mutants also carried insertions in ftsX . Introduction of the wild-type F . johnsoniae ftsX gene restored motility and normal cell morphology to each of the three ftsX mutants . CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate . In E . coli, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane . Mutations in F . johnsoniae ftsX may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes . Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division . The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.

Ann Acad Med Singapore, 1999 Nov, 28(6), 858 - 60
Chryseobacterium meningosepticum (Flavobacterium meningosepticum)--a report of five cases in a local hospital; Lim LC et al.; Chrysobacterium meningosepticum (Flavobacterium meningosepticum) is a known cause of meningitis in premature and newborn infants . Infection due to this organism in adults is uncommon . We report 5 cases of Chrysobacterium meningosepticum in adult patients . Most of these patients were elderly and had underlying co-morbidities.

Appl Environ Microbiol, 2000 Feb, 66(2), 499 - 508
Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis; Casamayor EO et al.; The microbial assemblages of Lake Ciso and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments . Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed . Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy . The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis . Sequences obtained from Lake Ciso samples were related to gram-positive bacteria and to members of the division Proteobacteria . Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria . Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously . The changes in the species composition from winter to spring were also marked . The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes . Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes . These euryarchaeal group sequences dominated the archaeal sequences in Lake Ciso but not in Lake Vilar . In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band . The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known . We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results . Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.

J Bacteriol, 2000 Feb, 182(4), 911 - 8
Cloning and characterization of the Flavobacterium johnsoniae gliding-motility genes gldB and gldC; Hunnicutt DW et al.; The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known . A large number of mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) with defects in gliding motility have been previously isolated, and genetic techniques to analyze these mutants have recently been developed . We complemented a nongliding mutant of F . johnsoniae (UW102-99) with a library of wild-type DNA by using the shuttle cosmid pCP26 . The complementing plasmid (pCP200) contained an insert of 26 kb and restored gliding motility to 4 of 50 independently isolated nongliding mutants . A 1.9-kb fragment which encompassed two genes, gldB and gldC, complemented all four mutants . An insertion mutation in gldB was polar on gldC, suggesting that the two genes form an operon . Disruption of the chromosomal copy of gldB in wild-type F . johnsoniae UW101 eliminated gliding motility . Introduction of the gldBC operon, or gldB alone, restored motility . gldB appears to be essential for F . johnsoniae gliding motility . It codes for a membrane protein that does not exhibit strong sequence similarity to other proteins in the databases . gldC is not absolutely required for gliding motility, but cells that do not produce GldC form colonies that spread less well than those of the wild type . GldC is a soluble protein and has weak sequence similarity to the fungal lectin AOL.

J Food Prot, 2000 Jan, 63(1), 102 - 5
The influence of divalent cations and chelators on aflatoxin B1 degradation by Flavobacterium aurantiacum; D'Souza DH et al.; The influence of divalent cations (Mg2+ and Ca2+) and chelators (EDTA and 1,10-phenanthroline) on aflatoxin B1 (AFB1) degradation by Flavobacterium aurantiacum was determined in an effort to elucidate the possible manner by which this organism degrades AFB1 . AFB1 (10 microg/ml) was added to 72-h cultures of F . aurantiacum that had been washed and resuspended in phosphate buffer (pH 7.0) . High-performance liquid chromatography was used to determine AFB1 concentration in these cultures . Incubating cells with 0.1, 1, and 10 mM Ca2+ for 48 h significantly increased AFB1 degradation by 11.8, 13.5, and 14.0%, respectively, compared with F . aurantiacum cells alone . Likewise, incubation with 0.1, 1, and 10 mM Mg2+ for 48 h significantly increased AFB1 degradation by 13.8, 13.3, and 13.1%, respectively . Incubating the bacterium with either divalent cation for 16 and 24 h did not significantly affect AFB1 degradation (P < or = 0.05) . Addition of 0.1, 1, and 10 mM EDTA and 0.1 and 1 mM 1,10-phenanthroline resulted in significant increases in AFB1 degradation after 24 h . Significantly less AFB1 degradation was observed using 10 mM 1,10-phenanthroline after 24-h incubation . These results suggest the involvement of Mg2+ and Ca2+ cations in AFB1 degradation by F . aurantiacum.

FEMS Microbiol Ecol, 2000 Feb 1, 31(2), 153 - 161
Investigation of 0.2 µm filterable bacteria from the Western Mediterranean Sea using a molecular approach: dominance of potential starvation forms; Haller CM et al.; Although the existence of 0.2 microm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class . According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 microm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 microm, but flexible enough to pass the nominal filter pore-size . In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 microm and the 0.2 microm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments . The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 microm filterable and the total bacterial populations within the samples . Additionally the 0.2 microm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction . According to the current knowledge concerning the status of 0.2 microm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 microm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria . The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 microm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.

Int J Food Microbiol, 1999 Dec 15, 53(2-3), 81 - 94
The taxonomy, ecology and cultivation of bacterial genera belonging to the family Flavobacteriaceae; Jooste PJ et al.; The group known as the 'flavobacteria' has previously been regarded as synonymous with the genus Flavobacterium . Today, however, flavobacteria refers to the family Flavobacteriaceae comprising 10 genera . This review deals with the rapid changes in the taxonomy of these bacteria, especially over the last decade . It also briefly reviews the ecology of the genera in this family and describes the media that have been utilized in the general and selective cultivation of these organisms.

Appl Environ Microbiol, 2000 Jan, 66(1), 29 - 35
Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum; Tkalec AL et al.; In medium supplemented with chondroitin sulfate, Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space . Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B) . The genes coding for both enzymes were isolated from F . heparinum and designated cslA (chondroitinase AC) and cslB (chondroitinase B) . They were found to be separated by 5.5 kb on the chromosome of F . heparinum, transcribed in the same orientation, but not linked to any of the heparinase genes . In addition, the synthesis of both enzymes appeared to be coregulated . The cslA and cslB DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively . Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues . The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively . Truncated cslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coli . Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from F . heparinum.

J Infect, 1999 Sep, 39(2), 146 - 52
Flavobacterium spp . organisms as opportunistic bacterial pathogens during advanced HIV disease; Manfredi R et al.; OBJECTIVE: To assess the role of Flavobacterium spp . infection in patients with HIV disease . METHODS: Clinical charts of 2412 consecutive HIV-infected patients hospitalized in a 8-year period were retrospectively reviewed, to identify all cases of Flavobacterium spp . infections, and to evaluate their occurrence and outcome according to several epidemiological, clinical, and laboratory parameters . RESULTS: Six patients out of 2412 (0.25%), developed Flavobacterium spp . complications: septicaemia in five cases, and pneumonia in the remaining patient, with F . meningosepticum and F . odoratum isolated in two cases and one case, respectively, and unnamed Flavobacterium spp . organisms in the remaining three cases . Flavobacterium spp . organisms were responsible for six out of 1939 overall episodes of non-mycobacterial bacterial diseases observed in our patient group (0.31%) . All patients were severely immunocompromised, showing a prior diagnosis of AIDS, a mean CD4+ lymphocyte count of 64.2 (range 12-187) cells/microl, and a mean neutrophil count of 1.143 (range 700-1600) cells (range 700-1600) cells/microl . Antibiotic, corticosteriod, or cotrimoxazole treatment was carried out during the month preceding disease onset by three, two and five patients, respectively . Community-acquired and nosocomial Flavobacterium spp . disease were equally frequent, but the latter occurred with a significantly lower mean neutrophil and CD4+ cell count . Antimicrobial susceptibility assays showed complete sensitivity to ciprofloxacin, and variable resistance to ureidopenicillins, ceftazidime, imipenem, aztreonam, and aminoglycosides . An appropriate antimicrobial regimen obtained clinical and microbiological cure in all cases, in absence of related mortality or relapses . CONCLUSIONS: Since only one episode of HIV-associated F . (Sphingobacterium) multivorum complication has been described to date, our series represents the largest one dealing with Flavobacterium spp . infection in the setting of HIV disease . Our experience suggests that Flavobacterium spp . organisms may play a pathogenic role in patients with advanced HIV disease, even when some commonly recognized risk factors are lacking (i.e . indwelling catheters, instrumentation, IV drug abuse), while a very low CD4+ lymphocyte count, leukopaenia-neutropaenia, and concurrent AIDS-related infectious complications may act as important predisposing factors . In view of the infrequent occurrence of these infections, early suspicion is essential for both clinicians and microbiologists facing immunocompromised patients at risk for invasive bacterial complications . Flavobacterium spp . organisms should be taken into consideration as nosocomial- or community-acquired opportunistic pathogens, due to their relationship with advanced immunodeficiency and their elevated resistance to many antimicrobial agents commonly used against Gram-negative bacterial pathogens.

Antimicrob Agents Chemother, 2000 Jan, 44(1), 1 - 9
Genetic-biochemical analysis and distribution of the Ambler class A beta-lactamase CME-2, responsible for extended-spectrum cephalosporin resistance in Chryseobacterium (Flavobacterium) meningosepticum; Bellais S et al.; In vitro synergy between extended-spectrum cephalosporins and either clavulanic acid or cefoxitin was found for Chryseobacterium meningosepticum isolates during a double-disk assay on an agar plate . An extended-spectrum beta-lactamase (ESBL) gene from a C . meningosepticum clinical isolate was cloned and expressed in Escherichia coli DH10B . Its protein conferred resistance to most beta-lactams including extended-spectrum cephalosporins but not to cephamycins or to imipenem . Its activity was strongly inhibited by clavulanic acid, sulbactam, and tazobactam, as well as by cephamycins and imipenem . Sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) of 891 bp with a G+C content of 33.9%, which lies close to the expected range of G+C contents of members of the Chryseobacterium genus . The ORF encoded a precursor protein of 297 amino acids, giving a mature protein with a molecular mass of 31 kDa and a pI value of 9.2 in E . coli . This gene was very likely chromosomally located . Amino acid sequence comparison showed that this beta-lactamase, named CME-2 (C . meningosepticum ESBL), is a novel ESBL of the Ambler class A group (Bush functional group 2be), being weakly related to other class A beta-lactamases . It shares only 39 and 35% identities with the ESBLs VEB-1 from E . coli MG-1 and CBL-A from Bacteroides uniformis, respectively . The distribution of bla(CME-2) among unrelated C . meningosepticum species isolates showed that bla(CME-2)-like genes were found in the C . meningosepticum strains studied but were absent from strains of other C . meningosepticum-related species . Each C . meningosepticum strain produced at least two beta-lactamases, with one of them being a noninducible serine ESBL with variable pIs ranging from 7.0 to 8.5.

J Mol Biol, 1999 Dec 17, 294(5), 1257 - 69
Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution; Huang W et al.; Glycosaminoglycans (GAGs) are a family of acidic heteropolysaccharides, including such molecules as chondroitin sulfate, dermatan sulfate, heparin and keratan sulfate . Cleavage of the O-glycosidic bond within GAGs can be accomplished by hydrolases as well as lyases, yielding disaccharide and oligosaccharide products . We have determined the crystal structure of chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, as well as its complex with a dermatan sulfate disaccharide product, both at 1.7 A resolution . Chondroitinase B adopts the right-handed parallel beta-helix fold, found originally in pectate lyase and subsequently in several polysaccharide lyases and hydrolases . Sequence homology between chondroitinase B and a mannuronate lyase from Pseudomonas sp . suggests this protein also adopts the beta-helix fold . Binding of the disaccharide product occurs within a positively charged cleft formed by loops extending from the surface of the beta-helix . Amino acid residues responsible for recognition of the disaccharide, as well as potential catalytic residues, have been identified . Two arginine residues, Arg318 and Arg364, are found to interact with the sulfate group attached to O-4 of N-acetylgalactosamine . Cleavage of dermatan sulfate likely occurs at the reducing end of the disaccharide, with Glu333 possibly acting as the general base .

J Appl Microbiol, 1999 Nov, 87(5), 668 - 75
Amine borate catabolism by bacteria isolated from contaminated metal-working fluids
Sherburn RE, Large PJ.
Four bacterial strains (tentatively identified as strains of Aeromonas, Pseudomonas, Flavobacterium and Bacillus) isolated from contaminated metal-working fluids were assayed for the capacity to utilize the borate derivatives of monoethanolamine (MEA), diethanolamine (DEA) and triethanolamine (TEA) . Two of these strains, isolates AV1 (Flavobacterium) and CL1 (Bacillus) were capable of growth on each of the borate esters with cell yields of 0.6 gl - 1 for AV1 cultured on DEA- and TEA-borate, 0.3-0.4 gl - 1 for CL1 cultured on DEA- and TEA-borate and approximately 1.4 gl - 1 for AV1 and CL1 cultured on MEA-borate . In the case of strain CL1, growth yields on TEA- or DEA-borate as substrates were doubled by the addition of potassium ions . Lower ethanolamines, glycolaldehyde, acetaldehyde and ammonia were identified as breakdown products . The enzymes produced during growth upon the alkanolamine borates were shown to possess similar properties to those seen for cells cultured upon alkanolamine hydrochlorides.

Glycoconj J, 1999 Jun, 16(6), 265 - 70
New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E . coli and 2-O-desulfated heparin; Nader HB et al.; The capsular polysaccharide from E . Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum . The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc . N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS . The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively . These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity . Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S . All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase . This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.

Biochim Biophys Acta, 1999 Nov 16, 1435(1-2), 117 - 26
Novel bacterial peroxidase without catalase activity from Flavobacterium meningosepticum: purification and characterization; Koga S et al.; A novel bacterial peroxidase co-produced intracellularly with H(2)O(2)-forming nucleoside oxidase, was purified from the cell-free extract of Flavobacterium meningosepticum to homogeneity with 10.3% overall recovery through simple purification procedures including successive DEAE-Sephacel, phenyl-Sepharose CL-4B and Sephacryl S-300 chromatography . The relative molecular mass of the native enzyme was 220 inverted question mark omitted inverted question mark000 Da, and that of its subunit was 54 inverted question mark omitted inverted question mark000 Da . In contrast to other major intercellular peroxidases of bacterial origin, the enzyme did not show any catalase activity . The amino acid sequences of the 92 NH(2)-terminal amino acids and three internal peptides showed no significant homology with known peroxidases . The enzyme was not sensitive to the typical peroxidase inhibitors NaCN, NaF and NaN(3), while mercuric ion strongly inhibited the enzyme activity, and some carbonyl reagents were also found to have inhibitory effects . The enzyme showed a small K(m) value for H(2)O(2) (9.5 microM) compared to other peroxidases . On the basis of its visible absorption spectrum, the enzyme contained about 1.3 mol of heme per molecule.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1551 - 7
Phylogenetic analysis of genus Marinilabilia and related bacteria based on the amino acid sequences of gyrB and emended description of Marinilabilia salmonicolor with Marinilabilia agarovorans as its subjective synonym; Suzuki M et al.; The detailed phylogenetic relationships for genus Marinilabilia and related taxa were analysed by using DNA gyrase B subunit gene (gyrB) sequences . Anaerobic bacteria in the Cytophaga-Flavobacterium-Bacteroides phylum, namely genera Marinilabilia, Bacteroides, Rikenella, Prevotella and Porphyromonas and Cytophaga fermentans, were clustered in the same branch and the facultative anaerobes Marinilabilia and Cytophaga fermentans formed a subcluster in the branch of the anaerobic bacteria . Phylogenetic analysis using 16S rDNA sequences gave a similar result but with a lower bootstrap value for each cluster . The gyrB sequences of Marinilabilia salmonicolor and Marinilabilia agarovorans were the same, and the relatedness of their chromosomal DNA, as determined by DNA-DNA hybridization, was greater than 70% . These genetic aspects led to the conclusion that M . salmonicolor IFO 15948T and M . agarovorans IFO 14957T belong to a single species . Since M . salmonicolor was described first, as Cytophaga salmonicolor, M . salmonicolor is a senior subjective synonym of M . agarovorans . Therefore, the name M . salmonicolor should be retained and strain IFO 14957T should be reclassified as M . salmonicolor . However, the agar-degrading ability of strain IFO 14957T is a prominent biochemical characteristic . It is therefore proposed that strain IFO 14957T should be renamed M . salmonicolor biovar agarovorans.

Annu Rev Microbiol, 1999, 53, 189 - 215
Poles apart: biodiversity and biogeography of sea ice bacteria; Staley JT et al.; This review introduces the subjects of bacterial biodiversity and biogeography . Studies of biogeography are important for understanding biodiversity, the occurrence of threatened species, and the ecological role of free-living and symbiotic prokaryotes . A set of postulates is proposed for biogeography as a guide to determining whether prokaryotes are "cosmopolitan" (found in more than one geographic location on Earth) or candidate endemic species . The term "geovar" is coined to define a geographical variety of prokaryote that is restricted to one area on Earth or one host species . This review discusses sea ice bacteriology as a test case for examining bacterial diversity and biogeography . Approximately 7% of Earth's surface is covered by sea ice, which is colonized principally by psychrophilic microorganisms . This extensive community of microorganisms, referred to as the sea ice microbial community (SIMCO), contains algae (mostly diatoms), protozoa, and bacteria . Recent investigations indicate that the sea ice bacteria fall into four major phylogenetic groups: the proteobacteria, the Cytophaga-Flavobacterium-Bacteroides (CFB) group, and the high and low mol percent gram-positive bacteria . Archaea associated with sea ice communities have also been reported . Several novel bacterial genera and species have been discovered, including Polaromonas, Polaribacter, Psychroflexus, Gelidibacter, and Octadecabacter; many others await study . Some of the gram-negative sea ice bacteria have among the lowest maximum temperatures for growth known, < 10 degrees C for some strains . The polar sea ice environment is an ideal habitat for studying microbial biogeography because of the dispersal issues involved . Dispersal between poles is problematic because of the long distances and the difficulty of transporting psychrophilic bacteria across the equator . Studies to date indicate that members of some genera occur at both poles; however, cosmopolitan species have not yet been discovered . Additional research on polar sea ice bacteria is needed to resolve this issue and extend our understanding of its microbial diversity.

Dis Aquat Organ, 1999 Sep 14, 37(3), 159 - 63
Flavobacterium psychrophilum in Baltic salmon Salmo salar brood fish and their offspring; Ekman E et al.; Baltic salmon brood fish were investigated for the presence of Flavobacterium psychrophilum in the kidney, spleen, brain and sexual products (ovarian fluid, unfertilised eggs and milt) . Samples for bacteriology were taken at capture, when the fish were ascending their native river to spawn, and after a period of captivity in indoor pools, at stripping . During captivity, abnormal wiggling behaviour was recorded in some of the fish . Bacterial samples were taken to determine if F . psychrophilum had any role in the aetiology of the condition . Furthermore, the presence of F . psychrophilum on egg surfaces during incubation was investigated . F . psychrophilum was isolated from internal organs and/or sexual products in 7 out of 50 (14.0%) fish sampled at capture and 63 out of 272 (23.2%) fish sampled at stripping . The bacteria was isolated from either spleen or gonads in 2 out of 19 (10.5%) fish with abnormal wiggling behaviour but no bacteria was isolated from the brain . No F . psychrophilum was isolated from eggs at the eyed stage . Just before hatching, the bacterium was isolated from 5 out of 15 (33.3%) family groups . The present study shows that Baltic salmon brood fish are carriers of F . psychrophilum during their spawning migration . The presence of the bacteria in sexual products from both females and males indicates that transmission from the brood fish to the offspring should be considered an important route of infection.

Appl Environ Microbiol, 1999 Nov, 65(11), 5050 - 8
Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil; Hengstmann U et al.; We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil . Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J . Chin, D . Hahn, U . Hengstmann, W . Liesack, and P . H . Janssen, Appl . Environ . Microbiol . 65:5042-5049, 1999) . Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences . Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g {dry weight} of soil) . This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45) . In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations . The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil . Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.

Appl Environ Microbiol, 1999 Nov, 65(11), 5042 - 9
Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms; Chin KJ et al.; Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants . The highest viable counts (up to 2.5 x 10(8) cells per g {dry weight} of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate . The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil . The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria . Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large . None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available . Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate) . The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U . Hengstmann, K.-J . Chin, P . H . Janssen, and W . Liesack, Appl . Environ . Microbiol . 65:5050-5058, 1999) . Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.

Acta Crystallogr D Biol Crystallogr, 1999 Nov, 55(11), 1961 - 4
Purification and crystallization of precursors and autoprocessed enzymes of Flavobacterium glycosylasparaginase: an N-terminal nucleophile hydrolase; Cui T et al.; Glycosylasparaginase (GA) represents a novel group of proteins that are activated by self-catalyzed peptide-bond cleavage from a single-chain precursor to yield the two subunits required for hydrolase activity . The wild-type GA precursor autoproteolyzes spontaneously into alpha and beta subunits . Strategies are reported here for purification to homogeneity of GA from Flavobacterium meningosepticum in both single-chain precursor and mature (autoprocessed) forms . The recombinant proteins crystallize in different space groups: P1 and P2(1) for the precursor and mature enzymes, respectively . The precursor crystals diffract to 1.9 A resolution with laboratory X-ray radiation.

J Comp Pathol, 1999 Oct, 121(3), 277 - 82
Nodular gill disease (amoebic gill infestation) in arctic char, Salvelinus alpinus; Speare DJ; Two groups of Arctic char (Salvelinus alpinus) and one of rainbow trout (Oncorhynchus mykiss) from a commercial fish farm in eastern Canada were found to have mixed infection of the gills with Flavobacterium branchiophilum (the causative agent of bacterial gill disease (BGD) and amoebae similar to those responsible for nodular gill disease (NGD) . The diagnoses were confirmed by immunofluorescence antibody testing and transmission electron microscopy . The gill lesions were typical for a mixed BGD and NGD infection and the extensive and dramatic hyperplasia of filament epithelium was characteristic of NGD . The diagnosis of NGD in Arctic char in eastern Canada represents both a geographical and species extension for this infection . To date, within commercially farmed salmonid populations, NGD has been reported only in central Canada in rainbow trout .

Appl Environ Microbiol, 1999 Sep, 65(9), 4163 - 70
Expression of the isoamylase gene of Flavobacterium odoratum KU in Escherichia coli and identification of essential residues of the enzyme by site-directed mutagenesis; Abe J et al.; The isoamylase gene from Flavobacterium odoratum KU was cloned into and expressed in Escherichia coli JM109 . The promoter of the gene was successful in E . coli, and the enzyme produced was excreted into the culture medium, depending on the amount of the enzyme expressed . The enzyme found in the culture medium showed almost the same M(r), heat-inactivating constant, and N-terminal sequence as those of the enzyme accumulated in the periplasmic space . This result indicated that the enzyme accumulated in an active form at the periplasm was transported out of the cell . The primary sequence of the enzyme, which was deduced from its nucleotide sequence, showed that the mature enzyme consisted of 741 amino acid residues . By changing five possible residues to Ala independently, it was found that Asp-374, Glu-422, and Asp-497 were essential . The sequences around those residues were highly conserved in isoamylases of different origins and the glycogen operon protein X, GlgX . The comparison of the distance between these essential residues with those of various amylases suggested that the bacterial and plant isoamylase but not GlgX had a longer fourth loop than the other amylases . This longer fourth loop had a possible role in accommodating the long branched chains of native glycogens and starches.

Antimicrob Agents Chemother, 1999 Sep, 43(9), 2193 - 9
Cloning of a Chryseobacterium (Flavobacterium) meningosepticum chromosomal gene (blaA(CME)) encoding an extended-spectrum class A beta-lactamase related to the Bacteroides cephalosporinases and the VEB-1 and PER beta-lactamases; Rossolini GM et al.; In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine beta-lactamase, named CME-1, with an alkaline isoelectric pH . The blaA(CME) gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones . Sequence analysis revealed that the CME-1 enzyme is a new class A beta-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER beta-lactamases and the Bacteroides chromosomal cephalosporinases . The blaA(CME) determinant is located on the chromosome and exhibits features typical of those of C . meningosepticum resident genes . The CME-1 protein was purified from an E . coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step . Kinetic parameters for several substrates were determined . CME-1 is a clavulanic acid-susceptible extended-spectrum beta-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems . The enzyme exhibits strikingly different kinetic parameters for different classes of beta-lactams, with both K(m) and k(cat) values much higher for cephalosporins than for penicillins and monobactams . However, the variability of both kinetic parameters resulted in overall similar acylation rates (k(cat)/K(m) ratios) for all types of beta-lactam substrates.

Vet Microbiol, 1999 Jul 1, 67(4), 287 - 98
The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops); Decostere A et al.; The ability of a high virulence strain (AJS 1) and a low virulence strain (AJS 4) of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of black mollies (Poecilia sphenops) was investigated . For that purpose, two groups of 25 black mollies each were immersed in a bath containing 10(6) CFU/ml of F . columnare AJS 1 or AJS 4 . At regular intervals from 1 to 12 h after the contact infection, fish were sacrificed and gills, skin, spleen and heart were sampled for bacteriology . Samples of the gills were taken for immunohistochemical and electron microscopic examination . Bacteriological examination proved that the number of gill-associated F . columnare was higher for AJS 1 than for AJS 4 . Strain AJS 1 was isolated from the heart and spleen of 6 and 1 of the 16 examined animals, respectively . Strain AJS 4 was not isolated from the internal organs of any fish . When examined immunohistochemically, strain AJS 1 was found closely associated with gill epithelium whereas this was not the case for strain AJS 4 . The adherence of bacteria to the gill tissue challenged with the virulent strain AJS 1 was also clearly demonstrated using scanning and transmission electron microscopy . These results indicate that adhesion of F . columnare to the gill tissue constitutes an important step in pathogenesis.

J Ind Microbiol Biotechnol, 1999 Jul, 23(1), 697 - 700
Influence of carbon and nitrogen sources on Flavobacteriumgrowth and zeaxanthin biosynthesis; Alcantara S et al.; Growth and production of zeaxanthin by Flavobacterium sp were studied using different carbon and nitrogen sources in a chemically defined medium . The best growth was supported by sucrose, but glucose yielded similar carotenoid concentrations . Both asparagine and glutamine stimulated growth and pigment formation . Carotenoid production and glucose consumption increased as a function of asparagine concentration . In the presence of asparagine, high glucose concentrations decreased pigment production without affecting biomass formation . In the absence of glucose, asparagine could not support growth and zeaxanthin production . When compared to the effect of 55 mM glucose, 10 mM oxaloacetate increased growth and carotenoid production . Pyruvate and other intermediates of the citric acid cycle showed a similar stimulatory effect . The intermediates of glycolysis: glucose 6-phosphate and fructose 1,6-diphosphate did not support growth . These results suggest that Flavobacterium sp utilizes asparagine primarily as a nitrogen source for growth and production of zeaxanthin.

Phytomedicine, 1999 Jul, 6(3), 197 - 203
Screening of crude drug extracts for prolyl endopeptidase inhibitory activity; Tezuka Y et al.; Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme to play a role in metabolism of proline-containing neuropeptides, such as vasopressin, substance P and thyrotropin-releasing hormone (TRH), which were suggested to be involved with learning and memory processes . Then, specific inhibitor of PEP is expected to have antiamnesic effects, and thus we screened forty-six water- and methanol-extracts from crude drugs selected on the basis of traditional Chinese medicine theory, for Flavobacterium prolyl endopeptidase inhibition . Among them, the water-extracts of Rhodiola sacra (IC50, 0.77 microgram/ml) and the methanol-extracts of Lycopodium clavatum (IC50, 1.3 micrograms/ml), Paeonia lactiflora var . trichocarpa (IC50, 5.7 micrograms/ml), Paeonia veitchii (IC50, 2.4 micrograms/ml) and Rhodiola sacra (IC50, 0.67 microgram/ml) showed strong inhibitory activity . In addition, we also examined the PEP inhibitory activity of eleven compounds from Salvia deserta, and found that in addition to a catechol group alpha-hydroxy-para-quinone group may be related to the PEP inhibition.

Biochim Biophys Acta, 1999 Aug 5, 1428(2-3), 273 - 83
Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis; Dietrich CP et al.; A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis . The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins . (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues . The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin . The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.

Appl Environ Microbiol, 1999 Aug, 65(8), 3721 - 6
Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization; Glockner FO et al.; Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples . An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea, and of these, about half could be identified at the subdomain level with a set of group-specific probes . Beta subclass proteobacteria constituted a dominant fraction in freshwater systems, accounting for 16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined . Members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in the marine systems, accounting for 18% (range, 2 to 72%) of the 4',6-diamidino-2-phenylindole (DAPI) counts, and they were also important in freshwater systems (7%, range 0 to 18%) . Furthermore, members of the alpha and gamma subclasses of Proteobacteria as well as members of the Planctomycetales were detected in both freshwater and marine water in abundances <7%.

Res Microbiol, 1999 Jun, 150(5), 351 - 8
Production of viable cultures of Flavobacterium psychrophilum: approach and control; Michel C et al.; Although the fish pathogen Flavobacterium psychrophilum is a major source of concern in salmonid hatcheries, few studies have been conducted on its pathogenicity . Difficulties are often experienced when trying to control or quantify standard procedures for in vitro culture of the bacterium . Plate enumeration and counting chamber enumeration combined with epifluorescent microscopy with fluorescent dyes determined that no more than 25% of the bacterial cells present in the cultures were able to produce colonies on agar media . This was strongly dependent upon different medium components . Tryptone-enriched Anacker and Ordal medium proved more suitable than tryptone-yeast extract-salts with skimmed milk . Adding horse serum and trace elements in controlled proportions offered the most reproducible results . Viable but nonculturable forms were apparently not responsible for the difficulties in production of F . psychrophilum, but the cells were highly susceptible to osmotic conditions . Improvements in the media and careful handling of the bacteria in isotonic suspension media resulted in predictable production of viable bacteria and allowed an absorbance/colony-forming-units relation curve to be established.

Braz J Med Biol Res, 1999 May, 32(5), 545 - 50
Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography; Aguiar JA et al.; Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC . The purpose of the present study was to optimize the preparation of F . heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates . We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost . Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved . Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction . Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose . A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.

Dis Aquat Organ, 1999 May 31, 36(3), 169 - 76
Reproducible methods for experimental infection with Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss; Madsen L et al.; Experiments were done in order to achieve a reproducible method that can be used to infect rainbow trout Oncorhynchus mykiss with Flavobacterium psychrophilum, the causal agent of coldwater disease and rainbow trout fry syndrome . The main method investigated was intraperitoneal injection, and this method was tested using isolates with different elastin-degrading profiles and representing different serotypes . Injecting trout, average weight 1 g, with 10(4) CFU (colony-forming units) per fish caused cumulative mortalities around 60 to 70% . The virulent strains belonged to certain serotypes and degraded elastin . The intraperitoneal injection challenge method could be used on larger fish, but the infection dose was 10(7) CFU per fish before mortalities occurred . Bath infection and bath infection in combination with formalin treatment (stress) seemed to be reproducible methods that could be used as alternatives to the intraperitoneal method, although the mortalities among infected trout were lower . The results of investigated methods were influenced by parameters such as the challenge isolate, number of fish in the tank affecting the infection pressure, origin of fish and weight of fish.

Syst Appl Microbiol, 1999 May, 22(2), 237 - 48
The response of the microbial community of marine sediments to organic carbon input under anaerobic conditions; Rossello-Mora R et al.; Cyanobacterial biomass was added to anaerobic sediment to simulate the natural input of complex organic substrate that occurs in nature after algae blooms . Sediments were incubated at 0 degree C, 8 degrees C and 24 degrees C for 13 days . Community dynamics were measured by fluorescence in situ hybridisation (FISH), denaturing gradient gel electrophoresis (DGGE), and sequencing of 16S rDNA PCR products . Metabolic changes were followed by the analysis of total carbon mineralisation, sulfate reduction, and ammonium production rates . The addition of organic material resulted in significant changes in the composition of the microbial community at all temperatures tested . Sulfate reduction was the main mineralisation process detected . However, not sulfate-reducers but rather members of the Cytophaga-Flavobacterium phylogenetic cluster showed the highest increase in the bacterial cells as detected by FISH . We conclude that these organisms play an important role in the anaerobic decomposition of complex organic material perhaps because they are the main catalysts of macromolecule hydrolysis and fermentation . The molecular methods also indicated a stimulation of ribosome synthesis . The detection of a large number of rRNA-rich cells belonging to the Cytophaga-Flavobacterium phylogenetic cluster further supports the importance of their role in the degradation of complex organic material in anaerobic marine sediments . Their detection in high numbers in the field may indicate recent deposition events.

J Am Vet Med Assoc, 1999 Jun 15, 214(12), 1833 - 8, 1792-3
Identification and management of an outbreak of Flavobacterium meningosepticum infection in a colony of South African clawed frogs (Xenopus laevis); Green SL et al.; During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a colony of South African clawed frogs (Xenopus laevis) . Clinical signs were consistent with septicemia: ascites, anasarca, dyspnea, extreme lethargy, congestion of web vessels, petechial hemorrhages, and sudden death . Mortality rate reached 35%, and all infections were fatal . The organism was resistant to most antibiotics but was susceptible to enrofloxacin, chloramphenicol, and trimethoprim-sulfadiazine . Treatment with trimethoprim-sulfadiazine was unsuccessful . Although the point source of the infection was not determined, several environmental reservoirs were identified, including a communal water barrel and various pieces of equipment . Molecular strain typing by pulsed-field gel electrophoresis and biochemical analyses revealed that frogs were infected with a single strain of F meningosepticum . Sanitation and management procedures were effective in controlling the outbreak.

Cell Mol Life Sci, 1999 May, 55(5), 812 - 8
Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide; Fanuel L et al.; Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts . The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O . anthropi SCRC C1-38 (ATCC49237) . The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification . Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities . The gene encoding the DmpA protein was cloned and sequenced . The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria . None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp . K172.

Planta, 1999 Apr, 208(2), 283 - 93
Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice; Fujita N et al.; Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion-exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography . Although the protein exhibited a molecular size of ca . 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm . The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan . The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 degrees C, respectively . The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM . These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase . Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G . James et al., 1995 . Plant Cell 7: 417-429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A . Amemura et al . 1988, J Biol Chem 263: 9271-9275) . The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase . Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv . Nipponbare which belongs to the Japonica rice group . Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium . Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes . Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type.

J Mol Biol, 1999 May 14, 288(4), 635 - 47
Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes; Fethiere J et al.; Glycosaminoglycans (GAGs), highly sulfated polymers built of hexosamine-uronic acid disaccharide units, are major components of the extracellular matrix, mostly in the form of proteoglycans . They interact with a large array of proteins, in particular of the blood coagulation cascade . Degradation of GAGs in mammalian systems occurs by the action of GAG hydrolases . Bacteria express a large number of GAG-degrading lyases that break the hexosamine-uronic acid bond to create an unsaturated sugar ring . Flavobacterium heparinum produces at least five GAG lyases of different specificity . Chondroitin AC lyase (chondroitinase AC, 75 kDa) is highly active toward chondroitin 4-sulfate and chondroitin-6 sulfate . Its crystal structure has been determined to 1.9 A resolution . The enzyme is composed of two domains . The N-terminal domain of approximately 300 residues contains mostly alpha-helices which form a doubly-layered horseshoe (a subset of the (alpha/alpha)6 toroidal topology) . The approximately 370 residues long C-terminal domain is made of beta-strands arranged in a four layered beta-sheet sandwich, with the first two sheets having nine strands each . This fold is novel and has no counterpart in full among known structures . The sequence of chondroitinase AC shows low level of homology to several hyaluronate lyases, which likely share its fold . The shape of the molecule, distribution of electrostatic potential, the pattern of conservation of the amino acids and the results of mutagenesis of hyaluronate lyases, indicate that the enzymatic activity resides primarily within the N-terminal domain . The most likely candidate for the catalytic base is His225 . Other residues involved in catalysis and/or substrate binding are Arg288, Arg292, Lys298 and Lys299 .

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 867 - 74
Coenonia anatina gen . nov., sp . nov., a novel bacterium associated with respiratory disease in ducks and geese; Vandamme P et al.; Taxon 1502 was originally described as a Riemerella anatipestifer-like bacterium causing exudative septicaemia in ducks and geese . In the present study, an integrated genotypic and phenotypic approach was used to elucidate the phylogenetic affiliation and taxonomic relationships of 12 strains of taxon 1502 . Whole-cell protein and fatty acid analyses and an extensive biochemical examination by using conventional tests and several API microtest systems indicated that all isolates formed a homogeneous taxon, which was confirmed by DNA-DNA hybridizations . 16S rDNA sequence analysis of a representative strain (LMG 14382T) indicated that this taxon belongs to the Cytophaga-Flavobacterium-Bacteroides phylum and revealed a moderate but distinct relationship to species of the genus Capnocytophaga (overall 16S rDNA sequence identities were 88.8-90.2%) . Taxon 1502 is concluded to represent a single species that should be allocated to a novel genus, and the name Coenonia anatina gen . nov., sp . nov . is proposed . The DNA G + C content of representative strains was 35-36 mol% and the type strain is LMG 14382T.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 479 - 81
Phylogenetic position of Chitinophaga pinensis in the Flexibacter-Bacteroides-Cytophaga phylum; Sly LI et al.; Comparison of the 16S rRNA gene sequence determined for Chitinophaga pinensis showed that this species is most closely related to Flexibacter filiformis in the Flexibacter-Bacteroides-Cytophaga phylum . These two chitinolytic bacteria, which are characterized by transformation into spherical bodies on ageing, belong to a strongly supported lineage that also includes Cytophaga arvensicola, Flavobacterium ferrugineum and Flexibacter sancti . The lineage is distinct from the microcyst-forming species Sporocytophaga myxococcoides.

Eur J Biochem, 1999 May, 262(1), 127 - 33
Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region . Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride; Tsuda H et al.; Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate . Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region . In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis . Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue . In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue . Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage . These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.

Biochem Genet, 1998 Dec, 36(11-12), 407 - 15
Expression and export of Pseudomonas putida NTU-8 creatinase by Escherichia coli using the chitinase signal sequence of Aeromonas hydrophila; Hong MC et al.; The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691 . The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp . An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E . coli JM109 . The amount of this fusion enzyme was about 50% exported into the periplasmic space of E . coli.

Appl Environ Microbiol, 1999 May, 65(5), 1980 - 90
Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase
Henckel T, Friedrich M, Conrad R.
Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4 . After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv) . However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv) . The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes . A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria {gamma-Proteobacteria}) and type II methylotrophs (members of the alpha-Proteobacteria) were used . Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs . The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used . The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4 . The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis . DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began . High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.

Zhonghua Yi Xue Za Zhi (Taipei), 1999 Mar, 62(3), 125 - 32
Flavobacterium meningosepticum bacteremia: an analysis of 16 cases; Liu CE et al.; BACKGROUND: Flavobacterium meningosepticum is an uncommon pathogen causing nosocomial pneumonia and meningitis in newborns . It is usually resistant to antimicrobial agents used to treat gram-negative bacilli . While the pathogen often results in high mortality and serious sequelae in newborns, it is also found to cause to disease in adults . Therefore, it is necessary to know the full spectrum of the infection in adults and to identify effective antimicrobial agents . METHOD: Microbiology logbooks were reviewed for F meningosepticum isolated from January, 1992, to March, 1996 . The medical records of these patients were reviewed . Special attention was paid to clinical manifestations, underlying diseases, risk factors, treatments, and prognosis . Twenty-four antimicrobial agents were tested using antimicrobial susceptibility tests . RESULTS: Eighteen isolates of F meningosepticum were identified from 16 patients . There were 10 men and six women, with a mean age of 63.7 years . The clinical features of infection included fever (> or = 38 degrees C) in 13 patients, chills in seven, shortness of breath in four, rales or rhonchi in four, shock in three and flank pain in two . All except one patient survived without sequelae . Fifteen patients contracted F meningosepticum from nosocomial sources . Of them, seven were suspected to have acquired the pathogen from diagnostic or therapeutic procedures . Bacteremia occurred in these patients within a mean period of 2.2 days . The other eight patients suffered nosocomial bacteremia within a mean period of 33.4 days after admission . The suspected infection route was not identified in only one patient . The organism was resistant to penicillins, cephalosporins, aztreonam, imipenem, aminoglycosides and macrolides . Testing with lomefloxacin, ciprofloxacin and ofloxacin yielded 72.2%, 83.3% and 94.4% susceptibility rates, respectively . Rifampin (61.1%) and trimethoprim-sulfamethoxazole (TMP-SMX) (88.9%) were effective . Vancomycin and minocycline were 100% effective . CONCLUSIONS: F meningosepticum is an opportunistic pathogen of low virulence and rarely causes serious infections in adults . Reducing the use of unnecessary residual devices and invasive procedures may help reduce the incidence of infection . Therapeutic options include vancomycin, TMP-SMX, minocycline, rifampin or fluoroquinolones.

J Nat Prod, 1999 Apr, 62(4), 631 - 2
A novel monoacyldiglycosyl-monoacylglycerol from Flavobacterium marinotypicum; Yagi H et al.; A monoacyldiglycosyl-monoacylglycerol was isolated from the Gram-negative bacterium Flavobacterium marinotypicum (ATCC 19260) . The structure was determined, mainly by spectral data, to be {alpha-glucopyranosyl-(1-->3)-O-(6-O-acyl-alpha-mannopyranosyl)}-O- acylglycerol.

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1055 - 7
Crystallization and preliminary X-ray analysis of chondroitinase B from Flavobacterium heparinum; Li Y et al.; Chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, has been crystallized by hanging-drop vapor diffusion in space group P21 with unit-cell parameters a = 50.6, b = 74.5, c = 58 . 7 A, beta = 92.9 degrees and one molecule in the asymmetric unit . This enzyme degrades dermatan sulfate, a glycosaminoglycan primarily made up of a disaccharide repeating unit of iduronic acid and N-acetylgalactosamine . A complete native data set has been collected from a single crystal to 2.2 A resolution using a rotating-anode source.

Lett Appl Microbiol, 1999 Apr, 28(4), 297 - 9
An improved growth medium for Flavobacterium psychrophilum; Daskalov H et al.; Supplementing cytophaga agar and broth with 0.5 g l-1 each of D(+) galactose, D(+) glucose, L-rhamnose and skimmed milk gave a dramatic improvement in the isolation of the fish pathogen Flavobacterium psychrophilum . By means of spread-plating, approximately double the number of colonies of larger size were obtained on the improved medium compared to cytophaga agar alone . In supplemented cytophaga broth, growth of Fl . psychrophilum was more rapid and generated greater biomass.

Vet Rec, 1999 Mar 6, 144(10), 251 - 4
Histopathological and electron microscopical observations on rainbow trout fry syndrome; Rangdale RE et al.; Rainbow trout (Oncorhynchus mykiss) fry and fingerlings with clinical signs of rainbow trout fry syndrome, and rainbow trout (0.5 to 3.5 g) with experimentally induced infections with Flavobacterium psychrophilum, were examined histopathologically and electron microscopically . Severe hypertrophy of the spleen and cellular degeneration were consistently observed . Distinctive features of the disease were the loss of definition of the splenic border and its replacement by a loosely structured eosinophilic layer, fibrinous inflammation and intercellular oedema within the spleen, and the presence of numerous filamentous bacteria interspersed throughout the organ.

Arch Microbiol, 1999 Feb, 171(3), 189 - 97
Diversity of chlorophenol-degrading bacteria isolated from contaminated boreal groundwater; Mannisto MK et al.; Chlorophenol-degrading bacteria from a long-term polluted groundwater aquifer were characterized . All isolates degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol at concentrations detected in the contaminated groundwater (< 10 mg 1(-1)) . Pentachlorophenol was degraded by three isolates when present alone . In two gram-positive isolates, 2,3,4,6-tetrachlorophenol was required as an inducer for the degradation of pentachlorophenol . The gram-positive isolates were sensitive to pentachlorophenol, with an IC50 value of 5 mg/l . Isolates belonging to the Cytophaga/Flexibacter/Bacteroides phylum had IC50 values of 25 and 63 mg/l . Isolates belonging to alpha-, beta- and gamma-Proteobacteria generally tolerated the highest pentachlorophenol concentrations (> 100 mg/l) . Polychlorophenol-degrading capacity was found in strains of Nocardioides, Pseudomonas, Ralstonia, Flavobacterium, and Caulobacter previously not known to degrade polychlorophenols . In addition, six polychlorophenol-degrading sphingomonads were found.

Appl Environ Microbiol, 1999 Apr, 65(4), 1746 - 52
Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ; Ouverney CC et al.; We propose a novel method for studying the function of specific microbial groups in situ . Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial "black box" should not be regarded as homogeneous . Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR) . Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup . The method was verified with pure cultures . In addition, in situ uptake by members of the alpha subdivision of the class Proteobacteria (alpha-Proteobacteria) and of the Cytophaga-Flavobacterium group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined ( approximately 60% of the universal probe-labeled cells and approximately 50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ . Nearly 90% of the total cells and 80% of the cells belonging to the alpha-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests . We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the "dissection" of microbial communities by type and function.

Toxicol Appl Pharmacol, 1999 Apr 1, 156(1), 56 - 63
Antagonism of paraoxon intoxication by recombinant phosphotriesterase encapsulated within sterically stabilized liposomes; Petrikovics I et al.; This investigation effort is focused on increasing organophosphate (OP) degradation by phosphotriesterase to antagonize OP intoxication . For these studies, sterically stabilized liposomes encapsulating recombinant phosphotriesterase were employed . This enzyme was obtained from Flavobacterium sp . and was expressed in Escherichia coli . It has a broad substrate specificity, which includes parathion, paraoxon, soman, sarin, diisopropylfluorophosphate, and other organophosphorous compounds . Paraoxon is rapidly hydrolyzed by phosphotriesterase to the less toxic 4-nitrophenol and diethylphosphate . This enzyme was isolated and purified over 1600-fold and subsequently encapsulated within sterically stabilized liposomes (SL) . The properties of this encapsulated phosphotriesterase were investigated . When these liposomes containing phosphotriesterase were incubated with paraoxon, it readily degraded the paraoxon . Hydrolysis of paraoxon did not occur when these sterically stabilized liposomes contained no phosphotriesterase . These sterically stabilized liposomes (SL) containing phosphotriesterases (SL)* were employed as a carrier model to antagonize the toxic effects of paraoxon by hydrolyzing it to the less toxic 4-nitrophenol and diethylphosphate . This enzyme-SL complex (SL)* was administered intravenously to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine intraperitoneally . These results indicate that this carrier model system provides a striking enhanced protective effects against the lethal effects of paraoxon . Moreover when these carrier liposomes were administered with 2-PAM and/or atropine, a dramatic enhanced protection was observed .

FEMS Microbiol Lett, 1999 Feb 15, 171(2), 127 - 32
Molecular characterization of a carbapenem-hydrolyzing beta-lactamase from Chryseobacterium (Flavobacterium) indologenes; Bellais S et al.; Chryseobacterium (Flavobacterium) indologenes 001 clinical strain was resistant to several beta-lactam classes including carbapenems . Shotgun cloning experiments of Sau3AI restricted genomic DNA of C . indologenes 001 into pBKCMV cloning vector followed by transformation into Escherichia coli DH10B gave one recombinant plasmid possessing a 4.2-kb DNA insert . It encoded a pI 7.2 beta-lactamase of 239 amino acids (IND-1) which is a metallo-enzyme with a broad spectrum beta-lactam hydrolysis profile . This class B carbapenem-hydrolyzing beta-lactamase shares the highest identity (43%) with BlaB from C . meningosepticum, thus showing heterogeneity of carbapenem-hydrolyzing beta-lactamases in Chryseobacterium spp.

Biol Pharm Bull, 1999 Feb, 22(2), 157 - 61
Prolyl endopeptidase inhibitors from the underground part of Rhodiola sacra S . H . Fu; Fan W et al.; Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme which plays a role in the metabolism of proline-containing neuropeptides, e.g., vasopressin, substance P and thyrotropin-releasing hormone (TRH), which have been suggested to be involved in learning and memory processes . In our systematic screening for PEP inhibitors from traditional Chinese medicines, we found that MeOH extract from the underground part of Rhodiola sacra S . H . Fu shows significant inhibitory activity against PEP from Flavobacterium meningosepticum . Examination of the constituents of the extract resulted in the isolation of nineteen known compounds, identified as hydroquinone (1), 4-hydroxybenzoic acid (2), caffeic acid (3), 4-hydroxycinnamic acid (4), suberic acid (5), protocatechuic acid (6), gallic acid (7), (-)-epigallocatechin 3-O-gallate (8), 2-phenylethyl beta-D-glucopyranoside (9), 3-O-galloylepigallocatechin-(4beta-->8)-epigallocatechin+ ++ 3-O-gallate (10), 2-phenylethyl alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside (11), sacranoside A (12), beta-D-glucopyranosyl 4-hydroxybenzoate (13), rhodiocyanoside A (14), rhodiooctanoside (15), sarmentosin (16), heterodendrin (17), arbutin (18) and 4-O-(beta-D-glucopyranosyl)-gallic acid (19) . Among these, 1, 2, 5, 8-10, 13, 16, 18 and 19 have been isolated for the first time from R . sacra, among which 5, 9, 10, 13, 16, 18 and 19 have been isolated from Rhodiola plants for the first time . On the PEP inhibition, seven compounds (6-8, 10, 12, 18, 19) showed inhibition with an 1C50 of 27.8, 487, 1.47, 0.437, 348, 391 and 215 microM, respectively . The kinetic study of these inhibitors indicated that they are noncompetitive inhibitors, except for 6 which is a competitive inhibitor.

South Med J, 1999 Feb, 92(2), 225 - 7
Flavobacterium meningosepticum sepsis in an infant with a diarrheal prodrome; Springer SC et al.; A full term, previously normal 2 1/2-month-old black boy was transferred to our hospital from an outlying facility on hospital day 5 for failure to thrive . Three weeks before transfer, the infant was hospitalized for a diarrheal illness with fever . The baby received 3 days of ceftriaxone empirically and was discharged home after the sepsis evaluation was negative . Mild diarrhea and steady weight loss continued and the baby was readmitted . Blood culture done on admission grew Flavobacterium meningosepticum, an organism previously described as an uncommon cause of sepsis in neonates and immunocompromised individuals . As it is water-borne, it has been associated with infection via contaminated water . This organism is usually resistant to antibiotics commonly used for empiric treatment . To our knowledge, this is the first reported case of Flavobacterium bacteremia associated with a prodromal and concurrent diarrheal illness.

Appl Environ Microbiol, 1999 Mar, 65(3), 1241 - 50
Morphological and compositional changes in a planktonic bacterial community in response to enhanced protozoan grazing; Jurgens K et al.; We analyzed changes in bacterioplankton morphology and composition during enhanced protozoan grazing by image analysis and fluorescent in situ hybridization with group-specific rRNA-targeted oligonucleotide probes . Enclosure experiments were conducted in a small, fishless freshwater pond which was dominated by the cladoceran Daphnia magna . The removal of metazooplankton enhanced protozoan grazing pressure and triggered a microbial succession from fast-growing small bacteria to larger grazing-resistant morphotypes . These were mainly different types of filamentous bacteria which correlated in biomass with the population development of heterotrophic nanoflagellates (HNF) . Small bacterial rods and cocci, which showed increased proportion after removal of Daphnia and doubling times of 6 to 11 h, belonged nearly exclusively to the beta subdivision of the class Proteobacteria and the Cytophaga-Flavobacterium cluster . The majority of this newly produced bacterial biomass was rapidly consumed by HNF . In contrast, the proportion of bacteria belonging to the gamma and alpha subdivisions of the Proteobacteria increased throughout the experiment . The alpha subdivision consisted mainly of rods that were 3 to 6 microm in length, which probably exceeded the size range of bacteria edible by protozoa . Initially, these organisms accounted for less than 1% of total bacteria, but after 72 h they became the predominant group of the bacterial assemblage . Other types of grazing-resistant, filamentous bacteria were also found within the beta subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster . We conclude that the predation regimen is a major structuring force for the bacterial community composition in this system . Protozoan grazing resulted in shifts of the morphological as well as the taxonomic composition of the bacterial assemblage . Grazing-resistant filamentous bacteria can develop within different phylogenetic groups of bacteria, and formerly underepresented taxa might become a dominant group when protozoan predation is the major selective pressure.

Lett Appl Microbiol, 1999 Jan, 28(1), 61 - 5
The degradation of sodium O,O-diethyl dithiophosphate by bacteria from metalworking fluids; Sherburn RE et al.; The breakdown of sodium O,O-diethyl dithiophosphate (O,O-diethyl phosphorodithioate) by four bacterial strains (tentatively identified as strains of Aeromonas, Pseudomonas, Flavobacterium and Bacillus) isolated from contaminated metalworking fluids was shown to involve the successive formation of ethanol, aldehyde and orthophosphate . An acid phosphodiesterase was identified in cell-free extracts that was five- to sevenfold enhanced in specific activity in bacteria grown on O,O-diethyl dithiophosphate as sole phosphorus source, compared with bacteria grown on orthophosphate . This is thought to initiate the breakdown process.

Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2388 - 95
A novel glycerol kinase from Flavobacterium meningosepticum: characterization, gene cloning and primary structure; Sakasegawa S et al.; A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum . The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE . The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively . The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks . The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced . The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids . The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E . coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.

J Biol Chem, 1999 Feb 12, 274(7), 4089 - 95
The calcium-binding sites of heparinase I from Flavobacterium heparinum are essential for enzymatic activity; Liu D et al.; In the accompanying paper (Shriver, Z., Liu, D., Hu, Y., and Sasisekharan, R . (1999) J . Biol . Chem . 274, 4082-4088), we have shown that calcium binds specifically to heparinase I and have identified two major calcium-binding sites (CB-1 and CB-2) that partly conform to the EF-hand calcium-binding motif . In this study, through systematic site-directed mutagenesis, we have confirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity . More specifically, we identified critical residues (viz . Asp210, Asp212, Gly213, and Thr216 in CB-1 and Asn375, Tyr379, and Glu381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity . Mutations in CB-1 resulted in a lower kcat, but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the kcat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides . Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I . Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I . These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism . Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.

J Biol Chem, 1999 Feb 12, 274(7), 4082 - 8
Biochemical investigations and mapping of the calcium-binding sites of heparinase I from Flavobacterium heparinum; Shriver Z et al.; The heparinases from Flavobacterium heparinum are lyases that specifically cleave heparin-like glycosaminoglycans . Previously, amino acids located in the active site of heparinase I have been identified and mapped . In an effort to further understand the mechanism by which heparinase I cleaves its polymer substrate, we sought to understand the role of calcium, as a necessary cofactor, in the enzymatic activity of heparinase I . Specifically, we undertook a series of biochemical and biophysical experiments to answer the question of whether heparinase I binds to calcium and, if so, which regions of the protein are involved in calcium binding . Using the fluorescent calcium analog terbium, we found that heparinase I tightly bound divalent and trivalent cations . Furthermore, we established that this interaction was specific for ions that closely approximate the ionic radius of calcium . Through the use of the modification reagents N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, we showed that the interaction between heparinase I and calcium was essential for proper functioning of the enzyme . Preincubation with either calcium alone or calcium in the presence of heparin was able to protect the enzyme from inactivation by these modifying reagents . In addition, through mapping studies of Woodward's reagent K-modified heparinase I, we identified two putative calcium-binding sites, CB-1 (Glu207-Ala219) and CB-2 (Thr373-Arg384), in heparinase I that not only are specifically modified by Woodward's reagent K, leading to loss of enzymatic activity, but also conform to the calcium-coordinating consensus motif.

Microb Ecol, 1999 Feb, 37(2), 77 - 85
Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing; Giuliano L et al.; > Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples . The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea) . The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment . Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups . The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate . Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes . A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions . The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures . While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively . We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 243 - 9
Sequence analysis of a cryptic plasmid from Flavobacterium sp . KP1, a psychrophilic bacterium; Ashiuchi M et al.; A cryptic plasmid found at high copy number was isolated from Flavobacterium sp . KP1, a psychrophilic Gram-negative bacterium, cloned, and sequenced . The sequence will appear in the DDBJ/EMBL/GenBank databases under the accession number AB007196 . The pFL1 plasmid is 2311 nucleotides in length with 32.7% GC content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length . The plasmid contains two open reading frames of significant length, ORFI and ORFII . ORFI encodes a protein similar to the replication proteins found in Gram-negative bacterial plasmids, Bacteroides fragilis plasmid pBI143 and Zymomonas mobilis plasmid pZM2 . The putative translation product of ORFII shows homologies with plasmid recombination proteins found mainly in Gram-positive bacterial plasmids such as Staphylococcus aureus plasmid pT181.

Appl Microbiol Biotechnol, 1998 Dec, 50(6), 669 - 75
Properties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104; Morita Y et al.; Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 degrees C, which was isolated from salmon (Oncorhynchus keta) intestine . The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographyies . The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5 . Maximal activity toward azocasein was observed at 40 degrees C and from pH 7.0 to 9.0 . The activity was strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease . The N-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu- Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Gly-Asn . A search through the database for sequence homology yielded no significant match . The initial cleavage sites for oxidized insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds . The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate.

J Food Prot, 1998 Dec, 61(12), 1666 - 9
The role of trace metal ions in aflatoxin B1 degradation by Flavobacterium aurantiacum; D'Souza DH et al.; Flavobacterium aurantiacum NRRL B-184 possesses the ability to degrade aflatoxin B1 in solution and in several food items . Aflatoxin B1 is a potent carcinogen that causes significant economic losses to the agricultural and food industry . The role of trace metal ions (Cu2+, Mn2+, Zn2+, and Co2+) were studied in an effort to understand the enzymatic system involved in aflatoxin B1 degradation by F aurantiacum . The effect of divalent chelators (EDTA and 1,10-phenanthroline {OPT}) in the presence of the trace metal ions was studied as well . Aflatoxin B1 (10 microg/ml) was added to 72-h cultures of F aurantiacum that had been washed and resuspended in phosphate buffer (pH 7.0) . HPLC was used to determine aflatoxin B1 concentration in these cultures . Incubating cells at 30 degrees C with 1 and 10 mM Cu2+, Mn2+, and Zn2+ significantly decreased aflatoxin B degradation after 4 and 24 h (P < 0.05) . Decreased degradation was also observed with 1 and 10 mM Cu2+ and Zn2+ after 48 h and with 0.1 mM Cu2+ after 24 and 48 h . Co2+ did not have a significant effect on aflatoxin B1 degradation . EDTA and OPT did not counter the inhibition in the presence of Cu2+ . The addition of 1 mM EDTA countered the inhibition by 1 mM Mn2+ after 4 and 24 h, but 1 mM OPT did not counter the inhibition by 10 mM Mn2+ after 4 and 24 h . OPT countered the inhibition by 1 mM Zn2+ after 4 and 48 h . These trace elements inhibit aflatoxin B1 degradation by F aurantiacum . In addition, their presence necessitates higher concentrations (>1 mM) of EDTA and OPT for the removal of their inhibitory effect.

Eur J Biochem, 1998 Dec 1, 258(2), 775 - 83
Structural determination of sulfated tetrasaccharides and hexasaccharides containing a rare disaccharide sequence, -3GalNAc(4,6-disulfate)beta1-4IdoAalpha1-, isolated from porcine intestinal dermatan sulfate; Yamada S et al.; In the course of structural studies on sulfated oligosaccharides isolated from porcine intestinal heparin after extensive digestion with Flavobacterium heparinase, we isolated several heparitinase-resistant unsaturated oligosaccharides . Amino sugar analysis of these oligosaccharides indicated that they contained galactosamine residues but no glucosamine residues . They were sensitive to chondroitinase ABC but resistant to chondroitinase AC-II, and therefore derived from dermatan sulfate, which was presumably contained as a minor component in the starting heparin preparation . The structures of these oligosaccharides were characterized by enzymatic digestions in conjunction with HPLC analysis of the digests and by one-dimensional and two-dimensional 500-MHz 1H-NMR spectroscopy . Structures of two tetrasaccharides and two hexasaccharides were determined as deltaHexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3GalNAc(4S), deltaHexAalpha1-3GalNAc(4S,6S)}beta1-4IdoAalpha1-3GalNAc(4S) , deltaHexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3GalNAc(4S)beta 1-4IdoAalpha1-3GalNAc(4S), and deltaHexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3GalNAc(4S,6S)b eta1-4IdoAalpha1-3GalNAc(4S), where deltaHexA, IdoA, GalNAc, 4S and 6S represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, N-acetyl-D-galactosamine, 4-O-sulfate and 6-O-sulfate, respectively . The latter three compounds have never been reported as discrete structures . Since the four isolated oligosaccharides contained an unsaturated uronic acid residue at the nonreducing terminus, they appear to have been generated by eliminative cleavage by the action of Flavobacterium chondroitinase that was probably present as a minor contaminant in the Flavobacterium heparinase preparation used . Two out of the four oligosaccharides shared the rare disulfated disaccharide sequence, -3GalNAc(4S,6S)beta1-4IdoAalpha1- . These oligosaccharides will be useful as authentic reference compounds for microanalyzing biologically active domains of dermatan sulfate.

Appl Environ Microbiol, 1999 Jan, 65(1), 25 - 35
Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla; Hahn MW et al.; A facultatively filamentous bacterium was isolated from eutrophic lake water and was identified as Flectobacillus sp . strain MWH38 (a member of the Cytophaga-Flavobacterium-Bacteroides phylum) by comparative 16S rRNA gene sequence analysis . Filament formation by Flectobacillus sp . strain MWH38 and filament formation by Flectobacillus major, the closest known relative of strain MWH38, were studied in chemostat cultures under grazing pressure by the bacterivorous flagellate Ochromonas sp . strain DS and without predation at several growth rates . The results clearly demonstrated that filament formation by the two flectobacilli is growth rate controlled and thus independent of the presence of a predator . However, flagellate grazing positively influenced bacterial growth rates by decreasing bacterial biomass and thus indirectly stimulated filament formation . The results of investigations of cell elongation and filament formation by Comamonas acidovorans PX54 (a member of the beta subclass of the class Proteobacteria) supported the recent proposal that in this species the mechanism of filament formation is growth rate controlled . The finding that the grazing defense mechanism consisting of filament formation is growth rate controlled in the flectobacilli investigated and C . acidovorans PX54 (i.e., in bacteria belonging to divergent evolutionary phyla) may indicate that this mechanism is a phylogenetically widely distributed defense strategy against grazing.

Syst Appl Microbiol, 1998 Aug, 21(3), 374 - 383
Hymenobacter roseosalivarius gen . nov., sp . nov . from continental Antartica soils and sandstone: bacteria of the Cytophaga/Flavobacterium/Bacteroides line of phylogenetic descent; Hirsch P et al.; Aseptically collected sandstone and soil samples from the antarctic Dry Valleys were inoculated into oligotrophic media and incubated under low light intensities . A total of 41 Gram-negative isolates were obtained with reddish colonies spreading on agar . A sandstone isolate and four soil strains were characterized further . They were nearly identical in morphological, physiological, biochemical and chemotaxonomic properties . They produced large amounts of extracellular polymer and utilized for growth: glucose, saccharose, mannitol, sorbitol, L-aspartate, malate and acetate, but not D-ribose, adonitol, DL-alanine, glutamate, glycolate, lactate or succinate . All strains hydrolyzed gelatin, starch, casein, xylan, Tweens 80 or 60 and dead or living yeast cells, but not cellulose or pectin . Nitrate was not reduced, ethanol was not oxidized and acid was not produced from maltose, mannitol or dulcitol . Ammonia was not produced from peptone . They were strictly aerobic . Major fatty acids were n 16:1 d 9, n 16:1 d 11, n 17:1 d 11, and i 15:0 . The strains contained the quinone MK-7 and phosphatidylethanolamine as the main phospholipid . The base ratio ranged from 55 to 61 mol% G+C . A 16S rRNA sequence analysis of strains AA-688 and AA-718 showed these to be identical and to represent a special phylogenetic group within the Cytophaga/Flavobacterium/Bacteroides major line of descent . Three soil strains labeled "Taxeobacter" Txc1, Txg1, and Txo1 (Reichenbach, 1992) belonged to the same group but had lower sequence similarities (<95%) . Some of their characteristics were different from those of the antarctic strains: the utilization of C-compounds, hydrolysis of polymers, temperature tolerances, major fatty acids and base ratios . Txc1 and Txg1 may later have to be considered as members of this group, possibly on the species level, while Txo1 could represent a different related genus . It is concluded that the five antarctic strains represent a new genus and species for which the name of Hymenobacter roseosalivarius is proposed . The type strain is AA-718T (DSM 11622T).

Dis Aquat Organ, 1998 Jul 30, 33(3), 167 - 77
Fingerprinting of Flavobacterium psychrophilum isolates by ribotyping and plasmid profiling; Chakroun C et al.; Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide . Ribosomal RNA gene restriction patterns (ribotypes) and plasmid profiles were determined on a collection of 85 strains isolated from different countries and fish species . Several ribotypes were obtained by using the restriction endonucleases Hinc II and Pvu II . Computer analysis of the ribotypes revealed that some of them were clearly associated with the fish species from which the strains were isolated, whereas no correlation with the geographical origin was found . Most of the strains harboured at least one plasmid and several different plasmid profiles were observed, even among strains sharing the same ribotype . These methods, used alone or in combination with other typing techniques, can be considered powerful tools for the epidemiological tracing of F . psychrophilum infections.

Bioorg Med Chem, 1998 Oct, 6(10), 1775 - 80
The specificity of prolyl endopeptidase from Flavobacterium meningoseptum: mapping the S' subsites by positional scanning via acyl transfer; Bordusa F et al.; The S1'-S3' subsite specificity of prolyl endopeptidase from Flavobacterium meningoseptum was studied by acyl transfer to libraries of amino acid amides and peptides . Whereas the S1' and S3' subsites influence the specificity for the amino component by approximately one order of magnitude, the S2' subsite possesses a markedly higher specificity . Besides the high specificity for hydrophobic residues at P1'-P3', proline was efficiently bound by the S2' and S3' subsites of the enzyme . In contrast, no binding of P1' proline-containing peptides was observed . It could be demonstrated that the specificity of the S' subsite is not restricted to L-amino acids . Effective P'-S' interactions were also found for beta- and gamma-amino acids indicating that the enzyme does not form close contacts to the backbone of P1' and P2' amino acid residues.

Gene, 1998 Nov 19, 222(2), 187 - 94
Cloning, functional expression and purification of endo-beta-galactosidase from Flavobacterium keratolyticus; Leng L et al.; Endo-beta-galactosidase (EC 3.2.1.103) is an enzyme that hydrolyzes internal endo-beta-galactosyl linkages in keratan sulfate, and glycoconjugates with N-acetyl-lactosamine repeating units . Here, we report the cloning of the endo-beta-galactosidase-encoding gene from Flavobacterium keratolyticus, its expression in Escherichia coli and the purification of the enzyme . The enzyme was purified over 15000-fold to apparent homogeneity . The purified endo-beta-galactosidase consists of a single band of about 43kDa on SDS-PAGE and has a specific activity of 148micro/mg . Based on peptide sequences derived from the purified enzyme, a full-length clone encoding endo-beta-galactosidase was isolated from F . keratolyticus genomic DNA . The gene contains a single open reading frame coding for a protein of 422 amino acid residues with a putative N-terminal signal peptide . Its authenticity was confirmed by colinearity of deduced amino acid sequences with the peptide sequences, and synthesis of enzyme in E . coli.

Eur J Gastroenterol Hepatol, 1998 Oct, 10(10), 897 - 8
A case of spontaneous peritonitis caused by Weeksella virosa; Boixeda D et al.; A case of spontaneous peritonitis caused by Weeksella virosa is reported . This Flavobacterium has never been reported as a cause of spontaneous bacterial peritonitis . The patient responded to antimicrobial therapy . Clinical and therapeutic implications are discussed.

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1405 - 12
Flavobacterium hibernum sp . nov., a lactose-utilizing bacterium from a freshwater Antarctic lake; McCammon SA et al.; Four freshwater Antarctic lakes were examined for the presence of beta-galactosidase-producing bacteria using mineral medium enrichments and lactose . Enrichments from only one of the lakes produced growth and two strains were isolated that were very similar in phenotype and fatty acid profile, and shared considerable homology in their DNA (DNA-DNA hybridization = 93 +/- 7%) . The strains were psychrotrophic with theoretical Tmax, Tmin and Topt of 30-31, -7 degrees and 26 degrees C, respectively . The beta-galactosidase in cell extracts had an optimal activity at 39 degrees C . The strains were Gram-negative rods, showed gliding motility, contained branched and hydroxy fatty acids, and menaquinone 6 as the major respiratory quinone . The strains did not form microcysts and utilized lactose while using ammonium ions as a source of nitrogen, and a range of other sugars . The G + C content of the DNA was 34 mol% . Phylogenetic analysis of one of the strains, by comparison of 16S rDNA sequences, showed that it was most similar, but not identical to, Flavobacterium columnare and '{Sporocytophaga} cauliformis' . Both species could be differentiated phenotypically from the Antarctic isolates . DNA-DNA hybridization of the Antarctic isolate with six different members of the Flavobacterium 16S rDNA cluster showed no strain with greater than 18% relatedness . The nearest type species to the Antarctic isolate in the phylogenetic analysis was Flavobacterium aquatile . The name Flavobacterium hibernum is proposed for the Antarctic strains, and the type strain is ATCC 51468T (= ACAM 376T).

Appl Environ Microbiol, 1998 Nov, 64(11), 4299 - 306
Seasonal community and population dynamics of pelagic bacteria and archaea in a high mountain lake
Pernthaler J, Glockner FO, Unterholzner S, Alfreider A, Psenner R, Amann R.
The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenkollesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy . Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters . Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%) . Bacteria of the Cytophaga-Flavobacterium cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass . Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively . A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences . Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH . About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes . The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn . Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.

Rev Argent Microbiol, 1998 Jul-Sep, 30(3), 105 - 14
Occurrence of heterotrophic bacteria and fungi in an aviation fuel handling system and its relationship with fuel fouling; Ferrari MD et al.; Clean, dry and contaminant-free fuel is necessary for safe and economical aircraft operation . Microbial growth in aviation fuel handling systems can alter the quality of the product . This paper reports the occurrence of heterotrophic bacteria and fungi in a handling system of jet A-1 aviation turbine fuel . A total of 350 samples were collected during 1990-1996 . The aerobic microorganisms in fuel samples were mainly fungi, 85% of samples containing < or = 100 cfu/l (range 0 (< 1 cfu/l) to 2000 cfu/l) . The predominant fungi were Cladosporium and Aspergillus . Water was observed mainly in samples extracted from the drainage pipes of two tanks used frequently as intermediate storage tanks . The aerobic heterotrophic microorganisms found in water samples were mostly bacteria, counts varying from 100 to 8.8 x 10(7) cfu/ml, with 85% of samples containing 10(4)-10(7) cfu/ml . There was a preponderance of Pseudomonas spp . Bacterial contaminants belonging to the genus Flavobacterium and Aeromonas were also identified . Sulphate reducing bacteria were detected in 80% of water samples . It was not possible to assign a maximum microbial contamination level above which maintenance is required and it is suggested that analysis of successive samples from the same site are necessary for this purpose . Microbial sludges produced in the laboratory and collected from a contaminated tank bottom were analysed chemically . The data are presented and discussed . Samples collected from the supply pipes of tanks and refueller trucks during the period surveyed always met the standard specifications.

J Physiol Paris, 1998 Oct-Dec, 92(5-6), 357 - 62
Enzymes hydrolyzing organophosphates as potential catalytic scavengers against organophosphate poisoning; Masson P et al.; Enzymes hydrolyzing organophosphates could be used as catalytic scavengers for treatment of organophosphate poisoning and for decontamination . Two organophosphorus hydrolases (OPH) were selected: the Flavobacterium sp/Pseudomonas diminuta phosphotriesterase (PTE) and human paraoxonase (HuPON) . Genes encoding these enzymes were cloned and functional recombinant enzymes expressed . PTE was expressed in E . coli . Natural HuPON was purified from human plasma; recombinant HuPON was expressed in human embryonic kidney 293 T cells . Although HuPON displays interesting catalytic properties, a site-directed mutagenesis program was undertaken to improve its catalytic efficiency . PTE has high efficiency in hydrolysis of organophosphates, including nerve agents . PTE injected in rat has a half-life of 100 min . However, to overcome pharmacokinetic problems of injected OPH and/or immunological incompatibility, the model enzyme (recombinant PTE) was immobilized onto a hollow-fiber reactor . This reactor designed for extracorporeal blood circulation is under experimentation for post-exposure detoxification.

Gene, 1998 Aug 17, 216(1), 207 - 13
Inducible expression of green fluorescent protein within channel catfish cells by a cecropin gene promoter; Zhang Q et al.; The activity of an insect promoter of the cecropin B gene (Cec B) was investigated using green fluorescent protein (gfp) as a reporter in cells of channel catfish (Ictalurus punctatus) . The expression vector pQZ-1 containing the Cec B promoter and a modified gfp cDNA sequence was delivered by lipofection to three catfish types: fibroblast and leukocyte cell lines, and primary cultures of leukocytes . No resistance genes were included in the vector for selection of GFP-expressing cells . The GFP mRNA was detected in all three cell types with 5 to 10 times higher concentrations observed in leukocytes than in fibroblasts . Expression was enhanced with the addition of irradiated Flavobacterium columnare (7.0 inverted question mark10(6) cells/ml) or Escherichia coli LPS (125microgram/ml) . Quantitative RT-PCR showed GFP mRNA reached maximum levels 24h after bacterial challenge in fibroblast cells, and at 10-12h after LPS challenge in fibroblasts and leukocytes . The number of fibroblasts expressing GFP increased by 0.8%, and the average of green fluorescence intensity increased by 52.8%, whereas the increase in leukocytes was 0.13% in cell number and 3.4% in fluorescence intensity . These results suggest that the transcription of the Cec B promoter in channel catfish cells exhibited an inducible pattern and could be placed under the control of the immune system (in vivo) . The mechanisms for endogenous activation of the Cec B promoter and for production of gfp RNA in unchallenged cells remain to be studied.

Res Microbiol, 1998 Jul-Aug, 149(7), 519 - 30
Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum; Urdaci MC et al.; A PCR-based method was developed to identify and detect Flavobacterium psychrophilum, the causative agent of "cold-water disease" and "rainbow trout fry syndrome" in salmonid fish . Two oligonucleotide primers were designed by comparing the 16S rRNA sequence of all taxa in the genus Flavobacterium and of representative species in most related genera within rRNA superfamily V . Purified chromosomal DNAs from all these bacterial species, from 25 F . psychrophilum isolates and from several other fish-pathogenic bacteria were used to assess the specificity of the reaction . Amplification products were generated only with F . psychrophilum DNA . The detection level, equivalent to approximately 10 to 100 bacterial cells, was increased 10-fold by hybridization with a radioactive probe . Preliminary experiments demonstrated that this procedure can also be applied to samples of infected tissue . This PCR assay is therefore a rapid, specific, and sensitive alternative to conventional plate culture methods for the identification and detection of F . psychrophilum.

Acta Crystallogr D Biol Crystallogr, 1998 Mar 1, 54 ( Pt 2), 279 - 80
Crystallization and preliminary analysis of chondroitinase AC from Flavobacterium heparinum; Fethiere J et al.; Chondroitinase AC (E.C . 4.2.2.5) overexpressed in its host, Flavobacterium heparinum, was crystallized by vapor diffusion using polyethylene glycol methyl ether as precipitant . It crystallizes in the space group P43212 or its enantiomorph with a = b = 87.1 and c = 193.1 A and one molecule in the asymmetric unit . Crystals diffract to a maximum of 2.5 A resolution on a rotating-anode source . Screening for heavy-atom derivatives identified a lead compound that binds to a single site on the protein . Further screening is in progress.

Arch Biochem Biophys, 1998 Oct 1, 358(1), 141 - 8
Prolyl endopeptidase from Sphingomonas capsulata: isolation and characterization of the enzyme and nucleotide sequence of the gene; Kabashima T et al.; Prolyl endopeptidase (prolyl oligopeptidase, EC 3.4.21.26) was purified from Sphingomonas capsulata IFO 12533, and its gene was cloned and expressed in Escherichia coli . The recombinant enzyme was markedly inhibited by diisopropyl phosphofluoridate and hardly affected by SH reagents or metal chelators, similar to the native enzyme purified from S . capsulata . Nucleotide sequencing analysis revealed an open reading frame of 2169 bp, coding for a protein of 723 amino acids with a predicted molecular weight of 78,433 . The amino acid sequence was 39.6, 45.3, 38.9, and 38.3% homologous to Flavobacterium meningosepticum, Aeromonas hydrophila, porcine brain, and human T cell prolyl endopeptidase, respectively . A region near the C-terminus and the region containing the putative catalytic triad residues were highly conserved . The enzyme was crystallized by the hanging drop vapor diffusion method, using ammonium sulfate as a precipitant .

Carbohydr Res, 1998 Jul, 309(3), 261 - 8
Conversion of N-sulfated glucosamine to N-sulfated mannosamine in an unsaturated heparin disaccharide by non-enzymatic, base-catalyzed C-2 epimerization during enzymatic oligosaccharide preparation; Yamada S et al.; A novel disaccharide was isolated beside the predominant trisulfated disaccharide, delta HexA(2-O-sulfate)(alpha 1-4)GlcN(2-N-,6-O-disulfate) (delta HexA and GlcN represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and D-glucosamine, respectively) after treatment of porcine intestinal heparin with Flavobacterium heparinase . It accounted for 18% of total disaccharides . The structure was characterized by secondary ion mass spectrometry, enzymatic digestions, amino sugar analysis, and 500 MHz one- and two-dimensional 1H NMR spectroscopy as delta HexA(2-O-sulfate) (alpha 1-4)ManN(2-N-,6-O-disulfate), where ManN represents D-mannosamine . The C-2 epimerization from delta HexA(2-O-sulfate) (alpha 1-4)GlcN(2-N-,6-O-disulfate) to delta HexA(2-O-sulfate) (alpha 1-4)ManN(2-N-,6-O-disulfate) was also demonstrated to take place in vitro under very mild alkaline conditions . Hence, the latter compound is not a biosynthetic product, but is most likely an artifact generated by non-enzymatic, base-catalyzed C-2 epimerization during enzymatic preparation of heparin oligosaccharides . The present results warn that the formation of the C-2 epimerized compound has to be circumvented in the structural analysis of heparin/heparan sulfate.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Mar, 119(3), 539 - 47
New pathway of heparan sulphate degradation . Iduronate sulphatase and N-sulphoglucosamine 6-sulphatase act on the polymer chain prior to depolymerisation by a N-sulpho-glucosaminidase and glycuronidases in the mollusc Tagelus gibbus; Medeiros MG et al.; It has previously been shown that in the mollusc Anomalocardia brasiliana the desulphation of chondroitin sulphate precedes its depolymerisation by beta-glucuronidase and beta-N-acetylgalactosaminidase (Sousa Jr . et al . J . Biol . Chem . 1990;265:20150-20155) . This led us to investigate whether in molluscs, sulphatases also act on heparan sulphate before its depolymerisation by glycosidases . Radioactively labelled {35S}heparan sulphate was extensively degraded by enzyme extracts prepared from the mollusc Tagelus gibbus . Several enzymes acting in concert degrade the compound to inorganic sulphate, glucosamine N-sulphate, N-acetylglucosamine-6 sulphate and other oligosaccharide products . These results indicate the presence of iduronate sulphatase, N-sulphoglucosamine 6-sulphatase alpha-N-sulphoglucosaminidase, beta-glucuronidase and alpha-L-iduronidase . The di- and mono-saccharide composition of the oligosaccharides were analysed with the aid of heparitinase II from Flavobacterium heparinum . These analyses led to the characterisation of two sulphatases that act on the polymer chain removing sulphates from the C-2 position of iduronic acid residues and the C-6 position of the glucosamine moieties, respectively . The different enzymes were partially fractionated by ion exchange chromatography and molecular sieving . These results led to the proposition of a new pathway of degradation of heparan sulphate where sulphatases act directly on the polymer chain which is then depolymerised by several glycosidases.

J Biol Chem, 1998 Sep 4, 273(36), 22904 - 12
Heparinase II from Flavobacterium heparinum . Role of cysteine in enzymatic activity as probed by chemical modification and site- directed mutagenesis; Shriver Z et al.; Heparinase II (no EC number) is one of three lyases isolated from Flavobacterium heparinum that degrade heparin-like complex polysaccharides . Heparinase II is unique among the heparinases in that it has broad substrate requirements and possesses the ability to degrade both heparin and heparan sulfate-like regions of glycosaminoglycans . This study set out to investigate the role of cysteines in heparinase II activity . Through a series of chemical modification experiments, it was found that one of the three cysteines in heparinase II is surface-accessible and possesses unusual chemical reactivity toward cysteine-specific chemical modifying reagents . Substrate protection experiments suggest that this surface-accessible cysteine is proximate to the active site, since addition of substrate shields the cysteine from modifying reagents . The cysteine, present in an ionic environment, was mapped by radiolabeling with N-{3H}ethylmaleimide and identified as cysteine 348 . Site-directed mutagenesis of cysteine 348 to an alanine resulted in loss of activity toward heparin but not heparan sulfate, indicating that cysteine 348 is required for heparinase II activity toward heparin but is not essential for the breakdown of heparan sulfate . Furthermore, we show in this study that cysteine 164 and cysteine 189 are functionally unimportant for heparinase II.

J Appl Microbiol, 1998 Jun, 84(6), 950 - 8
A relatively small change in sodium chloride concentration has a strong effect on adhesion of ocular bacteria to contact lenses; Cowell BA et al.; Adhesion of bacteria to hydrogel lenses is thought to be an initial step of ocular colonization allowing evasion of normal host defences . The salt concentration of media is an important parameter controlling microbial adhesion . Salinity varies from 0.97% NaCl equivalents in the open eye to 0.89% in the closed eye state . In this study, the effect of sodium chloride in the concentration range of 0.8-1.0% (w/v) NaCl on adhesion of ocular bacteria to soft contact lenses was investigated using a static adhesion assay . Pseudomonas aeruginosa was found to adhere to lenses in significantly greater amounts than Serratia marcescens, Flavobacterium meningosepticum, Stenotrophomonas maltophilia and Staphylococcus intermedius . Increasing NaCl from 0.8% to 1.0% (w/v) increased adhesion of all bacteria tested . This adhesion was strong since the organisms could not be removed by washing in low ionic buffer . Adhesion of these organisms did not correlate with their cell surface properties as determined by bacterial adhesion to hydrocarbons (BATH) and retention on sepharose columns.

J Biol Chem, 1998 Aug 7, 273(32), 20205 - 12
Crystal structures of Flavobacterium glycosylasparaginase . An N-terminal nucleophile hydrolase activated by intramolecular proteolysis; Guo HC et al.; Glycosylasparaginase (GA) is a member of a novel family of N-terminal nucleophile hydrolases that catalytically use an N-terminal residue as both a polarizing base and a nucleophile . These enzymes are activated from a single chain precursor by intramolecular autoproteolysis to yield the N-terminal nucleophile . A deficiency of GA results in the human genetic disorder known as aspartylglycosaminuria . In this study, we report the crystal structure of recombinant GA from Flavobacterium meningosepticum . Similar to the human structure, the bacterial GA forms an alphabetabetaalpha sandwich . However, some significant differences are observed between the Flavobacterium and human structures . The active site of Flavobacterium glycosylasparaginase is in an open conformation when compared with the human structure . We also describe the structure of a mutant wherein the N-terminal nucleophile Thr152 is substituted by a cysteine . In the bacterial GA crystals, we observe a heterotetrameric structure similar to that found in the human structure, as well as that observed in solution for eukaryotic glycosylasparaginases . The results confirm the suitability of the bacterial enzyme as a model to study the consequences of mutations in aspartylglycosaminuria patients . They also suggest that further studies are necessary to understand the detail mechanism of this enzyme . The presence of the heterotetrameric structure in the crystals is significant because dimerization of precursors has been suggested in the human enzyme to be a prerequisite to trigger autoproteolysis.

Glycobiology, 1998 Sep, 8(9), 869 - 77
Determination of the structure of oligosaccharides prepared from acharan sulfate; Kim YS et al.; The fine structure of acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica , was examined . This glycosaminoglycan has a major disaccharide repeating unit of -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1--> (where GlcNpAc is N -acetylglucosamine, IdoAp is iduronic acid, and S is sulfate) making it structurally related to both heparin and heparan sulfate . Using heparin lyases prepared from Flavobacterium heparinum and a newly isolated heparinase from Bacteroides stercoris , the controlled enzymatic depolymerization of acharan sulfate was undertaken to prepare a mixture of oligosaccharides . Fractionation of this mixture of oligosaccharides by strong-anion-exchange high performance liquid chromatography afforded oligosaccharides that capillary electrophoresis established were sufficiently pure for structural characterization . Electrospray ionization mass spectrometry identified two series of oligosaccharides, one derived from acharan sulfate's major repeating unit and a second minor group of undersulfated oligosaccharides . Proton nuclear magnetic resonance spectroscopy established the structure of these two classes of oligosaccharides to be DeltaUAp2S(1-->{4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S (1-->}n4)- D-GlcNpAcalpha,beta (where n = 0,1,2,3 and DeltaUAp is 4-deoxy-alpha-L- threo -hex-4-enopyranosyluronic acid) and DeltaUAp(1-->{4)- alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1-->}m-D-GlcNpAcal pha,beta (where m = 1,2,3) . These results suggest the presence of minor sequence variants in acharan sulfate containing unsulfated iduronic acid having the structure -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp(1-->.

J Environ Sci Health B, 1998 Jul, 33(4), 347 - 67
Characterization of a phosphotriesterase from genetically-engineered Escherichia coli; Cheng YD et al.; A phosphotriesterase (PTE) capable of hydrolyzing organophosphate esters was purified from Escherichia coli strain DH-5 alpha carrying a cloned opd gene from Flavobacterium . The effects of pH, temperature and metal ion concentrations on enzyme stability and activity were investigated . Optimum conditions for PTE's catalytic activity were determined to be 35 degrees C and pH 8.5 . Protein-metal equilibrium binding experiments showed that PTE could accommodate two equivalents of Co2+ or Zn2+ ions . PTE protein was found to have higher affinity for Co2+ . In addition, Co2+ was found to possess the most positive effects in maintaining and restoring PTE's stability and catalytic activity when compared to other divalent metal ions . Assessment of the feasibility of PTE operation in a practical environment was performed in a system designed to mimic a continuously stirred tank reactor (CSTR) with different solution compositions in the flow reservoir . PTE was deactivated in 24 hours when the inflow solution contained 5% ethanol or 1 mM EDTA, while it retained one third of its initial activity in a deionized water stream . When the inflow solution contained 1 mM Co2+, PTE was found to retain activity throughout the 24-hour experiment.

Vet Microbiol, 1998 Apr 30, 62(1), 35 - 45
Characterization of four Flavobacterium columnare (Flexibacter columnaris) strains isolated from tropical fish; Decostere A et al.; Four Flavobacterium columnare strains (AJS 1-4) were isolated from black mollies (Poecilia sphenops) and platies (Xiphophorus maculatus), showing white spots on the back, head and skin ulcers . The isolates developed characteristic rhizoid yellow pigmented colonies on Shieh agar and typical growth in Shieh broth . They were Gram-negative, filamentous bacteria exhibiting flexing movements . When compared to F . columnare strains isolated from temperate fish, it was noted that the four strains originating from tropical aquarium fish are more capable of growing at higher temperatures, the opposite being true for the strains isolated from temperate fish . Biochemical characterization and agglutination tests proved that the isolated strains could be classified as F . columnare . Low minimal inhibitory concentration (MIC) values were found for chloramphenicol, erythromycin, furazolidone, kanamycin, lincomycin, nalidixic acid, oxytetracycline and streptomycin . MIC values were high for colistin, sulfamethoxazole and neomycin . Pathogenicity studies were performed on black mollies . When these animals were submersed in an infective solution of the F . columnare strains, a marked difference in virulence was noted among the four isolated strains, strain AJS 1 being the most virulent one and strain AJS 4 being of low virulence.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2691 - 6
Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization; Llobet-Brossa E et al.; The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides . A large fraction (up to 73%) of the DAPI (4',6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes . Nearly 45% of the total cells could be further identified as belonging to known phyla . Members of the Cytophaga-Flavobacterium cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.

Appl Environ Microbiol, 1998 Jul, 64(7), 2439 - 42
Cytoplasmic membrane lipoprotein LppC of Streptococcus equisimilis functions as an acid phosphatase; Malke H; The function of the streptococcal cytoplasmic membrane lipoprotein, LppC, was identified with isogenic Streptococcus equisimilis H46A and Escherichia coli JM109 strain pairs differing in whether they contained {H46A and JM109(pLPP2)} or lacked (H46A lppC::pLPP10 and JM109) the functional lppC gene for comparative phosphatase determinations under acidic conditions . lppC-directed acid phosphatase activity was demonstrated zymographically and by specific enzymatic activity assays, with whole cells or cell membrane preparations as enzyme sources . LppC acid phosphatase showed optimum activity at pH 5, and the enzyme activity was unaffected by Triton X-100, L-(+)-tartaric acid, or EDTA . Database searches revealed significant structural homology of LppC to the Streptococcus pyogenes LppA, Flavobacterium meningosepticum OplA, Helicobacter pylori HP1285, and Haemophilus influenzae Hel {e (P4)} proteins . These results suggest a possible function for these proteins and establish a novel function of streptococcal cell membrane lipoproteins.

Biol Chem, 1998 Apr-May, 379(4-5), 567 - 8
Cloning and characterization of the gene encoding M.FauI DNA methyltransferase; Degtyarev SK et al.; The enzymes of the FauI restriction-modification system from the Flavobacterium aquatile strain recognize the non-palindromic sequence 5'-CCCGC-3'/3'-GG-GCG-5' . We have cloned the gene encoding the DNA modifying component of this system and determined its nucleotide sequence . The deduced amino acid sequence contains ten conserved motifs characteristic for {cytosine-5} DNA methyltransferases . Part of the gene sequence that encodes the putative target recognizing domain of the M.FauI shows some homology with the downstream region, thus indicating that duplication of the DNA segment was probably involved in the gene evolution.

Cell Mol Biol (Noisy-le-grand), 1998 May, 44(3), 417 - 29
Structure of heparan sulfate: identification of variable and constant oligosaccharide domains in eight heparan sulfates of different origins; Dietrich CP et al.; The use of specific enzymes (heparinase and heparitinases from Flavobacterium heparinum, endoglucuronidase, alphaN-acetylglucosaminidase and beta-glucuronidase from the mollusc Anomalocardia brasiliana) and chemical methods (nitrous acid degradation, hydrazine N-deacetylation and borohydride reduction), led to the proposal of the total sequence of a heparan sulfate derived from bovine pancreas and partial sequences of heparan sulfates from different origins (bovine: lung, liver, brain; hog: liver, brain; rabbit liver; dog liver) . It was shown that all the heparan sulfates contain common structural features such as: a N-acetylated and a N-sulfated domain made of glucuronic acid-containing disaccharides and a more sulfated region made of iduronic acid-containing disaccharides . Separating the two domains a peculiar tetrasaccharide made of GlcNAc-(alpha1-4)-IdoUA-(alpha1-4)-GlcNS-(alpha1-4)-IdoUA was identified in all the heparan sulfates analyzed . It was also shown that the non-reducing ends of the heparan sulfates contain the monosaccharides glucosamine N-sulfate and/or glucosamine 2,6 disulfate.

J Clin Microbiol, 1998 Jun, 36(6), 1646 - 52
Diverse and related 16S rRNA-encoding DNA sequences in prostate tissues of men with chronic prostatitis; Riley DE et al.; Treatment of chronic prostatitis/chronic pelvic pain syndrome is often empirical because clinical culture methods fail to detect prostate-associated pathogens in >90% of patients . Previously, we tested a variety of specific-microorganism PCRs and began a DNA sequence study after we found that 77% of prostatitis patients were PCR positive for prokaryotic rRNA-encoding DNA sequences (rDNAs) despite negative cultures using optimal techniques . In the present study, 36 rDNA clones from 23 rDNA-positive patients were sequenced . This study represents more than twice the total rDNA sequence and more than twice the number of patients in the previous study . The increased number of patients and clones sequenced allowed enhanced phylogenetic analyses and refinements in our view of rDNA species inhabiting the prostate . A continuum of related rDNAs that might be arbitrarily described as two major groups of rDNAs and several minor groups was found . Sequences termed Pros A, identified in 8 (35%) of 23 rDNA-positive patients, grouped with Aeromonas spp . in phylogenetic studies . Sequences termed Pros B, identified in 17 (74%) of 23 rDNA-positive patients, were distinct from previously reported sequences, although all were >90% similar to known gram-negative bacteria . Of the nine patients for whom multiple rDNAs were sequenced, six had biopsy specimens containing rDNAs from more than one species . Four (17%) patients had rDNAs different from those of the Pros A and Pros B groups . Of these four, one patient had rDNA similar to that of Flavobacterium spp., another had rDNA similar to that of Pseudomonas testosteroni, and two patients had rDNAs <70% similar to known rDNAs . These findings suggest that the prostate can harbor bacteria undetectable by traditional approaches . Most of these diverse sequences are not reported in environments outside the prostate . The sequence similarities suggest adaptation of limited groups of bacteria to the microenvironment of the prostate . Further studies may elucidate the relationship of prostate-associated bacteria to chronic prostatitis/chronic pelvic pain syndrome.

Biosci Biotechnol Biochem, 1998 Apr, 62(4), 655 - 60
Optimization for beta-mannanase production of a psychrophilic bacterium, Flavobacterium sp; Zakaria MM et al.; We found that a psychrophilic bacterium, Flavobacterium sp., characterized in this study, has a beta-mannanase (EC 3.2.1.78) activity in the culture medium . The mannanase activity was the highest in the culture medium, containing 1.0% (w/v) guar gum (as a carbon source), 0.3% (NH4)2SO4 (as a nitrogen source), and 0.06% (w/v) yeast extract, of five-days cultivation at 4 degrees C . No mannanase activity was found in the medium containing a monosaccharide or a disaccharide as a carbon source, although the psychrophile could use them . The enzyme activity was found only when the bacterium was cultured in the medium containing a polysaccharide . The enzyme preparation showed a single activity band on a washed gel of SDS-PAGE . The optimal temperature for the enzyme activity was 35 degrees C . When the reaction was done at 10 degrees C, the enzyme showed 25% of the optimal activity . The beta-mannanase preparation efficiently hydrolyzed guar gum, locust bean gum, and glucomannan as well as beta-mannan.

J Biol Chem, 1998 Apr 24, 273(17), 10160 - 7
Heparinase II from Flavobacterium heparinum . Role of histidine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis; Shriver Z et al.; The three heparinases derived from Flavobacterium heparinum are powerful tools for studying heparin-like glycosaminoglycans in major biological processes, including angiogenesis and development . Heparinase II is unique among the three enzymes because it is able to catalytically cleave both heparin and heparan sulfate-like regions of heparin-like glycosaminoglycans . Toward understanding the catalytic mechanism of heparin-like glycosaminoglycan degradation by heparinase II, we set out to investigate the role of the histidines of heparinase II in catalysis . We observe concentration-dependent inactivation of heparinase II in the presence of the reversible histidine-modifying reagent diethylpyrocarbonate (DEPC) . With heparin as the substrate, the rate constant of inactivation was found to be 0.16 min-1 mM-1; with heparan sulfate as the substrate, the rate constant was determined to be 0.24 min-1 mM-1 . Heparinase II activity is restored following hydroxylamine treatment . This, along with other experiments, strongly suggests that the inactivation of heparinase II by DEPC is specific for histidine residues and that three histidines are modified by DEPC . Substrate protection experiments show that heparinase II preincubation with heparin followed by the addition of DEPC resulted in a loss of enzymatic activity toward heparan sulfate but not heparin . However, heparinase II preincubation with heparan sulfate was unable to protect heparinase II from DEPC inactivation for either of the substrates . Proteolytic mapping studies with Lys-C were consistent with the chemical modification experiments and identified histidines 238, 451, and 579 as being important for heparinase II activity . Further mapping studies identified histidine 451 as being essential for heparin degradation . Site-directed mutagenesis experiments on the 13 histidines of heparinase II corroborated the chemical modification and the peptide mapping studies, establishing the importance of histidines 238, 451 and 579 in heparinase II activity.

Clin Infect Dis, 1998 May, 26(5), 1169 - 76
In vitro antibiotic synergy against Flavobacterium meningosepticum: implications for therapeutic options; Di Pentima MC et al.; Flavobacterium meningosepticum is an unusual, highly resistant, gram-negative bacillus that is associated with neonatal meningitis and nursery outbreaks of meningitis . The optimal therapy for this infection is not known . We successfully treated three neonates with intravenous vancomycin and rifampin . We determined the in vitro activity of meropenem, ciprofloxacin, vancomycin, linezolid (PNU-100766), and rifampin, alone and in combination, against four isolates of F . meningosepticum from neonates with sepsis and meningitis . MICs were determined by tube dilution, and fractional inhibitory concentrations were calculated with use of the checkerboard microtiter dilution technique . Synergy was observed between rifampin and vancomycin against three isolates, while combinations of vancomycin, ciprofloxacin, and linezolid showed an additive effect against all isolates . These results support the clinical evidence that the combination of vancomycin and rifampin is an appropriate regimen for neonatal meningitis due to F . meningosepticum . The combination of meropenem and vancomycin was antagonistic . The clinical efficacy of combinations including ciprofloxacin, newer quinolones, or linezolid for treating F . meningosepticum meningitis deserves further study.

J Biol Chem, 1998 Apr 17, 273(16), 9688 - 94
Site-directed mutagenesis of essential residues involved in the mechanism of bacterial glycosylasparaginase; Liu Y et al.; Flavobacterium glycosylasparaginase was cloned in an Escherichia coli expression system . Site-directed mutagenesis was performed at residues suggested to be important in the catalytic mechanism based on the crystal structure of the human enzyme and other biochemical studies . In vitro autoproteolysis allowed the mutant enzymes to be activated, including those that were slow to self-cleave . Based on the activity of the mutant enzymes, six catalytically essential amino acids were identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183 . Kinetic analysis of each mutant further defined the function of these residues in substrate specificity and reaction rate . Mutagenesis of the N-terminal nucleophile residue Thr-152 confirmed the key function of its side-chain hydroxyl group . Partial activities of mutants T152S/C were in agreement with the general mechanism of N-terminal nucleophile (Ntn)-amidohydrolases . The side-chain hydroxyl of Thr-170 contributes to the reaction rate based on studies of mutants T170S/C/A . Residues Asp-183 and Arg-180 were found to H-bond, respectively, with the charged alpha-amino and alpha-carboxyl group of the substrate (Asn-GlcNAc) . Mutants R180Q/L and D183E/N had greatly decreased substrate affinity and reduced reaction rates . Kinetic studies also showed that Trp-11 is involved in regulation of the enzyme reaction rate, contradictory to a previous suggestion that this residue is involved in substrate binding . Asp-66 is a new residue found to be important in enzyme activity . The overall active site structure involving these catalytic residues resembles the glutaminase domain of glucosamine 6-phosphate synthase, another member of the Ntn-amidohydrolase family of enzymes.

J Comp Pathol, 1998 Apr, 118(3), 245 - 56
Particle clearance from the gills of rainbow trout (Oncorhynchus mykiss); Derksen JA et al.; The rate of particle clearance from the gills was assessed in healthy rainbow trout (Oncorhynchus mykiss), challenged with the formalin-killed bacterium Flavobacterium branchiophilum, as well as in fish with altered ventilation levels produced by varying the concentrations of dissolved oxygen (DO) in the water . The clearance of F . branchiophilum from the gills was quantified by means of an enzyme-linked immunosorbent assay . Fish held under normoxic conditions (DO = 9.5 mg/l) showed an initial rapid reduction in bacterial antigen, with 50% of the bacteria being cleared in the first 12 h after exposure, followed by slower clearance for the remaining bacteria; total elimination was achieved by 40 h . Fish with reduced ventilation rates (hyperoxia; DO = 25 mg/l) and elevated ventilation rates (hypoxia; DO = 4.5 mg/l) had significantly impaired particle clearance (r < 0.05), achieving only 60 and 20% reduction, respectively, at 72 h after challenge.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4182 - 7
Direct evidence for a predominantly exolytic processive mechanism for depolymerization of heparin-like glycosaminoglycans by heparinase I; Ernst S et al.; Heparinase I from Flavobacterium heparinum has important uses for elucidating the complex sequence heterogeneity of heparin-like glycosaminoglycans (HLGAGs) . Understanding the biological function of HLGAGs has been impaired by the limited methods for analysis of pure or mixed oligosaccharide fragments . Here, we use methodologies involving MS and capillary electrophoresis to investigate the sequence of events during heparinase I depolymerization of HLGAGs . In an initial step, heparinase I preferentially cleaves exolytically at the nonreducing terminal linkage of the HLGAG chain, although it also cleaves internal linkages at a detectable rate . In a second step, heparinase I has a strong preference for cleaving the same substrate molecule processively, i.e., to cleave the next site toward the reducing end of the HLGAG chain . Computer simulation showed that the experimental results presented here from analysis of oligosaccharide degradation were consistent with literature data for degradation of polymeric HLGAG by heparinase I . This study presents direct evidence for a predominantly exolytic and processive mechanism of depolymerization of HLGAG by heparinase I.

Biochem J, 1998 May 15, 332 ( Pt 1), 145 - 52
Characterization and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B beta-lactamase showing a broad substrate profile; Rossolini GM et al.; The metallo-beta-lactamase produced by Chryseobacterium (formerly Flavobacterium) meningosepticum, which is the flavobacterial species of greatest clinical relevance, was purified and characterized . The enzyme, named BlaB, contains a polypeptide with an apparent Mr of 26000, and has a pI of 8.5 . It hydrolyses penicillins, cephalosporins (including cefoxitin), carbapenems and 6-beta-iodopenicillanate, a mechanism-based inactivator of active-site serine beta-lactamases . The enzyme was inhibited by EDTA, 1-10 phenanthroline and pyridine-2,6-dicarboxylic acid, with different inactivation parameters for each chelating agent . The C . meningosepticum blaB gene was cloned and sequenced . According to the G+C content and codon usage, the blaB gene appeared to be endogenous to the species . The BlaB enzyme showed significant sequence similarity to other class B beta-lactamases, being overall more similar to members of subclass B1, which includes the metallo-enzymes of Bacillus cereus (Bc-II) and Bacteroides fragilis (CcrA) and the IMP-1 enzyme found in various microbial species, and more distantly related to the metallo-beta-lactamases of Aeromonas spp . (CphA, CphA2 and ImiS) and of Stenotrophomonas maltophilia (L1).

Biochim Biophys Acta, 1998 Mar 6, 1391(1), 79 - 91
A novel sulfonic-acid analogue of ceramide is the major extractable lipid of the gram-negative marine bacterium Cyclobacterium marinus WH; Batrakov SG et al.; The extractable lipids of the gram-negative, sea-water bacterium Cyclobacterium marinus strain WH contain about 94% of polar components which consist of two phospholipids, phosphatidylethanolamine (29% of the total lipids) and phosphatidylcholine (7%), and two phosphorus-free lipids . One of the latter has been shown to be a novel sulfonic-acid analogue of ceramide, 2-D-(2'-D-hydroxy-13'-methyltetradecanoyl) amino-3-D-hydroxy-15-methylhexadec-4 (E)-en-1-sulfonic acid (48%), and other is a lipodipeptide, N-{3-d-(13'-methyltetradecanoyloxy)-15-methylhexadecanoyl} glycyl-L-serine (11%), which has so far been found only in a Flavobacterium sp . strain . The dominant fatty acid residues of the phospholipids are iso-15:0, n-16:0, 16:1 and 18:1, the acyl residues linked to the sn-1 carbon of the glycerol moiety being somewhat more saturated as compared with those located at the sn-2 position . A new procedure for determination of the absolute configuration of 2- and 3-hydroxy fatty acids is briefly described .

Int J Syst Bacteriol, 1998 Jan, 48 Pt 1, 223 - 35
Polaribacter gen . nov., with three new species, P . irgensii sp . nov., P . franzmannii sp . nov . and P . filamentus sp . nov., gas vacuolate polar marine bacteria of the Cytophaga-Flavobacterium-Bacteroides group and reclassification of 'Flectobacillus glomeratus' as Polaribacter glomeratus comb . nov; Gosink JJ et al.; Several psychrophilic, gas vacuolate strains of the Cytophage-Flavobacterium-Bacteroides (CFB) phylogenetic group were isolated from sea ice and water from the Arctic and the Antarctic . The closest taxonomically defined species by 16S rRNA sequence analysis is 'Flectobacillus glomeratus' . However, 'Flc . glomeratus' is phylogenetically distant from the Flectobacillus type species, Flc . major . On the basis of phenotypic, genotypic and 16S rRNA sequence analyses we propose a new genus, Polaribacter, with three new species, Polaribacter irgensii strain 23-P (ATCC 700398), Polaribacter franzmannii strain 301 (ATCC 700399) and Polaribacter filamentus strain 215 (ATCC 700397) . P . filamentus is the type species of the genus . None of these species exhibits a cosmopolitan or bipolar distribution . This is the first taxonomic description of gas vacuolate bacteria in the CFB group . Additionally, we propose that 'Flc . glomeratus' be reclassified to the genus Polaribacter as P . glomeratus, comb . nov.

Protein Sci, 1998 Mar, 7(3), 774 - 81
Crystal structure of glycosylasparaginase from Flavobacterium meningosepticum; Xuan J et al.; The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 angstroms resolution . This enzyme is a glycoamidase that cleaves the link between the asparagine and the N-acetylglucosamine of N-linked oligosaccharides and plays a major role in the degradation of glycoproteins . The three-dimensional structure of the bacterial enzyme is very similar to that of the human enzyme, although it lacks the four disulfide bridges found in the human enzyme . The main difference is the absence of a small random coil domain at the end of the alpha-chain that forms part of the substrate binding cleft and that has a role in the stabilization of the tetramer of the human enzyme . The bacterial glycosylasparaginase is observed as an (alphabeta)2-tetramer in the crystal, despite being a dimer in solution . The study of the structure of the bacterial enzyme allows further evaluation of the effects of disease-causing mutations in the human enzyme and confirms the suitability of the bacterial enzyme as a model for functional analysis.

J Biochem (Tokyo), 1998 Feb, 123(2), 283 - 8
Characterization of heparinase from an oral bacterium Prevotella heparinolytica; Watanabe M et al.; Heparinase was purified to homogeneity from the cell extract of an oral bacterium, Prevotella heparinolytica, by a combination of anion exchange chromatography, gel filtration chromatography, and hydroxyapatite chromatography . Properties of the purified P . heparinolytica heparinase (P . heparinase) were investigated . The enzyme exhibited a maximum activity in 50 mM Tris-HCl buffer, pH 7.5-8.0, containing 75 mM sodium acetate, 0.1 M NaCl, and 1 mM CaCl2 . Optimum conditions for the maximum activity of P . heparinase were similar to those of the heparinase from Flavobacterium heparinum (F . heparinase) . The two enzymes also yielded similar digestion profiles of various glycosaminoglycans and heparin tetrasaccharides, suggesting that they have a similar substrate specificity . Kinetic study of the P . heparinase reaction using porcine intestinal heparin as substrate gave a Km value of 3.8 x 10(-5) M and a Vmax value of 11.4 micromol/min x mg protein . The Michaelis constant of P . heparinase was slightly larger than but not significantly different from that of F . heparinase . The amino acid composition of P . heparinase was also similar to that of F . heparinase, but its N-terminal sequence of 20 amino acid residues was different and hitherto unreported . These results together indicate that these heparinases are different proteins with closely similar enzymatic properties . Since F . heparinum produces not only heparinase but also heparitinase II, which has a broad substrate specificity, F . heparinase may be contaminated with this enzyme . In contrast, P . heparinolytica does not produce heparitinase II, and P . heparinase should prove a useful tool for degrading heparin without the risk of contamination with heparitinase II.

Folia Microbiol (Praha), 1997, 42(4), 337 - 43
Studies on mercury-detoxicating enzymes from a broad-spectrum mercury-resistant strain of Flavobacterium rigense; Gachhui R et al.; Flavobacterium rigense strain PR2, a broad-spectrum mercury-resistant bacterium abundantly present in soil exhibited multiple metal resistance properties . Mercury resistance was due to the sequential action of two mercury-detoxicating enzymes, organomercurial lyase and mercuric reductase . The levels of these enzyme activities were determined using different mercury compounds as inducers and substrates . Mercuric reductase was partially purified from the bacterium and the physicochemical properties of the enzyme were studied . The effect of several enzyme inhibitors and heavy metal ions on the enzyme activity was also studied.

Biochim Biophys Acta, 1998 Mar 4, 1396(1), 39 - 46
Sequence analysis of the Porphyromonas gingivalis dipeptidyl peptidase IV gene; Kiyama M et al.; We previously constructed a Porphyromonas gingivalis genomic library and isolated the 2.9 kb EcoRV fragment which specified glycylprolyl dipeptidyl aminopeptidase (GPase) . Nucleotide sequencing of this fragment identified the single 2169 bp open reading frame which coded for a 723 amino acid protein . The amino acid sequencing of the NH2-terminal domain of the native and recombinant mature enzymes suggested that the protease possessed a 16 amino acid residue signal peptide . The calculated mass of the precursor and mature proteases were 82,018 and 80,235 daltons, respectively . The homology search of this enzyme in registered protein sequences revealed that this enzyme was homologous to dipeptidyl peptidase (DPP) IV from the Flavobacterium meningosepticum and that from eukaryotic cells, including the human, mouse, and rat . Three amino acid residues, Ser-593, Asp-668, and His-700, were identified as a putative catalytic triad, a common feature of eukaryotic serine proteases . In addition, this enzyme showed a broad proteolytic spectrum toward synthetic substrates capable of splitting not only Gly-Pro-derivative but also Ala-Pro, Lys-Pro, and Phe-Pro-derivatives . Therefore, we conclude that this enzyme belongs to DPP IV rather than GPase.

Eur J Biochem, 1998 Feb 1, 251(3), 899 - 906
The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase M.FokI is a tandem enzyme of two independent domains with very different kinetic properties; Leismann O et al.; The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase, M.FokI, modifies both adenine residues within its asymmetric recognition sequence 5'-GGATG/CATCC-3' . It is a fusion protein comprising two independent enzymes . We have cloned, overexpressed and purified full-length M.FokI as well as both individual domains and analyzed their kinetics of DNA methylation using unmethylated and hemimethylated oligodeoxynucleotide substrates . Our data show that both domains of M.FokI methylate DNA independently of each other but cooperate in DNA binding . In agreement to former studies, the N-terminal domain of M.FokI modifies the upper strand of the recognition sequence (GGATG) . It strongly prefers hemimethylated (5'-GGATG/CmATCC-3'; mA = N6-methyladenosine) over unmethylated substrates . In contrast, the C-terminal domain prefers unmethylated DNA substrates . Surprisingly, in addition to methylating the lower strand of the recognition sequence (CATCC), M.FokI-(335-647) also modifies the upper strand (GGATG), albeit with a lower activity . In addition, methylation was detected at CACCC sites, but not at sites in which a central AT dinucleotide is flanked by at least one A x T or T x A base pair . These results suggests that M.FokI-(335-647) either has a very degenerate specificity for (G/C)AT(G/C) and sites similar to CATCC or GGATG or, alternatively, that it has a dual specificity for CATCC and GGATG.

Appl Environ Microbiol, 1998 Feb, 64(2), 535 - 42
Characterization of marine temperate phage-host systems isolated from Mamala Bay, Oahu, Hawaii; Jiang SC et al.; To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts . We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii . Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis and Flavobacterium sp . All of the phage isolates were tailed phages and contained double-stranded DNA . Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae and Podoviridae . The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm . The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb . The members of the Siphoviridae, T-phi HSIC, and T-phi D0, and the member of the Myoviridae, T-phi D1B, were found to form lysogenic associations with their bacterial hosts, which were isolated from the same water samples . Hybridization of phage T-phi HSIC probe with lysogenic host genomic DNA was observed in dot blot hybridization experiments, indicating that prophage T-phi HSIC was integrated within the host genome . These phage-host systems are available for use in studies of marine lysogeny and transduction.

J Biol Chem, 1998 Jan 2, 273(1), 248 - 55
Heparinase I from Flavobacterium heparinum . Role of positive charge in enzymatic activity; Godavarti R et al.; Heparinases are bacterial enzymes that are powerful tools to study the physiological roles of heparin-like complex polysaccharides . In addition, heparinases have significant therapeutic applications . We had proposed earlier that cysteine 135 and histidine 203 together form the catalytic domain in heparinase I . We had also identified a heparin binding domain in heparinase I containing two positively charged clusters HB-1 and HB-2 in a primary heparin binding site and other positively charged residues in the vicinity of cysteine 135 . In this study, through systematic site-directed mutagenesis studies, we show that the alteration of the positive charge of the HB-1 region has a pronounced effect on heparinase I activity . More specifically, site-directed mutagenesis of K199A (contained in HB-1) results in a 15-fold reduction in catalytic activity, whereas a K198A mutation (also in HB-1) results in only a 2- to 3-fold reduction in heparinase I activity . A K132A mutation, in close proximity to cysteine 135, also resulted in reduced (8-fold) activity . Heparin affinity chromatography experiments indicated moderately lowered binding affinities for the K132A, K198A, and the K199A mutant enzymes . The above results, taken together with our previous observations, lead us to propose that the positively charged heparin binding domain provides the necessary microenvironment for the catalytic domain of heparinase I . The dominant effect of lysine 199 suggests an additional, more direct, role in catalysis for this residue.

J Biol Chem, 1998 Jan 23, 273(4), 1863 - 71
A major common trisulfated hexasaccharide core sequence, hexuronic acid(2-sulfate)-glucosamine(N-sulfate)-iduronic acid-N-acetylglucosamine-glucuronic acid-glucosamine(N-sulfate), isolated from the low sulfated irregular region of porcine intestinal heparin; Yamada S et al.; The major structure of the low sulfated irregular region of porcine intestinal heparin was investigated by characterizing the hexasaccharide fraction prepared by extensive digestion of the highly sulfated region with Flavobacterium heparinase and subsequent size fractionation by gel chromatography . Structures of a tetrasaccharide, a pentasaccharide, and eight hexasaccharide components in this fraction, which accounted for approximately 19% (w/w) of the starting heparin representing the major oligosaccharide fraction derived from the irregular region, were determined by chemical and enzymatic analyses as well as 1H NMR spectroscopy . Five compounds including one penta- and four hexasaccharides had hitherto unreported structures . The structure of the pentasaccharide with a glucuronic acid at the reducing terminus was assumed to be derived from the reducing terminus of a heparin glycosaminoglycan chain and may represent the reducing terminus exposed by a tissue endo-beta-glucuronidase involved in the intracellular post-synthetic fragmentation of macromolecular heparin . Eight out of the 10 isolated oligosaccharides shared the trisaccharide sequence, -4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-, and its reverse sequence, -4GlcA beta 1-4GlcNAc alpha 1-4IdceA alpha 1-, was not found . The latter has not been reported to date for heparin/heparan sulfate, indicating the substrate specificity of the D-glucuronyl C-5 epimerase . Furthermore, seven hexasaccharides shared the common trisulfated hexasaccharide core sequence delta HexA(2-sulfate)alpha 1-4GlcN(N-sulfate)alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcN(N-sulfate) which contained the above trisaccharide sequence (delta HexA, IdceA, GlcN, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, D-glucosamine, and D-glucuronic acid, respectively) and additional sulfate groups . The specificity of the heparinase used for preparation of the oligosaccharides indicates the occurrence of the common pentasulfated octasaccharide core sequence, -4GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-4GlcN(N-sulfate) alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4 GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-, where the central hexasaccharide is flanked by GlcN(N-sulfate) and HexA(2-sulfate) on the nonreducing and reducing sides, respectively . The revealed common sequence constituted a low sulfated trisaccharide representing the irregular region sandwiched by highly sulfated regions and should reflect the control mechanism of heparin biosynthesis.

Microbiology, 1997 Dec, 143 ( Pt 12), 3693 - 701
Acetate acts as a protonophore and differentially affects bead movement and cell migration of the gliding bacterium Cytophaga johnsonae (Flavobacterium johnsoniae); Dzink-Fox JL et al.; Cells of Cytophaga johnsonae (now Flavobacterium johnsoniae) are able to translocate on solid surfaces but are unable to swim in liquid media . Organelles that may be involved in this gliding motility have not been detected, and the mechanism(s) responsible remains unknown . The movement of latex beads attached to the cell surface is considered by some to be a manifestation of the gliding machinery . In this study, acetate (in nutrient-level quantity, 45 mM) was found to inhibit bead movement on cell surfaces, whilst formation and movement of groups of cells (rafts) and typical colony spread were not affected; generation time (in liquid culture) was only slightly increased . Since acetate is a weak acid and is recognized as a protonophore, various electron-transport-associated features were assessed in an effort to understand the differential effects of acetate on bead movement and cell motility . Selected protonophores and electron transport inhibitors were tested to compare their effects on cell translocation and metabolic activities with those of acetate . Although O2 consumption was not significantly affected in the presence of acetate and the protonmotive force decreased only minimally, ATP levels were markedly decreased . Arsenate and cyanide were also shown to inhibit bead movement but did not inhibit either movement of rafts of cells or colony spreading . Cyanide lowered O2 consumption, while arsenate did not; both compounds effected substantial decreases in cellular ATP content, but little or no decrease in protonmotive force . The inhibitory effects of these compounds on bead movement over cell surfaces contrasted with the continued ability of cells to form rafts, to glide and to form spreading colonies and led to the conclusion that bead movement is not a complete correlate of the gliding machinery of C . johnsonae . In addition, it seems likely that bead movement is more affected by the level of cellular ATP than it is by the protonmotive force, which has been assumed to provide the energy (derived from the transmembrane gradients) for the gliding machinery.

Antimicrob Agents Chemother, 1997 Dec, 41(12), 2738 - 41
Reappraisal of the antimicrobial susceptibilities of Chryseobacterium and Flavobacterium species and methods for reliable susceptibility testing; Fraser SL et al.; Several Flavobacterium species, comprising a heterogeneous group of gram-negative bacilli that are capable of causing opportunistic infections in humans, have recently been reclassified as Chryseobacterium or Myroides species . Intrinsically resistant to a number of antibiotics, these organisms have been reported to be susceptible to vancomycin and certain other drugs that are normally active against gram-positive bacteria . By using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution procedure, 58 clinical isolates of former flavobacteria (36 Chryseobacterium meningosepticum isolates, 11 C . indologenes isolates, 3 C . gleum isolates, 4 unspeciated former members of Flavobacterium group IIb, and 4 Myroides odoratum isolates) were tested with 23 antibiotics, including conventional and investigational agents . In addition, the broth microdilution results were compared to those generated by agar dilution, E-test, and disk diffusion for vancomycin and piperacillin-tazobactam . Compared to the NCCLS microdilution results, there were 7.1 and 17.9% very major errors with piperacillin-tazobactam by agar dilution and E-test, respectively . In addition, there were from 12.1 to 48.3% minor errors with both procedures with vancomycin and piperacillin-tazobactam . The very major and minor error rates were unacceptably high with disk testing of piperacillin-tazobactam; the use of enterococcal vancomycin disk breakpoints (zone diameter of > or =17 mm = susceptible) resulted in >20% minor errors but only one very major error . All of the isolates were susceptible to minocycline; over 90% were susceptible to sparfloxacin, levofloxacin, and clinafloxacin; and 88% were susceptible to rifampin . None was susceptible to vancomycin . When Chryseobacterium or Myroides species are isolated from serious infections, susceptibility testing by broth microdilution should be performed and therapy should be guided by those results.

Parassitologia, 1997 Mar, 39(1), 71 - 5
Phylogenetically distant intracellular symbionts in termites; Bandi C et al.; Cockroaches are known to harbour intracellular bacteria in specialised cells (mycetocytes, or bacteriocytes) of the fat body . In termites, mycetocyte bacteria have been observed only in Mastotermes darwiniensis . These symbionts are thought to have originated from a bacterium that infected an ancestor common to cockroaches and termites . Thus, loss of the infection should have occurred during evolution in all termite lineages, with the exception of that leading to M . darwiniensis . One might suspect that traces of the ancient infection may be present in some termites, in the form of non-mycetocyte intracellular bacteria (e.g . a small number of bacteria within normal cells) . Indeed, circumstantial evidence for the presence of intracellular bacteria in two termite species has been reported . However, no data are available on the actual distribution of these bacteria in termites, or on their relationships with the mycetocyte bacteria of cockroaches and M . darwiniensis . In this paper we report results indicating that non-mycetocyte intracellular bacteria are widespread in termites . These results were obtained by electron microscopy on representatives of nine termite species . In addition, sequence analysis of the 16S rRNA genes indicated that the non-mycetocyte bacteria of termites belong to the wolbachia group of the alpha-2 subclass of the proteobacteria . These latter bacteria are not related to the mycetocyte bacteria of cockroaches and M . darwiniensis, which belong to the blattabacterium group of the flavobacteria-bacteroides . PCR analyses with primers specific for wolbachia or blattabacterium provided further support for the identification of the observed non-mycetocyte bacteria as members of the wolbachia group.

Appl Environ Microbiol, 1997 Dec, 63(12), 4833 - 8
Mutational analysis of pcpA and its role in pentachlorophenol degradation by Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723; Chanama S et al.; Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723 degrades pentachlorophenol (PCP) through a catabolic pathway encoded by multiple genes . One gene required for PCP degradation is pcpA, which encodes information for a 30-kDa polypeptide, PcpA, found in the periplasm of the bacterium . The biological role of PcpA has remained unknown . We disrupted pcpA by replacing it with a defective copy through homologous recombination . The pcpA recombinant, mutant strains accumulated 2,6-dichlorohydroquinone (2,6-DiCH) as a metabolite of PCP . This work confirms that pcpA is essential for degradation of PCP by S . chlorophenolica ATCC 39723 and suggests that it encodes a protein involved in hydrolytic dehalogenation of 2,6-DiCH, an already established primary metabolite of the PCP catabolic pathway.

Biosci Biotechnol Biochem, 1997 Oct, 61(10), 1754 - 6
Prolyl endopeptidase inhibitors derived from actinomycetes; Kimura K et al.; Four prolyl endopeptidase inhibitors isolated from actinomycetes, named propeptin, SNA-8073-B, staurosporine, and enduracidin were classified into 3 groups on the basis of their inhibition potency against prolyl endopeptidase from a bacterium (Flavobacterium) and a mammal (human placenta) . Staurosporine inhibited the enzyme from Flavobacterium more strongly than that from human placenta . Enduracidin inhibited the enzyme from human placenta more strongly than that from Flavobacterium . Propeptin and SNA-8073-B, both new compounds, inhibited the enzymes from both origins to the same extent.

J Am Anim Hosp Assoc, 1997 Nov-Dec, 33(6), 509 - 12
Flavobacterium breve meningitis in a dog; Haburjak JJ et al.; An unusual, gram-negative rod was isolated in significant numbers (4+) from the cerebrospinal fluid (CSF) of a dog . This isolate, Flavobacterium breve, has not been identified previously as a pathogen in the dog . The case and the characteristics of the organism are described.

Biol Pharm Bull, 1997 Oct, 20(10), 1047 - 50
Cloning and expression of the cDNA encoding prolyl oligopeptidase (prolyl endopeptidase) from bovine brain; Yoshimoto T et al.; Prolyl oligopeptidase (EC 3.4.21.26, prolyl endopeptidase) cDNA from bovine brain was cloned by PCR, and the amplified fragment was used as a probe to screen the cDNA library from bovine brain . The obtained clone contained a 2.7 kb DNA fragment with an open reading frame of 2130 nucleotides, and encoded a protein of 710 amino acids with a deduced molecular weight of 80640 Da . The deduced amino acid sequence is 95, 94, 51 and 48% homologous to those of human T-cell, porcine brain, Aeromonas hydrophila, and Flavobacterium meningosepticum prolyl endopeptidases, respectively . The bovine brain prolyl endopeptidase-encoding cDNA was expressed using an expression vector bearing a tac promoter, with an approximate yield of 20 micrograms/ml of cell culture.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 12139 - 44
Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA; Agarwal S et al.; The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known . A large number of nonmotile mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) have been previously isolated, and genetic techniques to analyze these mutants have recently been developed . We complemented a nonmotile mutant of F . johnsoniae (UW102-09) with a library of wild-type DNA by using the shuttle cosmid pCP17 . The complementing plasmid (pCP100) contained an insert of 13 kbp, and restored motility to 4 of 61 independently isolated nonmotile mutants . A 1.3-kbp fragment that encompassed a single ORF, gldA, complemented all four mutants . Disruption of the chromosomal copy of gldA in wild-type F . johnsoniae UW101 eliminated gliding motility . The predicted protein produced by gldA has strong sequence similarity to ATP binding cassette transport proteins.

J Biol Chem, 1997 Oct 24, 272(43), 27058 - 64
Detailed studies on substrate structure requirements of glycoamidases A and F; Fan JQ et al.; Glycoamidases (peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side chain . The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A) . To study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB) . The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH2 and COOH termini . Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F . GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides . Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity . Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase F on this substrate is minimal . While glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer . The Km and Vmax values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 mM and 0.66 micromol/min/mg, respectively . A natural glycodipeptide, Man9-GlcNAc2-Asn-Phe, was also completely hydrolyzed by glycoamidase A.

Klin Monatsbl Augenheilkd, 1997 Jun, 210(6), 388 - 91
{Post-traumatic endophthalmitis with multiple water pathogens}; Janknecht P et al.; HISTORY AND SIGNS: A 37-y-old worker for a recycling company suffered from a corneal perforation by a wire which had been in contact with dirt and moisture . THERAPY AND OUTCOME: Initially, the typical signs of an endophthalmitis were missing so that we only irrigated and instilled antibiotics into the anterior chamber . However, later on two pars-plana vitrectomies became necessary . At each operation a bacteriological examination was performed and we found four different species-Pseudomonas aeruginosa . Klebsiella oxytoca, Aeromonas caviae, Flavobacterium odoratum . CONCLUSION: The latter two species are very rare in ophthalmology . This fact and some peculiarities in the clinical course of the patient's disease make him an unusual case . After unsuccessful irrigation of the anterior chamber vitrectomy ought to be performed immediately.

Int J Syst Bacteriol, 1997 Jul, 47(3), 670 - 7
Psychroserpens burtonensis gen . nov., sp . nov., and Gelidibacter algens gen . nov., sp . nov., psychrophilic bacteria isolated from antarctic lacustrine and sea ice habitats; Bowman JP et al.; Psychrophilic, yellow-pigmented, seawater-requiring bacteria isolated from the pycnocline of meromictic Burton Lake and from sea ice cores obtained in the Vestfold Hills (68 degrees S, 78 degrees E) in eastern Antarctica were characterized . Phenotypic analysis showed that the strains isolated formed two distinct taxa . The first taxon included nonmotile, nutritionally fastidious strains that were isolated from the pycnocline of Burton Lake . The cells of these strains were morphologically variant, ranging from vibrioid to ring shaped to coiled and filamentous; in addition, the strains were unable to metabolise carbohydrates or polysaccharides and had DNA G + C contents of 27 to 29 mol% . The strains of the second taxon, which were isolated from sea ice cores and from ice aigal biomass, were saccharolytic, exhibited rapid gliding motility, were rodlike to filamentous, and had DNA G + C contents of 36 to 38 mol% . A 16S ribosomal DNA (rDNA) sequence analysis revealed that the two Antarctic taxa formed related but distinct lineages within the {Flexibacter} maritimus rRNA branch of the family Flavobacteriacrae . The levels of 16S rDNA sequence similarity between the taxa were 90.5 to 91.3%, while the levels of similarity to other members of the {F.} maritimus rRNA branch were 85 to 90% . The whole-cell lipid profiles of the Antarctic strains were mainly comprised of branched and unbranched monounsaturated C15 to C17 fatty acids . The presence of significant levels of the lipids a 15:1 omega 10c and a17:1 omega 7c appeared to be useful biomarkers for the new Antarctic taxa and for differentiating these organisms from other members of the family Flavobacteriaceae . On the basis of polyphasic taxonomic data we propose that the new taxa are novel bacterial species designated Psychroserpens burtonensis gen . nov., sp . nov . (type strain, ACAM 188) and Gelidibacter algens gen . nov., sp . nov . (type strain, ACAM 536).

Appl Environ Microbiol, 1997 Jul, 63(7), 2957 - 60
Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences; Hiorns WD et al.; Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA . Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%) . Additionally, a number of the sequences were similar to those of the order Verrucomicrobiales . However, few of the sequence types are closely related to those of characterized species . The relative contributions of the groups of sequences differed among the lakes, suggesting that bacterial population structure varies and that it may be possible to relate aquatic bacterial community structure to water chemistry.

Antimicrob Agents Chemother, 1997 Jun, 41(6), 1301 - 6
Antimicrobial susceptibility of flavobacteria as determined by agar dilution and disk diffusion methods; Chang JC et al.; A total of 106 clinical isolates of flavobacteria, including 41 isolates of Flavobacterium meningosepticum, 59 of Flavobacterium indologenes, and 6 of Flavobacterium odoratum were collected from January 1992 to December 1995 from patients in Taiwan . The in vitro activities of antimicrobial agents were determined concomitantly by the standard agar dilution and disk diffusion methods . More than 90% of the flavobacterial isolates were resistant to cephalothin, cefotaxime, ceftriaxone, moxalactam, aztreonam, imipenem, aminoglycosides, erythromycin, and glycopeptides . The majority of F . meningosepticum isolates were susceptible to piperacillin and to minocycline but resistant to ceftazidime, with MICs at which 90% of the isolates are inhibited being 8, 4, and > 128 microg/ml, respectively . Approximately half of the F . indologenes isolates were susceptible to piperacillin, cefoperazone, ceftazidime, and minocycline, with MICs at which 50% of the isolates are inhibited being 4, 16, 8, and 4 microg/ml, respectively . The majority of F . odoratum isolates were resistant to all the antimicrobial agents tested except minocycline, to which five of six isolates were susceptible . With least-squares regression analysis and error rate-bounded analysis methods, the following resistant and susceptible zone diameter breakpoints were established: < or = 12 and > or = 17 mm, respectively, for piperacillin against F . meningosepticum and F . indologenes; < or = 13 and > or = 18 mm, respectively, for ceftazidime against F . meningosepticum and F . indologenes, < or = 17 and > or = 21 mm, respectively, for ofloxacin against F . indologenes; < or = 16 and > or = 20 mm, respectively, for ciprofloxacin against F . meningosepticum . Valid breakpoints for the disk diffusion method could not be established for cefoperazone and ofloxacin against F . meningosepticum and for minocycline against F . meningosepticum and F . indologenes due to a poor correlation coefficient for the regression line or for cefoperazone and ciprofloxacin against F . indologenes due to the presence of remarkable error rates.

Mol Gen Genet, 1997 May 20, 254(5), 469 - 78
An isoamylase with neutral pH optimum from a Flavobacterium species: cloning, characterization and expression of the iam gene; Krohn BM et al.; The gene encoding an isoamylase with neutral pH optimum (iam) from a Flavobacterium species was cloned using a PCR probe generated from highly conserved regions of amylolytic enzymes . Active isoamylase was expressed from a 4.9-kb Pst I fragment in Escherichia coli, and was detected in the extracellular medium by a plate assay . The iam nucleotide sequence has an open reading frame of 2334 nucleotides (778 amino acids) with a GC content of 69% . Sequence analysis suggests that transcriptional control of the Flavobacterium sp . iam gene is mediated through the product of a malT regulatory gene . The deduced amino acid sequence of iam contained an N-terminal signal peptide of 32 amino acids, and was 61% homologous with Pseudomonas amyloderamosa isoamylase . The mature enzyme, which was engineered for overexpression in E . coli and purified to homogeneity, has a relative molecular mass of 83 kDa, a pH optimum of 6-7, and a highest rate of hydrolysis for glycogen (but did not cleave pullulan) . Polyclonal antiserum generated from purified donor isoamylase cross-reacted with crude and purified recombinant isoamylase from E . coli . This is the first report of the cloning, characterization, and sequence of an novel isoamylase that has a neutral pH optimum . A comparison of the sequence of Flavobacterium sp . iam with acidic isoamylase from Pseudomonas sp . identified putative residues which may be associated with the pH for optimal activity of isoamylases.

J Antibiot (Tokyo), 1997 May, 50(5), 384 - 9
Lipohexin, a new inhibitor of prolyl endopeptidase from Moeszia lindtneri (HKI-0054) and Paecilomyces sp . (HKI-0055; HKI-0096) . II . Inhibitory activities and specificity; Christner C et al.; The new proline-containing lipohexapeptide lipohexin (I) isolated from three fungal strains, Moeszia lindtneri (HKI-0054) and Paecilomyces sp . (HKI-0055 and HKI-0096) is a competitive inhibitor of prolyl endopeptidase (PEP) from human placenta with IC50 of 3.5 microM . Specificity of lipohexin (I) is indicated by the much weaker inhibitory activity against bacterial prolyl endopeptidase from Flavobacterium meningosepticum (IC50 25 microM) . No effect of lipohexin (I) was found on the activity of mechanistically related proteases such as proline specific proteases and other serine proteases.

J Antibiot (Tokyo), 1997 May, 50(5), 373 - 8
Propeptin, a new inhibitor of prolyl endopeptidase produced by Microbispora . I . Fermentation, isolation and biological properties; Kimura K et al.; Propeptin, an inhibitor of the prolyl endopeptidase isolated from the mycelium of Microbispora sp . SNA-115, is an atypical cyclic peptide antibiotic . It was purified by column chromatographies on silica gel and Sephadex LH-20 and high performance liquid chromatography using an ODS column . Propeptin has the molecular formula of C113H142N26O27 and consists of nineteen amino acids . Propeptin inhibited prolyl endopeptidase of the genus Flavobacterium competitively when Z-Gly-Pro-pNA was used as a substrate . The inhibitor constant (Ki) was 0.70 microM.

J Antibiot (Tokyo), 1997 Apr, 50(4), 291 - 6
SNA-8073-B, a new isotetracenone antibiotic inhibits prolyl endopeptidase . I . Fermentation, isolation and biological properties; Kimura K et al.; SNA-8073-B, an inhibitor of prolyl endopeptidase isolated from the broth filtrate of Streptomyces sp . SNA-8073, is a new isotetracenone antibiotic . It was purified by ethyl acetate extraction, silica gel column chromatography and high performance liquid chromatography on ODS column . SNA-8073-B has the molecular formula of C20H16O5 and is a stereoisomer of SNA-8073-A (fujianmycin B, rubiginone A2) . SNA-8073-B inhibited prolyl endopeptidase of Flavobacterium non-competitively (IC50 = 8.9 microM) when Z-Gly-Pro-pNA was used as a substrate, but SNA-8073-A did not show any inhibition even at 60 microM.

J Clin Microbiol, 1997 Apr, 35(4), 1021 - 3
Comparison of Etest and agar dilution method for antimicrobial susceptibility testing of Flavobacterium isolates; Hsueh PR et al.; The Etest was evaluated as a possible alternative to the standard agar dilution method for susceptibility testing of nine antimicrobial agents against Flavobacterium species . In studies of 100 clinical isolates, the agreement between the MICs (+/-1 log2 dilution) obtained by the two methods was acceptable for cefotaxime, ceftazidime, amikacin, minocycline, ofloxacin, and ciprofloxacin (> 90%) . Conversely, the agreement between the results obtained for piperacillin was limited (84%) . The overall agreement was 92.5%.

Eur J Biochem, 1997 Apr 1, 245(1), 40 - 6
Uncoupling of actomyosin adenosinetriphosphatase by heparin and its fragments; Tersariol IL et al.; Heparin and its enzymatic fragments, prepared by degradation of heparin with heparinase from Flavobacterium heparinum, were capable of inhibiting the actomyosin-ATPase activity obtained from striated and smooth vascular muscles . Heparin did not inhibit the myosin-ATPase activity in absence of actin . The results show that heparin changes the step of ATP hydrolysis of the complex actomyosin-ATPase by uncoupling the conformational transition on the myosin-head induced by actin upon the nucleotide-binding site . This mechanism is cooperative and dependent on conformational states of actomyosin complex which in turn is regulated by ATP and calcium levels . It was observed that in the presence of ATP, actin does not compete with heparin for binding to myosin showing that heparin and actin have different binding sites on myosin . The binding of heparin and ATP is cooperative suggesting that the nucleotide binding leads to an exposition of a second heparin-binding site . However, in the absence of ATP, actin competes with heparin for a binding site on the myosin . These results strongly suggest that in the weakly binding state of actin to myosin, the binding of heparin is powerful and in the rigor state its binding is decreased.

Int J Syst Bacteriol, 1997 Apr, 47(2), 562 - 5
Phylogenetic position of Riemerella anatipestifer based on 16S rRNA gene sequences; Subramaniam S et al.; Riemerella anatipestifer, the causative agent of septicemia anserum exsudativa (also called new duckling disease), belongs to the family Flavobacteriaceae of gram-negative bacteria . We determined the DNA sequences of the rrs genes encoding the 16S rRNAs of four R . anatipestifer strains by directly sequencing PCR-amplified rrs genes . A sequence similarity analysis confirmed the phylogenetic position of R . anatipestifer in the family Flavobacteriaceae in rRNA superfamily V and allowed fine mapping of R . anatipestifer on a separate rRNA branch comprising the most closely related species, Bergeyella zoohelcum, as well as Chryseobacterium balustinum, Chryseobacterium indologenes, and Chryseobacterium gleum . The sequences of the rrs genes of the four R . anatipestifer strains varied between 0.5 and 1.0%, but all of the strains occupied the same position on the phylogenetic tree . In general, differences in rrs genes were observed among R . anatipestifer strains, even within a given serotype, as shown by restriction fragment length polymorphism of PCR-amplified rrs genes.

J Bacteriol, 1997 Mar, 179(5), 1521 - 4
Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp . strain ATCC 39723; Lee JY et al.; The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp . strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized . In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723 . The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps . Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da . The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect . The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture . The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH . The enzyme required Fe2+ for activity and was severely inhibited by metal chelating agents . The optimal conditions for activity were pH 7.0 and 40 degrees C . The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.

Arch Biochem Biophys, 1997 Feb 1, 338(1), 22 - 8
Cloning, sequencing, and expression of Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase in Escherichia coli; Takegawa K et al.; The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was determined . A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found . The primary structure of Endo-A exhibited significant homology with FO1F.10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis . However, the enzyme had no significant homology with any previously reported endo-beta-N-acetylglucosaminidases . Transformed Escherichia coli cells carrying the 4.5-kb fragment expressed Endo-A activity . This enzyme activity was detected in the medium as well as in the periplasmic space of cells under the control of the Endo-A gene promoter . The recombinant Endo-A was efficiently isolated from the periplasmic space of the cells . N-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus . Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity.

Gene, 1997 Jan 31, 185(1), 35 - 41
Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp . strain R1534; Pasamontes L et al.; The Gram-negative bacterium Flavobacterium sp . strain R1534 is a natural producer of zeaxanthin . A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced . The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons . The five genes are necessary and sufficient for the synthesis of zeaxanthin . The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms . Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp . strain R1534 carotenoid biosynthesis cluster.

Dev Biol Stand, 1997, 90, 391 - 400
Vaccination strategies in freshwater salmonid aquaculture; Larsen JL et al.; The focus of this chapter is antibacterial vaccines . The main salmonid species in freshwater aquaculture is the rainbow trout . Other salmonid species are produced on a limited scale . The most important bacterial fish diseases in European freshwater aquaculture are the rainbow trout fry syndrome-RTFS-(Flavobacterium psychrophilum) and enteric redmouth disease-ERM-(Yersinia ruckeri) which are widespread and cause serious epizootics, while furunculosis (Aeromonas salmonicida) is endemic, only giving overt disease under extremely stressing conditions . In the hatchery, there is a need for vaccination against RTFS (not commercially available) and ERM; in the ponds it is urgent to vaccinate against ERM, while the importance of furunculosis vaccination is not clear . The fish for ongrowing in saltwater should be vaccinated against ERM, furunculosis and vibriosis . Commercial vaccines are available against these diseases, either as single component or combination vaccines for immersion and injection-and oral vaccines are under registration . Hitherto, there has not been much strategic research on vaccination in freshwater; however recent results suggest that with the regime of vaccines available (and soon available), fish should be vaccinated with an ERM immersion vaccine in the hatchery approximately four weeks before transfer to the ponds . To cover the growth period in fresh water an oral booster should be given two to three months later . There is a need for development and research in strategic use of an RTFS and a furunculosis vaccine in freshwater aquaculture.

Dev Biol Stand, 1997, 90, 179 - 88
Immunization with bacterial antigens: Flavobacterium and Flexibacter infections; Bernardet JF; Seven bacterial species belonging to the Flavobacterium-Cytophaga group are currently considered to be pathogenic for fish . Because they were only recently described and/or because the disease they provoke has little economic significance, no study has been performed concerning immunization against Chryseobacterium scophthalmum, Flavobacterium johnsoniae and Flexibacter ovolyticus . Immunization with Flexibacter maritimus has not been investigated, and fish surviving a natural infection appear to remain equally susceptible to the disease during subsequent outbreaks . On the other hand, interesting data are available concerning immunization against Flavobacterium branchiophilum, Flavobacterium columnare and Flavobacterium psychrophilum because these bacterial species have been known for many years and are responsible for heavy losses in many countries . Surviving fish are usually protected against further infection by the same pathogens . Extensive serological studies have been performed and virulence mechanisms have also been investigated . The review of immunization trials against these three bacterial species shows important variations depending on the species of fish and the route of administration, but in several cases vaccinated fish were successfully protected by high titre of specific antibodies . However, no vaccine is commercially available.

Ophthalmologica, 1997, 211(2), 98 - 100
Flavobacterium indologenes keratitis; Lu PC et al.; Flavobacterium indologenes has recently been implicated in nosocomial or opportunistic infection . It has been isolated from lids, conjunctiva, and lacrimal system, and is resistant to most antibiotics . No previous cases of F . indologenes corneal ulcer have been reported in the literature . The natural habitat of Flavobacteria is soil, water, plants, and foodstuffs . In the hospital environment, these bacteria exist in water systems and on wet surfaces . They are not part of the normal ocular flora.

Microbiol Immunol, 1997, 41(2), 101 - 6
Adjuvanticity of an ornithine-containing lipid of Flavobacterium meningosepticum as a candidate vaccine adjuvant; Kato H et al.; Ornithine-containing lipids (OrnL) extracted from Flavobacterium meningosepticum have been reported to have various biological activities such as B-cell mitogenicity and macrophage activation to generate interleukin-1 and prostaglandin E2 . We, using ovalbumin (OVA) as an antigen, evaluated the adjuvant activity of OrnL as an immunological adjuvant in BALB/c mice . OrnL showed the function of forming liposome-like vesicles retaining biological activities when prepared as either small unilamellar or dehydration-rehydration vesicles . Although OrnL was not shown to have enough entrapping efficacy for use as a vaccine adjuvant, phosphatidylglycerol (PG) and cholesterol (CHOL) added to stabilize the vesicle membrane increased the entrapping efficacy to the same extent as that of conventional liposomes . Furthermore, the stabilized OrnL vesicles tolerated centrifugation to remove non-entrapped antigens . Completely antigen-entrapped OrnL vesicles including Pg and CHOL induced a significantly greater enhancement of IgG antibody production than did aluminum hydroxide gel in BALB/c mice from week 6 . These results indicate that OrnL can be utilized as an immunological adjuvant for vaccines.

Mol Ecol, 1997 Jan, 6(1), 39 - 49
PCP degradation is mediated by closely related strains of the genus Sphingomonas; Ederer MM et al.; There have been numerous reports in the literature of diverse bacteria capable of degrading pentachlorophenol (PCP) . In order to gain further insight into the phylogenetic relationships of PCP-degrading bacteria, we examined four strains: Arthrobacter sp . strain ATCC 33790, Flavobacterium sp . strain ATCC 39723, Pseudomonas sp . strain SR3, and Sphingomonas sp . strain RA2 . These organisms were isolated from different geographical locations and all of them degrade high concentrations (100-200 mg/L) of PCP . Southern blot analyses determined that these bacteria all harbour DNA that encodes similar, if not identical, genes involved in PCP degradation . Comparison of the 16S rRNA nucleotide sequences revealed that these organisms were very closely related and, in fact, represent a monophyletic group . The 16S rRNA analyses together with fatty acid and sphingolipid analyses strongly suggest that the four strains are members of the genus Sphingomonas . The close relationship of the four organisms is supported by nucleotide sequence analysis data of the pcpB locus encoding PCP-4-monooxygenase, the first enzyme in the PCP degradative pathway.

J Clin Microbiol, 1997 Jan, 35(1), 322 - 4
Shieh medium supplemented with tobramycin for selective isolation of Flavobacterium columnare (Flexibacter columnaris) from diseased fish; Decostere A et al.; Tobramycin was found to be less inhibitory to Flavobacterium columnare (formerly Flexibacter columnaris) than to other fish-associated bacteria . The selective capacity of Shieh medium, an isolation medium described for this species, was markedly enhanced by adding tobramycin at a concentration of 1 microgram/ml.

Biochem Biophys Res Commun, 1996 Dec 24, 229(3), 770 - 7
A comparative analysis of the primary sequences and characteristics of heparinases I, II, and III from Flavobacterium heparinum; Godavarti R et al.; Heparinases I, II and III from F . heparinum cleave heparin-like molecules, with a high degree of substrate specificity, at the glucosamine-uronate linkage by elimination, leaving an unsaturated C4-C5 bond in the uronic acid . The primary sequences of these enzymes have been reported earlier . In this study we perform a comparative analysis of the properties and primary sequences of heparinase I, II and III . Alignment of the primary sequences revealed little sequence homology (15% residue identity in a LALIGN alignment) at both DNA and amino acid levels . There are three basic clusters in heparinase II satisfying the heparin binding consensus sequence with one of the sequences sharing homology with a consensus sequence in the heparin binding site of heparinase I and two basic clusters in heparinase III . Similar to heparinase I, there are two putative 'EF-hand' calcium coordinating motifs in heparinase III, while heparinase II does not contain any such motifs . Recombinant heparinases II and III's degradation of the substrate and the subsequent separation of the oligosaccharide products by POROS anion exchange chromatography were identical to those obtained from native heparinases II and III from F . heparinum.

Biosci Biotechnol Biochem, 1996 Dec, 60(12), 2032 - 7
Dipeptidyl aminopeptidase IV from Pseudomonas sp . WO24; Ogasawara W et al.; Dipeptidyl aminopeptidase IV from Pseudomonas sp . WO24 was purified as two molecular forms of 84 and 82-kDa by SDS-PAGE . Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus . The DAP IV gene of Pseudomonas sp . WO24 was cloned and expressed in E . coli . The enzyme expressed in E . coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24% . The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1:1) was different from that of the native ones (about 1:4) . The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity . The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others.

Protein Sci, 1996 Dec, 5(12), 2617 - 22
Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases; Grueninger-Leitch F et al.; Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures . A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins . A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins . Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization . First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale . Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward . To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase . The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation . We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.

Lab Invest, 1996 Dec, 75(6), 771 - 81
Differences in the nonreducing ends of heparan sulfates excreted by patients with mucopolysaccharidoses revealed by bacterial heparitinases: a new tool for structural studies and differential diagnosis of Sanfilippo's and Hunter's syndromes; Toma L et al.; Enzymatic and chemical analyses of the structures of heparan sulfates excreted in the urine by patients with Sanfilippo's and Hunter's syndromes revealed that their nonreducing ends differ from each other and reflect the enzyme deficiency of the syndromes . The heparan sulfates from the different syndromes were treated with heparitinase II, crude enzyme extracts from Flavobacterium heparinum, and nitrous acid degradation . The heparan sulfates from patients with Sanfilippo A (deficient in heparan N-sulfatase) and Sanfilippo B (deficient in alpha-N-acetylglucosaminidase) were degraded with heparitinase II producing, besides unsaturated disaccharides, substantial amounts of glucosamine N-sulfate and N-acetylglucosamine, respectively . The heparan sulfate from patients with Hunter's syndrome (deficient in iduronate sulfatase) were degraded by heparitinase II or crude enzyme extracts to several products, including two saturated disaccharides containing a sulfated uronic acid at their nonreducing ends . The heparan sulfate from patients with Sanfilippo's C syndrome (deficient in acetyl Co-A: alpha-glucosaminide acetyltransferase) produced, by action of heparitinase II, among other products, two sulfated trisaccharides containing glucosamine with a nonsubstituted amino group . In addition to providing a new tool for the differential diagnosis of the mucopolysaccharidoses, these results bring new insights into the specificity of the heparitinases from Flavobacterium heparinum.

Appl Environ Microbiol, 1996 Dec, 62(12), 4433 - 40
Identifying numerically abundant culturable bacteria from complex communities: an example from a lignin enrichment culture; Gonzalez JM et al.; Culturable bacteria that were numerically important members of a marine enrichment community were identified and characterized phylogenetically . Selective and nonselective isolation methods were used to obtain 133 culturable bacterial isolates from model marine communities enriched with the high-molecular-weight (lignin-rich) fraction of pulp mill effluent . The culture collection was screened against community DNA from the lignin enrichments by whole-genome hybridization methods, and three marine bacterial isolates were identified as being numerically important in the communities . One isolate was in the alpha-subclass of Proteobacteria, and the other two were in the gamma-subclass of Proteobacteria . Isolate-specific 16S rRNA oligonucleotide probes designed to precisely quantify the isolates in the lignin enrichment communities indicated contributions ranging from 2 to 32% of enrichment DNA, values nearly identical to those originally obtained by the simpler whole-genome hybridization method . Two 16S rRNA sequences closely related to that of one of the isolates, although not identical, were amplified via PCR from the seawater sample originally used to inoculate the enrichment medium . Partial sequences of 14 other isolates revealed significant phylogenetic diversity and unusual sequences among the culturable lignin enrichment bacteria, with the Proteobacteria, Cytophaga-Flavobacterium, and gram-positive groups represented.

Appl Environ Microbiol, 1996 Dec, 62(12), 4318 - 22
Complete microbial degradation of both enantiomers of the chiral herbicide mecoprop {(RS)-2-(4-chloro-2-methylphenoxy)propionic acid} in an enantioselective manner by Sphingomonas herbicidovorans sp . nov; Zipper C et al.; Sphingomonas herbicidovorans MH (previously designated Flavobacterium sp . strain MH) was able to utilize the chiral herbicide (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) as the sole carbon and energy source . When strain MH was offered racemic mecoprop as the growth substrate, it could degrade both the (R) and the (S) enantiomer to completion, as shown by biomass formation, substrate consumption, and stoichiometric chloride release . However, the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer . These results suggest the involvement of specific enzymes for the degradation of each enantiomer . This view was substantiated by the fact that resting cells of strain MH grown on (S)-mecoprop were able to degrade the (S) but not the (R) enantiomer of mecoprop . Accordingly, resting cells of strain MH grown on (R)-mecoprop preferentially metabolized the (R) enantiomer . Nevertheless, such cells could transform (S)-mecoprop at low rates . Oxygen uptake rates with resting cells confirmed the above view, as oxygen consumption was strongly dependent on the growth substrate . Cells grown on (R)-mecoprop showed oxygen uptake rates more than two times higher upon incubation with the (R) than upon incubation with the (S) enantiomer and vice versa.

Arch Biochem Biophys, 1996 Dec 1, 336(1), 35 - 41
Prolyl aminopeptidase gene from Flavobacterium meningosepticum: cloning, purification of the expressed enzyme, and analysis of its sequence; Kitazono A et al.; In spite of the numerous studies regarding prolyl aminopeptidase, little is known about its mechanism and the significance of its similarity to a number of hydrolases of diverse specificity that belong to the alpha/beta hydrolase-fold family (Pseudomonas 2-hydroxymuconic semialdehyde hydrolase, atropinesterase, and 2-hydroxy-6-oxophenylhexa-2,4-dienoic acid hydrolase; human and rat epoxide hydrolases) . We report the cloning and sequencing of the novel prolyl aminopeptidase gene from Flavobacterium meningosepticum (FPAP) which allowed a more comprehensive sequence comparison . FPAP was found to be a 35-kDa monomeric enzyme, releasing N-terminal proline but not hydroxyproline residues from small peptides and naphthylamide esters . Using the unweighted pair group method with arithmetic mean method, an evolutionary tree that depicts the probable relationship between the prolyl aminopeptidases and the alpha/beta hydrolase-fold enzymes was constructed . Since the alpha/beta hydrolase-fold family might also include the members of the prolyl oligopeptidase family (prolyl oligopeptidase, dipeptidyl peptidase IV, and prolyl carboxypeptidase), this proposal links all the known Pro-Y bond-cleaving proline-specific peptidases (prolyl oligopeptidase family, prolyl aminopeptidases, and prolinase) as enzymes with similar scaffolds and hydrolytic mechanisms . On the other hand, the enzymes that cleave X-Pro bonds are metalloenzymes grouped within the "pita-bread" fold family (aminopeptidase P and prolidase) . Although the latter two enzymes show significant sequence homology, prolyl aminopeptidase, prolinase, and the members of the prolyl oligopeptidase family do not, and might share the alpha/beta hydrolase-fold scaffold . This rationale would explain the failure in finding a common "proline-recognizing motif" in the primary structures of these proline-specific peptidases.

Int J Syst Bacteriol, 1996 Oct, 46(4), 866 - 70
Emendation of the genus Planococcus and transfer of Flavobacterium okeanokoites Zobell and Upham 1944 to the genus Planococcus as Planococcus okeanokoites comb . nov; Nakagawa Y et al.; The taxonomic position of Flavobacterium okeanokoites IFO 12536T (T = type strain) was determined by 16S rRNA gene sequencing and chemotaxonomic methods . Phylogenetic evidence derived from a 16S rRNA sequence analysis indicated that F . okeanokoites, which forms rod-shaped cells, belongs to the genus Planococcus, which forms spherical cells . A phylogenetically close relationship was supported by chemotaxonomic characteristics, such as the presence of menaquinone 7 and menaquinone 8 as major isoprenoid quinones, the presence of phosphatidylglycerol, bisphosphatydylglycerol, and phosphatidylethanolamine as cellular polar lipids, and the G + C content of the DNA (46.3 mol%) . These data suggest that whether a cell is a rod or a coccus is not a generic criterion . Accordingly, we propose that F . okeanokoites should be transferred to the genus Planococcus and that the description of the genus Planococcus should be emended.

Clin Infect Dis, 1996 Sep, 23(3), 550 - 5
Flavobacterium indologenes bacteremia: clinical and microbiological characteristics; Hsueh PR et al.; To our knowledge, Flavobacterium indologenes has never been reported as a cause of bacteremia in humans . F . indologenes bacteremia was diagnosed in 12 patients at a tertiary referral center in southern Taiwan between 1 January 1992 and 31 December 1994 . Six of these patients had ventilator-associated pneumonia, two had primary bacteremia, and one patient each had pyonephrosis, peritonitis, biliary tract infection, and surgical wound infection . Five patients (42%) had malignancies, and three (25%) had multiple burns . Polymicrobial bacteremia was diagnosed in eight patients (67%) . Two (17%) of the patients in this study died; both had polymicrobial bacteremia . Antimicrobial susceptibility testing of the blood isolates from the 12 patients showed that > 90% of the isolates were susceptible to piperacillin, cefoperazone, ceftazidime, and minocycline . The chromatograms of esterified fatty acids for the isolates were identical . F . indologenes should be considered an etiologic agent of bloodstream infection, especially in hospitalized patients with severe underlying diseases.

Appl Environ Microbiol, 1996 Sep, 62(9), 3548 - 50
A selective and differential medium for Vibrio harveyi; Harris L et al.; A new medium, termed Vibrio harveyi agar, has been developed for the isolation and enumeration of V . harveyi . It is possible to differentiate V . harveyi colonies from the colonies of strains representing 15 other Vibrio species with this medium . This medium has been shown to inhibit the growth of two strains of marine Pseudomonas spp . and two strains of marine Flavobacterium spp . but to allow the growth of Photobacterium strains . Colonies displaying typical V . harveyi morphology were isolated from the larval rearing water of a commercial prawn hatchery with V . harveyi agar as a primary isolation medium and were positively identified, by conventional tests, as V . harveyi . This agar displays great potential as a primary isolation medium and offers significant advantages over thiosulfate-citrate-bile salts-sucrose agar as a medium for differentiating V . harveyi from other marine and estuarine Vibrio species.

Biochem Biophys Res Commun, 1996 Aug 23, 225(3), 751 - 8
Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli; Godavarti R et al.; Heparinase III (E.C . 4.2.2.8), formerly heparinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides . In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR) . Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a approximately 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template . The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II . The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to approximately 18% of the protein . Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system . This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate.

Eur J Biochem, 1996 Aug 15, 240(1), 143 - 9
Characterization and ion channel activities of novel antibacterial proteins from the skin mucosa of carp (Cyprinus carpio); Lemaitre C et al.; A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers . A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31-kDa and 27-kDa proteins . In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A . The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus . The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave Pcl/Pk ratios of 0.6 and 1.0, respectively . These proteins had large potent microbicidal activities (0.018-0.18 microM) against different strains of gram-negative and gram-positive bacteria . This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane . To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp.; Aeromonas hydrophila . Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities . Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence . This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins . These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.

J Clin Microbiol, 1996 Aug, 34(8), 1908 - 13
Clinical and microbiological characteristics of Flavobacterium indologenes infections associated with indwelling devices; Hsueh PR et al.; Clinical infections caused by Flavobacterium indologenes have never been documented . Thirteen isolates derived from seven patients with indwelling device-associated F . indologenes infections were identified from 1 April through 30 November 1995 . The antimicrobial susceptibilities to 20 antimicrobial agents of the isolates, the cellular fatty acid chromatograms for the isolates, and the random amplified polymorphic DNA (RAPD) patterns generated by arbitrarily primed PCR of the isolates were studied . The antibiotypes and RAPD patterns differed among the isolates recovered from different patients . However, both antibiotypes and RAPD patterns were identical among the five isolates from one patient with multiple episodes of central venous catheter-associated bacteremia within a 1.5-month period and between the two isolates from another patient suffering from two episodes of catheter-related bacteriuria at an interval of 14 days . It is documented that the recurrent infections in each of these two patients were caused by a single F . indologenes clone, respectively . Identical antibiotypes and RAPD patterns were also demonstrated between two isolates from a patient with ventilator-associated pneumonia, one recovered from an endotracheal aspirate and the other derived from a blood specimen 10 days later, indicating the invasive nature of F . indologenes . Two cellular fatty acid chromatograms were identified among these isolates . All of the isolates showed in vitro resistance to cephalothin, cefotaxime, ceftriaxone, moxalactam, aztreonam, aminoglycosides, erythromycin, clindamycin, vancomycin, and teicoplanin . F . indologenes should be included as an etiologic agent of infections associated with the use of indwelling devices.

Eur J Biochem, 1996 Aug 1, 239(3), 865 - 70
Specificity studies of bacterial sulfatases by means of structurally defined sulfated oligosaccharides isolated from shark cartilage chondroitin sulfate D; Sugahara K et al.; Chondro-4-sulfatase and chondro-6-sulfatase from Proteus vulgaris and delta-hexuronate-2-sulfatase from Flavobacterium heparinum are potentially useful tools for structural studies of chondroitin sulfate and dermatan sulfate . Their substrate specificities were investigated with various structurally defined, sulfated hexasaccharides isolated from chondroitin sulfate as described in the accompanying report {Sugahara, K., Nadanaka, S., Takeda, K . & Kojima, T . (1996) Eur . J . Biochem . 239, 871-880} . The results indicated that delta-hexuronate-2-sulfatase released an ester sulfate from the C2 position of the delta-hexuronate residue located at the non-reducing terminus, while chondro-6-sulfatase removed an ester sulfate from the C6 position of the GalNAc residue at the reducing end of the hexasaccharides . Chondro-4-sulfatase acted preferentially on an ester sulfate on the C4 position of the GalNAc residue at the reducing end under mild incubation conditions, but also released a sulfate group under harsh conditions from the C4 position of the GalNAc residue at the internal positions of the hexasaccharide chains, unless the GalNAc residue had another ester sulfate on its C6 position . The results demonstrated the usefulness of the sulfatases as tools for the structural characterization of chondroitin sulfate oligosaccharides.

Appl Environ Microbiol, 1996 Aug, 62(8), 2723 - 34
Isolation and expression in Escherichia coli of hepB and hepC, genes coding for the glycosaminoglycan-degrading enzymes heparinase II and heparinase III, respectively, from Flavobacterium heparinum; Su H et al.; Upon induction with heparin, Flavobacterium heparinum synthesizes and secretes into its periplasmic space heparinase I (EC 4.2.2.7), heparinase II, and heparinase III (heparitinase; EC 4.2.2.8) . Heparinase I degrades heparin, and heparinase II degrades both heparin and heparan sulfate, while heparinase III degrades heparan sulfate predominantly . We isolated the genes encoding heparinases II and III (designated hepB and hepC, respectively) . These genes are not contiguous with each other or with the heparinase I gene (designated hepA) . hepB and hepC were found to contain open reading frames of 2,316 and 1,980 bp, respectively . Enzymatic removal of pyroglutamate groups permitted sequence analysis of the amino termini of both mature proteins . It was determined that the mature forms of heparinases II and III contain 746 and 635 amino acids, respectively, and have calculated molecular weights of 84,545 and 73,135, respectively . The preproteins have signal sequences consisting of 26 and 25 amino acids . Truncated hepB and hepC genes were used to produce active, mature heparinases II and III in the cytoplasm of Escherichia coli . When these enzymes were expressed at 37 degrees C, most of each recombinant enzyme was insoluble, and most of the heparinase III protein was degraded . When the two enzymes were expressed at 25 degrees C, they were both present predominantly in a soluble, active form.

J Biol Chem, 1996 Jul 19, 271(29), 16991 - 4
Selective loss of cerebral keratan sulfate in Alzheimer's disease; Lindahl B et al.; Proteoglycans, especially heparan sulfate-substituted species, are known to be associated with the deposition of amyloid in Alzheimer's disease . We previously found that heparan sulfate from afflicted brains, and from control subjects, differed minimally in quantity and structure (Lindahl, B., Eriksson, L., and Lindahl, U.(1995) Biochem . J . 306, 177-184) . In the present study, a glycosaminoglycan fraction, shown to contain heparan sulfate and keratan sulfate, was radiolabeled by partial N-deacetylation (hydrazinolysis) followed by re-N-acetylation using {3H}acetic anhydride . Quantitation of the 3H-labeled polysaccharides, based on digestion with heparitinase I from Flavobacterium heparinum and keratanase from Pseudomonas sp., revealed that the amounts of keratan sulfate in Alzheimer cerebral cortex are reduced to less than half of control values . Moreover, a monoclonal antibody against a highly sulfated keratan sulfate epitope bound to the majority of the neurons in normal cortex but not in the diseased tissue . The lack of highly sulfated keratan sulfate structures may reflect a specific functional defect of the cells.

Glycobiology, 1996 Jul, 6(5), 527 - 36
Purification, biochemistry and molecular cloning of an insect glycosylasparaginase from Spodoptera frugiperda; Liu Y et al.; Glycosylasparaginase (EC 3.5.1.26) from Sf9 cells (Spodoptera frugiperda) was purified to homogeneity with a specific activity of 2.1 unit/mg . The enzyme is composed of two non-identical alpha/beta subunits joined by strong non-covalent forces and has one glycosylation site located in the alpha subunit . Molecular masses of the subunits were determined to be 28 kDa and 17 kDa by SDS-PAGE . Native enzyme existed in quaternary structures of either heterodimer (alpha beta) or heterotetramer (alpha 2 beta 2) . These forms exhibited different ionic characteristics during DE52 anion exchange chromatography, and their molecular masses were determined to be 47 kDa and 101 kDa by gel filtration . The enzyme was thermostable, requiring 65-70 degrees C to be denatured, and it had a broad pH optimum from 4-10.5 with a pKa around 6 . SDS easily inactivated the enzyme . The K(m) of glycosylasparaginase for its normal substrate GlcNAc-Asn was 0.88 mM . The enzyme also exhibited asparaginase activity with a K(m) of 3.0 mM for asparagine . N-terminal amino acids of the denatured subunits were sequenced and degenerate primers were designed for cloning its cDNA using PCR and 5' and 3' RACE . Glycosylasparaginase cDNAs from bovine and rat were also cloned using similar strategies, and primary structures of glycosylasparaginases from six species (human, bovine, rat, mouse, Sf9 cells and Flavobacterium) have been compared and related to a recent crystal structure of the human enzyme.

Biochem Mol Biol Int, 1996 Jul, 39(4), 823 - 31
Opposite effects of basic fibroblast growth factor and heparin on heparin/heparan sulfate degrading enzymes from Flavobacterium heparinum; Cavari S et al.; Heparinase I is normally inhibited by an excess or substrate, in this paper we describe that bFGF counteracts the inhibiting effect of the substrate and that heparinase I activity is increased in the presence of bFGF . This effect was observed using heparin concentrations ranging from 10(-9) to 10(-7) M with either a ten fold molar excess or an equimolar concentration of bFGF . In addition, the degree of depolymerization of heparan sulfate produced by heparitinase from Flavobacterium heparinum was increased in the presence of bFGF.

Forensic Sci Int, 1996 Jun 28, 80(1-2), 49 - 61
Investigations on the mechanism of adipocere formation and its relation to other biochemical reactions; Takatori T; In the adipocere, which is one of the postmortem changes, some specific fatty acids possessing higher melting points, together with soap, play an important role in the formation of adipocere . These fatty acids were shown to be mainly 10-hydroxystearic and 10-hydroxypalmitic acids . Moreover, slight amounts of 10-oxostearic and 10-oxopalmitic acids, which have higher melting points than those of hydroxy fatty acids, exist in the adipocere as well . The substantial adipocere is formed and stabilized by these specific fatty acids together with the soap . The hydroxy fatty acid (OHFA) and oxo fatty acid (OXOFA) are biosynthesized by some bacterial enzymes . Various aerobic and anaerobic bacteria are involved in the formation of adipocere . For example, microbial conversion of various unsaturated fatty acids to 10-OHFA by Micrococcus luteus was investigated . It turned out that 10-OHFA was synthesized only from fatty acids possessing cis-9-unsaturation . It was also shown that 10-OHFAs were converted to the corresponding 10-OXOFAs but the 10-OXO compounds were inactive as substrates . Furthermore, it was found that the enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxystearic acid, but also dehydrogenation of this product in the presence of deuterium . On the other hand, we found out that there was 10-hydroxy-12-octadecenoic acid (10-OHODA) from linoleic acid in some kinds of adipocere . Recently, 10-epoxy-12-octadecenoic acid (leukotoxin, LTx), which is one of the lipid peroxides, was found not only in rice plants but in polymorphonuclear leukocytes . Since LTx was found in leukocytes related to inflammatory response, interest has been focused on its involvement, not only in the basic mechanism of biological defense, but also on the mechanism of shock as a vasoactive substance . A postmortem change itself is only remotely associated with a phenomenon in a living body . However, 10-OHODA found in adipocere seemed to exist also in polymorphonuclear leukocytes, suggesting that this compound might be closely related to a biological reaction.

Res Microbiol, 1996 Jun, 147(5), 415 - 25
Ribotyping of Chryseobacterium meningosepticum: its use as an epidemiological tool and its correlation with serovars; Quilici ML et al.; Chryseobacterium meningosepticum (basonym, Flavobacterium meningosepticum King 1959) is associated with neonatal meningitis and is isolated from normal and immunocompromised adults . AAF-labelled Escherichia coli 16 + 23S rRNA was used as a probe for ribotype analysis of 92 clinical isolates from tracheal exsudate, blood culture, cerebrospinal fluid, urine and pus . The 92 isolates belonged to the 15 described serovars of C . meningosepticum, and included 21 strains isolated during an outbreak in an intensive care unit, all belonging to serovar G . Three restriction endonucleases, EcoRI, HindIII and PstI, were selected for use in ribotyping after preliminary experiments . Epidemiologically unrelated isolates were discriminated by ribotyping and could be classified into 48 ribotypes according to the hybridization banding patterns obtained after restriction with the three enzymes . Strains which were not discriminated by combined ribotype analysis belonged to the same serovar, and were of identical geographic origin . In one case, analysis with an additional enzyme, PvuII, was necessary for separating strains from two different serovars . However, three strains from different serovars (two isolated from the same place and one elsewhere within eight years) showed the same combined ribotype . Analysis of the rRNA gene patterns revealed 6 different patterns for clinical isolates of the outbreak, suggesting unrelated sources of infection . In three patients, isolation of C . meningosepticum with different combined ribotypes suggested superinfection . Ribotyping enabled differentiation between isolates belonging to the same serovar as well as between isolates of different serovars and provided a useful molecular epidemiological tool for the study of C . meningosepticum . Combined ribotype analysis with several restriction endonucleases increased the discriminating power of the method . However, there was only a partial correlation between serovars and the extent of DNA relatedness.

Biochemistry, 1996 May 28, 35(21), 6846 - 52
Heparinase I from Flavobacterium heparinum . Identification of a critical histidine residue essential for catalysis as probed by chemical modification and site-directed mutagenesis; Godavarti R et al.; We recently identified cysteine-135 as an important amino acid for heparinase I (EC 4.2.2.7) activity . In this study, we have identified a second residue critical for enzymatic activity . We observe concentration-dependent inactivation of heparinase I in the presence of reversible histidine-modifying diethyl pyrocarbonate (DEPC); 0.3 mM DEPC results in 95% of heparinase I inactivation in less than 3 min, and as low as 10 microM DEPC results in a 85% loss of heparinase I activity in 15 min . Heparinase I activity is restored following hydroxylamine treatment . This, along with other experiments, strongly suggests that the inactivation of heparinase I by DEPC is specific for histidine residues . Chemical modification, under nondenaturing conditions, of the histidines using nonradiolabeled and {14C}DEPC indicates that between one and two histidine residues are modified . Chemical modification of the surface-accessible histidines, in the presence and absence of heparin, suggests that the histidine(s) lie(s) in or near the active site of heparinase I . The wild-type heparinase I has four histidine residues; site-directed mutagenesis of H129A, H165A, and H339A did not affect enzyme activity and the kinetic parameters, suggesting that these residues are not essential for heparinase I activity . However, H203A inactivates heparinase I while a H203D mutant has residual activity, indicating a role of this residue in catalysis . We propose that histidine-203, contained in the heparin binding site, is immediately adjacent to cysteine-135, and these residues together form the catalytic domain of heparinase I.

J Biol Chem, 1996 May 3, 271(18), 10495 - 502
Structures of five sulfated hexasaccharides prepared from porcine intestinal heparin using bacterial heparinase . Structural variants with apparent biosynthetic precursor-product relationships for the antithrombin III-binding site; Tsuda H et al.; Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography . Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions . Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region . The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G . (1995) J . Biol . Chem . 270, 22914-22923) . In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy . The tetrasaccharide has the hexasulfated structure typical of heparin . The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively) . Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) . Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue . Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site . Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway . Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.

Antibiot Khimioter, 1996 May, 41(5), 6 - 12
{A new metabolite with fungistatic and bacteriostatic activity, produced by strain L-30 of Flavobacterium sp.}; Shenin IuD et al.; Flavocin, an agent with fungistatic and bacteriostatic activities, was isolated from the culture of Flavobacterium sp . L-30 . The data on the physico-chemical and biological properties of flavocin A, the major active component, are presented . The component was identified with metabolites of the genus Flavobacterium and other genera of nonsporulating bacteria . Flavocin was highly efficient in the treatment of various farm crops . The antagonistic effect of flavocin was observed under field conditions in growing sugar beet, barley, spring wheat, potato and grape . Presowing and nonradical exposure to flavocin lowered the affection of the plants with brown patch, scab, soft rot and oidium.

Biochem J, 1996 Apr 15, 315 ( Pt 2), 589 - 97
Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum; Ernst S et al.; The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum is used for the determination of plasma heparin and for elimination of heparin from circulation . Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme . Heparinase I was expressed with an N-terminal histidine tag . The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography . The cumulative yield was 43%, and the recovery of purified heparinase I was 14.4 mg/l of culture . The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure . The reverse-phase HPLC profile of the tryptic digest, the Michaelis-Menten constant Km (47 micrograms/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme . Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F . heparinum . We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media . Separation of characteristic di-, tetra- and hexa-saccharide products is performed in 10 min . These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I-based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix.

FEMS Microbiol Lett, 1996 Mar 1, 136(3), 305 - 8
Isolation of a gene encoding cysteine synthase from Flavobacterium K3-15; Muller R et al.; The cysteine synthase gene (cysK) from Flavobacterium K3-15 was cloned and sequenced . The gene exhibits 30-50% identity to known cysteine synthases on both the DNA and the amino acid levels . The pyridoxal phosphate binding site of the enzyme is part of a conserved motif comprising seven amino acids (SIKDRIA) . The lys31 residue of the flavobacterial enzyme is conserved in all known cysteine synthases . The cysK gene from Flavobacterium K3-15 was heterologously expressed and the gene product identified by immunoblotting and determination of the enzyme activity.

Microbiol Res, 1996 Mar, 151(1), 37 - 41
Isolation and characterization of a new arsenic methylating bacterium from soil; Honschopp S et al.; An arsenic resistant and arsenic methylating bacterium belonging to the Flavobacterium-Cytophaga group was isolated from soil with an arsenic content of 1.5 ppm . The growth of the bacterium is enhanced in the presence of As compounds in concentrations up to 200 ppm in the cultural media with a stronger effect of As(V) than of As(III) compounds . As a volatile product of the methylation of both NaH2AsO3 and NaH2AsO4 exclusively, Me3As was formed and detected by mass spectrometry . Quantitative aspects of the methylation were studied with GC/MS . The intracellular accumulation of arsenic in the methylating strain was compared with two non methylating strains from the same soil.

Anal Biochem, 1996 Mar 1, 235(1), 98 - 101
Porcine fibrinogen glycopeptides: substrates for detecting endo-beta-N-acetylglucosaminidases F2 and F3(1); Plummer TH Jr et al.; Two different glycopeptides were isolated in high yield from a thermolytic digest of porcine fibrinogen . Edman analysis established their sequences as Val-Glu-Asn(CHO)-Lys and Val-Gly-Glu-Asn(CHO)-Arg . These sequences are nearly identical to the two human fibrinogen glycopeptides, Val-Glu-Asn(CHO)-Lys (gamma-chain), and Met-Gly-Glu-Asn(CHO)-Arg (beta-chain) . The predominant carbohydrate moiety of both asialoglycopeptides was a biantennary oligosaccharide with a core alpha(1-->6)-linked fucose as reported earlier (Da Silva et al . (1994) Arch . Biochem . Biophys . 312, 151-157) . Both glycopeptides can be dansylated and used as sensitive substrates for Flavobacterium meningosepticum endo-beta-N-acetylglucosaminidases F2 and F3 . Porcine fibrinogen represents the best source for substrates with this oligosaccharide type that can be reliably produced in multimicromole quantities.

J Biol Chem, 1996 Mar 1, 271(9), 5095 - 100
Cloning and expression of the unique Ca2+-ATPase from Flavobacterium odoratum; Peiffer WE et al.; The 60-kDa Ca2+-ATPase from Flavobacterium odoratum is kinetically and mechanistically similar to other P-type ATPases, suggesting its use as a model system for structure-function studies of ion transport . A portion of the gene was amplified by polymerase chain reaction of genomic DNA with degenerate oligonucleotide primers, one based on the N-terminal amino acid sequence of the purified protein and the other based on a consensus sequence for the phosphorylation site of P-type ATPases . This gene fragment was used to screen a lambda library of F . odoratum 29979 DNA . Clone "C" is 3.3 kilobases in length and contains one complete and part of a second open reading frame, the first of which encodes a 58-kDa protein containing the exact N-terminal amino acid sequence of the purified protein . We have named this gene cda, for calcium-dependent ATPase . Escherichia coli, transformed with clone C, demonstrates high levels of calcium-dependent and vanadate-sensitive ATP hydrolysis activity, forms a 60-kDa phosphointermediate, and cross-reacts with antibodies to the purified Ca2+-ATPase . The gene has almost no sequence homology to even the highly conserved regions characteristic of P-type ATPases but does possess significant homology to a protein with alkaline phosphatase activity (PhoD) from Zymomonas mobilis . The putative phosphorylation site is a Walker A (P-loop) ATP binding sequence and is modified relative to P-type ATPases, suggesting that the F . odoratum Ca2+-ATPase may represent an ancestral link between the F- and the P-type ATPases or perhaps a new class of ATPases.

J Biol Chem, 1996 Feb 16, 271(7), 3945 - 51
Purification and characterization of the Ca2+-ATPase of Flavobacterium odoratum; Desrosiers MG et al.; The P-type Ca2+-ATPase from Flavobacterium odoratum has been purified to homogeneity and characterized . Inside-out membrane vesicles were extracted with C12E8, followed by ammonium sulfate fractionation, centrifugation through two successive 32-48% glycerol gradients, and DE52 ion exchange chromatography . The purified Ca2+-ATPase consists of a single polypeptide . It migrates electrophoretically with an apparent molecular mass of 60,000 Da, consistent with the phosphorylation pattern originally reported in membrane vesicles . This single polypeptide is functional and capable of calcium-dependent vanadate-sensitive ATP hydrolysis and of forward and reverse phosphorylation . Maximum hydrolysis activity occurs at pH 8.0, with a specific activity of approximately 75 micromol of ATP hydrolyzed min-1 mg-1 protein . The purified Ca2+-ATPase has an apparent Km for calcium of 1.5 microM and for ATP of 90 microM . Vanadate strongly inhibits the activity with an IC50 of 0.6 microM . The prokaryotic Ca2+-ATPase is insensitive to the SR Ca2+-ATPase inhibitors fluorescein isothiocyanate, thapsigargin, and cyclopiazonic acid . It is rapidly phosphorylated by {gamma-32P}ATP in a calcium-dependent vanadate-inhibited manner and can be phosphorylated by Pi in both the presence and absence of calcium.

Gene, 1996 Feb 12, 168(2), 157 - 63
A mouse kidney- and liver-expressed cDNA having homology with a prokaryotic parathion hydrolase (phosphotriesterase)-encoding gene: abnormal expression in injured and polycystic kidneys; Hou X et al.; To investigate abnormalities in gene expression associated with cyst formation in polycystic kidney disease, differential cDNA library screening was carried out using RNA from normal and cystic kidneys of the C57BL/6J-cpk mouse . Among a number of genes found to be abnormally expressed was one (cDNA clone 56-1) that was significantly underexpressed in cystic kidneys . Hybridization analyses revealed that the 56-1 mRNA is expressed primarily in kidney and liver, and that the kidney expression begins postnatally and continues in the adult . Expression of this mRNA was found to be significantly decreased upon acute renal injury induced by a single intraperitoneal injection of folic acid, and to return to normal levels upon recovery of kidney function . Analysis of the cDNA sequence predicted a protein of 349 amino acids (aa), which was confirmed by in vitro translation of a sense-strand transcript, producing a protein of approx . 39 kDa . The aa sequence shows similarity to Flavobacterium sp . and Pseudomonas diminuta parathion hydrolase (phosphotriesterase or PTE), an enzyme that hydrolyzes toxic organophosphates and other phosphotriesters, and to the predicted product of an Escherichia coli open reading frame of unknown function (phosphotriesterase homology protein or PHP) . Use of optimal alignment programs demonstrated a significant overall homology between the bacterial and mouse sequences, with greater than 50% aa sequence similarity . This cDNA represents the first eukaryotic sequence showing similarity to these prokaryotic genes . Based on this apparent homology, it has been named mpr56-1 (for mouse phosphotriesterase-related 56-1).

J Biol Chem, 1996 Feb 9, 271(6), 3124 - 31
Heparinase I from Flavobacterium heparinum . Mapping and characterization of the heparin binding domain; Sasisekharan R et al.; In this study we have identified the primary heparin binding site of heparinase I (EC 4.2.2.7) . Chemical and proteolytic digests of heparinase I were used in direct binding and competition assays, to map the regions of heparinase I that interact specifically with heparin . We find the heparin binding site contains two Cardin-Weintraub heparin binding consensus sequences and a calcium co-ordination consensus motif . We show that heparin binding to heparinase I is independent of calcium (Kd of 60 nm) and that calcium is able to activate heparinase I catalytically . We find that sulfhydryl selective labeling of cysteine 135 of heparinase I protects the lysines of the heparin binding sequence from proteolytic cleavage, suggesting the close proximity of the heparin binding site to the active site . Site-directed mutagenesis of H203A (contained in the heparin binding site) inactivated heparinase I; however, a H203D mutant retained marginal activity, indicating a role for this residue in catalysis . The above results taken together suggest that histidine 203 (hence the heparin binding site) is immediately adjacent to the scissile bond . We propose that the heparin binding site and active site are in close proximity to each other and that the calcium coordination motif, contained in the heparin binding site, may bridge heparin to heparinase I through calcium in a ternary complex during catalysis.

Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 146 - 9
Verification of the role of PCP 4-monooxygenase in chlorine elimination from pentachlorophenol by Flavobacterium sp . strain ATCC 39723; Lange CC et al.; The bacterial enzyme PCP 4-monooxygenase from Flavobacterium sp . strain ATCC 39723 catalyzes the oxygenolytic removal of the first chlorine from pentachlorophenol . PCP 4-monooxygenase is an FAD binding, NADPH requiring oxygenase, with similar functional domains as other bacterial flavoprotein monooxygenases specific for phenolic substrates . However, the definitive proof for the singular role of an oxygenolytic elimination of the primary chlorine from pentachlorophenol by Flavobacterium sp . has awaited the development of a genetic system whereby targeted mutagenesis via allelic exchange could be carried out with the corresponding gene from PCP 4-monooxygenase, pcpB . We report the development of a genetic system for Flavobacterium sp . strain ATCC 39723, and its application in targeted mutagenesis of the pcpB allele for elimination of PCP 4-monooxygenase activity.

J Biol Chem, 1996 Jan 19, 271(3), 1732 - 7
Activation of glycosylasparaginase . Formation of active N-terminal threonine by intramolecular autoproteolysis; Guan C et al.; The activation mechanism of glycosylasparaginase of Flavobacterium meningosepticum has been analyzed by site-directed mutagenesis and activation of purified precursors in vitro . Mutation of Thr-152 to Ser or Cys leads to gene products that are not activated in vivo but are activated in vitro because processing of the mutant precursors is inhibited by certain amino acids in the cell . Kinetic studies reveal that activation is an intramolecular autoproteolytic process . The involvement of His-150 and Thr/Ser/Cys-152 in activation suggests that autoproteolysis resembles proteolysis by serine/cysteine proteases . Multiple functions of the highly conserved active threonine residue are implicated.

Thromb Res, 1996 Jan 1, 81(1), 91 - 100
Platelet lysis and functional perturbation by 13-methyl myristate . The major fatty acid in Flavobacterium ranacida; Faung ST et al.; Flavobacterium ranacida consisted of 75% of 13-methyl myristate in total fatty acids . The acid at > 60 microM caused the lysis of gel-filtered platelets (GFP) in both time- and concentration-dependent manners . Scanning electron microscopy showed that: 1) . GFP in 40 microM of the acid changed the morphology to speculate discoid shape at 15 sec, and to ellipsoids after 30 sec; and 2), the cells gradually swelled to spherical forms as the concentration of the acid increased . At nonlytic concentration, the acid inhibited platelet responses to various agonists with differential concentrations . The order of inhibitory potency was U46619 > low dose collagen > ADP-fibrinogen > phorbol ester > high dose collagen . The results demonstrated that 13-methyl myristate exhibited both cell lytic activity and perturbation on membrane function.

Int J Syst Bacteriol, 1996 Jan, 46(1), 119 - 22
"Candidatus comitans," a bacterium living in coculture with Chondromyces crocatus (myxobacteria); Jacobi CA et al.; We describe the phylogenetic position and some taxonomically relevant characteristics of a small pleomorphic gram-negative bacterium that was cocultured with some strains of the myxobacterium Chondromyces crocatus that were isolated from the same geographic and ecological habitat . A 16S ribosomal DNA analysis revealed that the companion was a member of the "Cytophaga-Flavobacterium-Bacteroides" complex and was most closely related to members of the genus Sphingobacterium . The results of a fatty acid analysis, an isoprenoid composition analysis, and a DNA G+C content analysis and the presence of sphingolipids confirmed that this bacterium is affiliated with the genus Sphingobacterium . As the companion bacterium survived for only a few generations on solid media and could not be maintained in pure culture, we assign to this novel taxon that lives in close association with the myxobacterium C . crocatus Candidatus status as "Candidatus comitans."

Biochem J, 1995 Dec 1, 312 ( Pt 2), 569 - 77
Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum; Gu K et al.; The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates . Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism . There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics . The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity . This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies . Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described . Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase {chondro-(1-->3)-glycuronidase, no EC number} and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively . Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation . Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences . The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity . The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages . The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers.

Biochemistry, 1995 Nov 7, 34(44), 14441 - 8
Heparinase I from Flavobacterium heparinum: the role of the cysteine residue in catalysis as probed by chemical modification and site-directed mutagenesis; Sasisekharan R et al.; Heparinase I (heparin lyase I, EC 4.2.2.7), a heparin-degrading enzyme produced by Flavobacterium heparinum, is used to deheparinize blood following extracorporeal procedures in surgery and in other applications . The present study of mapping and characterization of the cysteines of heparinase I represents the first structural characterization of a heparinase . {3H}Iodoacetic acid labeling demonstrated that heparinase I has two free cysteines . One of the two cysteines is surface accessible and lies in a hydrophilic environment while the other is in a hydrophobic environment . Chemical modification of the cysteines, both in the presence and in the absence of heparin, suggests that the surface-accessible cysteine lies in or near the active site of heparinase I . Preferential reactivity of this cysteine with negatively charged sulfhydryl-modifying reagents and the cysteines' high reactivity to iodoacetic acid at pH 6.5 indicate that the surface-accessible cysteine is in a positively charged region . The surface-accessible cysteine (cysteine-135) was mapped as the active-site cysteine by radiolabeling with {3H}iodoacetic acid and by tryptic digestion and peptide sequencing . Site-directed mutagenesis of cysteine-135 to a serine or an alanine in r-heparinase I demonstrates that this cysteine is essential for enzymatic activity . However, replacement of the surface-inaccessible cysteine by a serine or alanine has no effect.

Clin Infect Dis, 1995 Oct, 21(4), 997 - 1000
Relapse of catheter-related Flavobacterium meningosepticum bacteremia demonstrated by DNA macrorestriction analysis; Sader HS et al.; A 6-year-old boy with non-Hodgkin's lymphoma presented because of recurrent episodes (on 13 September 1993 and 12 January 1994) of possibly catheter-related bacteremia . The strains isolated during both episodes and seven epidemiologically unrelated control strains were typed by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA . The similarity of the PFGE patterns of the two isolates suggests that both episodes of bacteremia were caused by the same strain . The antimicrobial susceptibility of the nine strains was tested against 32 antimicrobial agents . The antimicrobial susceptibility patterns of the two isolates from the referred case were identical and differed from those of other clinical isolates . The best in vitro activity (associated with 100% susceptibility) was demonstrated by ofloxacin, clinafloxacin, minocycline, and rifampin.

J Vet Diagn Invest, 1995 Oct, 7(4), 500 - 5
Examination of gills from salmonids with bacterial gill disease using monoclonal antibody probes for Flavobacterium branchiophilum and Cytophaga columnaris; Speare DJ et al.; Bacterial diseases of the gills of commercially reared salmonids in freshwater are common problems . They accounted for 18% of all diagnostic submissions to the Atlantic Veterinary College from commercial fish hatcheries . Definitive diagnosis is difficult because of the growth characteristics of the putative bacteria in culture . Research into the pathogenesis of these diseases has also been similarly limited . Monoclonal antibodies (MAbs) were developed to 2 globally significant gill bacterial pathogens, Flavobacterium branchiophilum, the causative agent of bacterial gill disease, and Cytophaga columnaris, the causative agent of columnaris disease of salmonids . These MAbs were then used as the basis for an indirect fluorescent antibody test to assess archived cases of gill disease in our study region . Of the cases tentatively diagnosed based on histopathology as bacterial gill disease, 76.2% tested positively with the MAbs to F . branchiophilum . Also present within 18.7% of these cases were bacteria which reacted positively to the MAbs for C . columnaris . We conclude that the MAbs produced are valuable diagnostic and research probes for common bacterial disease of the gills of salmon and trout in Atlantic Canada . This study also adds further proof that F . branchiophilum acting alone can be sufficient cause for bacterial gill disease.

Microbiology, 1995 Oct, 141 ( Pt 10), 2585 - 90
A plasmid encoding enzymes for nylon oligomer degradation: nucleotide sequence and analysis of pOAD2; Kato K et al.; The entire nucleotide sequence of nylon oligomer degradative plasmid pOAD2 from Flavobacterium sp . KI723T1 was determined . pOAD2 comprises 45519 bp, with a 66.6 mol% G+C content . The precise loci of the four nylon oligomer degradation genes, namely nylA (6-aminohexanoate-cyclic-dimer hydrolase gene), nylB (6-aminohexanoate-dimer hydrolase), nylB' (a gene having 88% homology to nylB) and nylC (endo-type 6-aminohexanoate oligomer hydrolase), and five IS6100 elements were identified on this plasmid . Comparison of the sequence of pOAD2 with those in the GenBank and EMBL databases revealed that the deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (oligopeptide permeases), ftsX (filamentation temperature sensitive), penDE (isopenicillin N-acyltransferase) and rep (plasmid incompatibility) . A functional map of pOAD2 is presented.

J Biol Chem, 1995 Sep 29, 270(39), 22914 - 23
Structure determination of the octa- and decasaccharide sequences isolated from the carbohydrate-protein linkage region of porcine intestinal heparin; Sugahara K et al.; Previously we isolated a tetrasaccharide-serine and a hexasaccharide-serine from the carbohydrate-protein linkage region of porcine intestinal heparin after digestion with a mixture of Flavobacterium heparinase and heparitinases I and II (Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J.F.G . (1992) J . Biol . Chem . 267, 1528-1533) . In this study four longer carbohydrate sequences (I-IV) attached to Ser or a dipeptide (Ser-Gly or Gly-Ser), which accounted for at least 18.2% of the total linkage region, were isolated from the same heparin preparation after digestion with heparinase only . IV was successfully isolated only after subsequent digestion with glycuronate-2-sulfatase . Their structures were determined by chemical and enzymatic analyses and 1H NMR spectroscopy and found to be the following octa- and decasaccharide sequences attached to Ser in a molar ratio of 1.1:2.3:1.0:1.3: delta HexA(2S)alpha 1-4GlcN(NS,6S)alpha 1-4GlcA beta 1-4GlcNAc alpha 1-4- GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (I), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1- 3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (II), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1- 4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcNAc-alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (III), delta HexA alpha 1-4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc(6S)alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (IV) (delta HexA, GlcA, IdoA, and GlcN represent 4,5-unsaturated hexuronic acid, D-glucuronic acid, L-iduronic acid, and D-glucosamine, whereas 2S, 6S, and NS stand for 2-sulfate, 6-sulfate, and N-sulfate, respectively) . I and II contained 1 mol of Gly in addition to Ser . The four structures indicate that sulfation in heparin chains takes place on the monosaccharide residues located in closer vicinity to the core protein than found for heparan sulfate chains and that there exist at least several heparin subclass chains with different linkage region structures . The significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of heparin.

Tidsskr Nor Laegeforen, 1995 Sep 10, 115(21), 2646 - 7
{Meningitis after myelography}; Bo SH et al.; Lumbar myelography is still frequently used in cases of suspected lumbar radiculopathy . Since 1984, iohexol has been the contrast medium of choice in myelography, and so far only a few cases of chemical meningitis have been reported . Bacterial meningitis cannot be distinguished from chemical meningitis on the basis of clinical findings . A cerebrospinal fluid Gram stain and culture are the only truly reliable tests in deciding the etiology of the meningitis . We describe two patients who developed meningitis following myelography with iohexol . In one of the patients, the cerebrospinal fluid culture was positive with subsequent identification of Flavobacterium meningosepticum, a species not previously reported as an infectious agent in meningitis after myelography.

Nihon Kyobu Shikkan Gakkai Zasshi, 1995 Sep, 33(9), 1024 - 9
{Hypersensitivity pneumonitis caused by a home humidifier}; Hagiwara S et al.; A 33-year-old man was admitted complaining of a fever, dyspnea, and a dry cough almost every night since December of 1992 . He had been using an ultrasonic humidifier at home . The chest CT scan and roentgenogram showed bilateral reticulonodular shadows . After admission, the symptoms resolved spontaneously . These findings were suggestive of hypersensitivity pneumonitis . After analysis of fluid obtained by bronchoalveolar lavage and of a specimen obtained by transbronchial biopsy, "humidifier lung" was diagnosed . Ten species of microorganisms were isolated from the water left in the patient's humidifier . On precipitation and complement fixation tests of the patients serum, the results were positive for three of those microorganisms: Flavobacterium multivorum, Yersinia pseudotuberculosis, and Aureobacterium liquefaciens . The titer on the complement fixation test increased immediately after a provocation test . The laboratory results suggest that at least one of these three microorganisms was the causative antigen in this case.

J Antibiot (Tokyo), 1995 Sep, 48(9), 924 - 8
Sulfobacins A and B, novel von Willebrand factor receptor antagonists . I . Production, isolation, characterization and biological activities; Kamiyama T et al.; Sulfobacins A and B, novel von Willebrand factor (vWF) receptor antagonists, have been isolated from the culture broth of Chryseobacterium sp . (Flavobacterium sp.) NR 2993 by ethyl acetate extraction, and by Sephadex LH-20 and silica gel column chromatographies . The physico-chemical properties of the sulfobacins indicate that their structures are completely different from that of aurintricarboxylic acid, the one known vWF receptor antagonist . Sulfobacins A and B inhibit the binding of vWF to its receptor with IC50S of 0.47 and 2.2 microM, respectively . Sulfobacin A also inhibits ristocetin-induced agglutination in human platelets fixed with paraformaldehyde with an IC50 of 0.58 microM.

Appl Environ Microbiol, 1995 Sep, 61(9), 3486 - 9
Biodiversity of gas vacuolate bacteria from Antarctic sea ice and water; Gosink JJ et al.; Psychrophilic, gas vacuolate, heterotrophic bacteria indigenous to sea ice communities in Antarctica have been isolated . Phylogenetic analysis of representative members of these bacteria shows that they belong to the alpha, beta, and gamma Proteobacteria and the Flavobacteria-Cytophaga group . This is the first report of gas vacuolate bacteria from the beta Proteobacteria and the Flavobacteria-Cytophaga groups.

J Pharmacol Exp Ther, 1995 Sep, 274(3), 1370 - 8
JTP-4819: a novel prolyl endopeptidase inhibitor with potential as a cognitive enhancer; Toide K et al.; JTP-4819 ((S)-2-{{(S)-2-(hydroxyacetyl)-1-pyrrolidinyl}carbonyl}-N- phenylmethyl)-1-pyrrolidinecarboxamide) is a potent (IC50: 0.83 +/- 0.09 nM in rat brain supernatant; 5.43 +/- 0.81 nM in Flavobacterium meningosepticum) and specific inhibitor of prolyl endopeptidase (PEP) . JTP-4819 (3 mg/kg p.o.) exhibited a strong and durable ex vivo inhibitory effect on PEP in various regions of the rat brain . In addition, JTP-4819 inhibited the degradation of substance P, arginine-vasopressin, thyrotropin-releasing hormone, neurotensin, oxytocin, bradykinin, and angiotensin II by purified PEP with IC50 values of 9.6, 13.9, 10.7, 14.0, 4.5, 7.6 and 10.6 nM, respectively . In the one-trial passive avoidance test in rats with scopolamine-induced amnesia, JTP-4819 significantly prolonged the retention time when administered orally at doses of 1 and 3 mg/kg 1 hr before acquisition or at 3 and 10 mg/kg 1 hr before retention . In addition, coadministration of JTP-4819 and substance P, arginine-vasopressin or thyrotropin-releasing hormone (at doses at which each drug alone did not prolong the retention time) improved the retention time of rats with scopolamine-induced amnesia . Microdialysis studies demonstrated that JTP-4819 caused a significant increase in ACh release in the frontal cortex and hippocampus of young rats at oral doses of 1 and 3 mg/kg, as well as in both brain regions of aged rats at a dose of 3 mg/kg . These results indicate that JTP-4819 potentiates neuropeptide functions inhibiting PEP, that it activates cholinergic transmission and that it enhances learning and memory.

Avian Dis, 1995 Jul-Sep, 39(3), 622 - 6
Detection of proteolytic bacteria in the upper respiratory tract flora of poultry; Byrum BR et al.; Proteolytic bacteria were readily demonstrated among the upper respiratory tract flora of poultry in two chicken flocks and two turkey flocks . Between 20% and 50% of birds in each flock had highly proteolytic bacteria in the upper respiratory tract flora, and the amount of the bacterial flora determined to be highly proteolytic in any given bird ranged from none to a majority . Highly proteolytic bacteria recovered included five species of staphylococci, as well as two gram-negative bacterial species, Flavobacterium sp . and Vibrio alginolyticus . All species of staphylococci isolated have previously been recovered from animal sources, whereas Flavobacterium sp . and V . alginolyticus are usually reported to be associated with soil and surface-water origins . The protocols used to screen for proteolytic bacteria among the flora and to assess protease activity required minimal supplies and equipment.

Biochem Mol Biol Int, 1995 Jul, 36(3), 627 - 31
Plasmid mediated organophosphate pesticide degradation by Flavobacterium balustinum; Somara S et al.; A bacterium capable of degrading methyl parathion, an organophosphorus insecticide into paranitrophenol (as evidenced by TLC) and other metabolites, was isolated from the agricultural soils of Anantapur district, Andhra Pradesh, India . This bacterium, identified as Flavobacterium balustinum was found to harbour an indigenous plasmid of approximately 86 kb in size . The degradative enzyme, parathion hydrolase, was found to be encoded by this plasmid . No enzyme activity was observed in plasmid cured strain.

Arch Biochem Biophys, 1995 Jun 20, 320(1), 123 - 8
Cloning, sequencing, and expression of the dipeptidyl peptidase IV gene from Flavobacterium meningosepticum in Escherichia coli; Kabashima T et al.; The dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) gene from Flavobacterium meningosepticum was cloned by Southern and colony hybridizations using probes amplified by PCR, and expressed in Escherichia coli DH1 . E . coli DH1 harboring pFDP-H1, which was a subclone derived from the positive clone pFDP-1, showed 3.5-fold higher activity than F . meningosepticum . Nucleotide sequencing analysis revealed an open reading frame of 2133 bp, coding for a protein of 711 amino acids with a predicted molecular weight of 80,626 . The expressed enzyme in E . coli DH1/pFDP-H1 was purified about 345-fold with an activity recovery of 12.3% . The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160,000 by gel filtration, respectively, suggesting a dimeric form of the native enzyme . The deduced amino acid sequence of DP IV was homologous to those of the serine proteases of the "prolyl endopeptidase family." A sequence near the C-terminal region and the putative catalytic triad residues were well conserved among these enzymes.

J Biol Chem, 1995 Jun 2, 270(22), 13197 - 203
Detailed structural analysis of a novel, specific O-linked glycan from the prokaryote Flavobacterium meningosepticum; Reinhold BB et al.; In the preceding paper, preliminary analysis revealed a new type of O-linked oligosaccharide of 1244 Da at each of two proposed glycosylation sites on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum (Plummer, T . H., Jr., Tarentino, A . L., and Hauer, C . R . (1995) J . Biol . Chem . 270, 13192-13196) . In this report we detail the linkage, sequence, and branching of this unusual heptasaccharide by electrospray (ES) ionization mass spectrometry (MS), and collision-induced dissociation (CID) . The proposed structure was supported by a combination of isotopic labeling, composition and methylation analysis, and the preparation of several chemical analogs and derivatives with each product evaluated by MS and CID . The singly branched structure contained seven residues, including three different uronyl analogs: a methylated rhamnose and mannose, a glucose, and a reducing terminal mannose . Only pyranose ring forms were detected ((2-OMe)Man1-4GlcNAcU1-4GlcU1-4Glc1-4(2-OMe)G lcU-4 {(2-OMe)Rham1-2}Man).

J Biol Chem, 1995 Jun 2, 270(22), 13192 - 6
Novel, specific O-glycosylation of secreted Flavobacterium meningosepticum proteins . Asp-Ser and Asp-Thr-Thr consensus sites; Plummer TH Jr et al.; A new type of O-linked oligosaccharide has been discovered on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum, including Endo F2 (three sites), Endo F3 (one site), and a P40 protease (one site) . The oligosaccharide moiety is covalently attached via a mannose residue to a serine or threonine at consensus sites corresponding to Asp-Ser* or Asp-Thr*-Thr . Preliminary characterization by mass spectroscopy revealed an oligosaccharide of 1244 Da at each of the proposed glycosylation sites . Collision-associated dissociation analysis showed a characteristic daughter ion series of m/z 218, 394, and 556, indicative of a common Flavobacterium oligosaccharide . Compositional analysis demonstrated an unusual profile of monosaccharides, including hexoses, methylated hexoses, and uronic acid derivatives.

Can J Microbiol, 1995 Jun, 41(6), 470 - 6
Degradation of polycyclic aromatic hydrocarbons (PAHs) by a mixed culture and its component pure cultures, obtained from PAH-contaminated soil; Trzesicka-Mlynarz D et al.; A mixed culture, isolated from soil contaminated with polycyclic aromatic hydrocarbons (PAHs), grew on and degraded fluoranthene in aqueous media supplemented with glucose, yeast extract, and peptone . Increased complex nitrogen levels in the medium promoted bacterial growth and a greater extent of fluoranthene degradation . Amendment of the media with high glucose levels also diminished specific fluoranthene degradation . The mixed culture was capable of degrading a range of other PAHs, including benzo{a}pyrene, anthracene, phenanthrene, acenaphthene, and fluorene . The mixed culture contained four predominant isolates, all of which were Gram-negative rods, three of which were identified as Pseudomonas putida, Flavobacterium sp., and Pseudomonas aeruginosa . Better degradation of a defined PAH mixture was observed with the mixed culture than with individual isolates . A reconstituted culture, prepared by combining the four individual isolates, manifested a similar PAH biodegradation performance to the original mixed culture . When compared with the mixed culture, individual isolates exhibited a relatively good capacity to remove more water-soluble PAHs (acenaphthene, fluorene, phenanthrene, fluoranthene) . In contrast, removal of less water-soluble PAHs (anthracene and pyrene) was low or negligible with isolated cultures compared with the mixed culture.

Med J Malaysia, 1995 Jun, 50(2), 158 - 61
Anti-lymphocyte globulin therapy in aplastic anaemia--a university hospital experience; Leong KW et al.; Antilymphocyte globulin (ALG) was given every other day for 5 doses with platelet transfusions immediately following ALG administration in 6 patients with aplastic anaemia . Four patients responded and 3 durable remissions were achieved . One patient relapsed and further treatment with anti-thymocyte globulin and cyclosporin also failed . One patient died of Flavobacterium septicaemia 6 days after completion of ALG . Our data suggests that using an alternate day regimen, a response rate similar to a daily regimen can be obtained.

APMIS, 1995 Jun, 103(6), 475 - 6
Self-induced bacteremia . Case report; Kristensen B et al.; We describe a patient with perforated appendicitis who postoperatively suffered repeated episodes of shaking chills and temperature spikes . Initial blood cultures yielded growth of Flavobacterium meningosepticum, Pseudomonas putida and Pseudomonas paucimobilis, and succeeding blood cultures growth of Pseudomonas acidoverans . These bacteria in combination led to a suspicion of self-inoculation of contaminated water through an intravenous catheter . Antibiotic treatment had no effect on the symptoms, which only ceased when the intravenous catheter was removed.

Structure, 1995 May 15, 3(5), 449 - 57
Crystal structure of endo-beta-N-acetylglucosaminidase H at 1.9 A resolution: active-site geometry and substrate recognition; Rao V et al.; BACKGROUND: Endo-beta-N-acetylglucosaminidase H (Endo H), an endoglycosidase secreted by Streptomyces plicatus, hydrolyzes the glycosidic bond between the core N-acetyglucosamine residues of asparagine-linked high-mannose oligosaccharides . Endo H is a commonly used reagent in glycobiology research, including the characterization of oligosaccharides in glycoproteins . On-going crystallographic studies of Endo H and related endoglycosidases are aimed at identifying the molecular features that determine the different substrate specificities of these enzymes . RESULTS: The three-dimensional structure of Endo H has been determined to 1.9 A resolution . The overall fold of the enzyme is that of an irregular (alpha/beta)8-barrel comprising eight beta-strand/loop/alpha-helix units . Units 5 and 6 have very short loop sections at the top of the molecule and their alpha-helices are replaced by sections of extended geometry . The loop of unit 2 includes a small two-stranded antiparallel beta-sheet . A shallow curved cleft runs across the surface of the molecule from the area of units 5 and 6, over the core of the beta-barrel to the area of the beta-sheet of loop 2 . This cleft contains the putative catalytic residues Asp130 and Glu132 above the core of the beta-barrel . These residues are surrounded by several aromatic residues . The loop 2 area of the cleft is formed by neutral polar residues, mostly asparagines . CONCLUSIONS: The structure of Endo H is very similar to that of Endo F1, a closely related endoglycosidase secreted by Flavobacterium meningosepticum . Detailed comparison of the structures of Endo H and Endo F1 supports the model previously proposed for substate binding and recognition, in which the area of loop 2 determines the substrate specificity and the alpha-helices of units 5 and 6 are missing to accommodate the protein moiety of the substrate.

Arch Biochem Biophys, 1995 May 10, 319(1), 281 - 5
Molecular cloning and sequence analysis of flavastacin: an O-glycosylated prokaryotic zinc metalloendopeptidase; Tarentino AL et al.; A new zinc metalloendopeptidase that cleaves peptides on the amino-terminal side of aspartic acid was isolated from the cultural filtrate of Flavobacterium meningosepticum . The gene for this new enzyme was cloned into pBluescript, and the complete nucleotide sequence was determined . Over 40% of the deduced amino acid sequence was verified independently by direct protein microsequencing . The most important structural features of this new enzyme include (i) the presence of an unusual O-linked oligosaccharide of unknown function located at a unique consensus site near the C-terminus and (ii) a characteristic extended zinc-binding site and corresponding Met-turn that places this metalloendopeptidase in the astacin family . This is the first example of a prokaryotic enzyme related to the eukaryotic astacin group; it is being designated hereafter as flavastacin.

J Biol Chem, 1995 Apr 14, 270(15), 8696 - 705
Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate . Heparinase cleaves glucuronate 2-O-sulfate-containing disaccharides in highly sulfated blocks in heparin; Yamada S et al.; Eleven tetrasaccharides were isolated from the repeating disaccharide region of porcine intestinal heparin after strong digestion with Flavobacterium heparinase . Their structures were determined by composition analysis, enzymatic analysis, and 1H NMR spectroscopy . Nine of them have the common tetrasaccharide backbone, delta HexA alpha 1-4GlcN alpha 1-4IdoA alpha 1-4GlcN, where delta HexA and IdoA represent 4,5-unsaturated hexuronic acid and L-iduronic acid, respectively, and their structural variations are based upon the positions of sulfate groups . The nine compounds include one hexasulfated, three pentasulfated and five tetrasulfated compounds, and four of them have not been isolated previously as discrete structures . The other two of the 11 tetrasaccharides have the following hitherto unreported structures with novel glucuronate 2-O-sulfate at the internal position: delta HexA(2-sulfate) alpha 1- 4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N-sulfate) and delta HexA(2-sulfate) alpha 1-4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N,6-disulfate) . Thus, 2-O-sulfated glucuronate in the highly sulfated tetrasaccharide structures typical of heparin has been demonstrated . The former and the latter tetrasaccharides account for 0.31 and 0.32% (w/w) of the starting heparin, respectively . Their yield, however, is an underestimation, since these tetrasaccharide structures in longer sequences will be degraded by heparinase . Although the latter tetrasaccharide described above was unexpectedly cleaved by heparinase into two disaccharide units, the former was not degraded by the enzyme most likely due to the lack of the 6-O-sulfate group on the GlcN residue at the reducing terminus . The results indicate its capability of catalyzing both anti and syn elimination, a property shared by heparitinases I and II and chondroitinase ABC . Both tetrasaccharides were degraded into disaccharides by heparitinase II . Therefore, it is necessary to reevaluate the disaccharide composition of heparin/heparan sulfate or oligosaccharide structures, which were previously determined after heparinase or heparitinase II digestion . It is no longer possible to conclude that the 2-O-sulfated unsaturated uronic acid residues obtained from heparin/heparan sulfate by lyase digestions are always derived from iduronate 2-O-sulfate residues in the original polymer . It is quite possible that the novel glucuronate 2-O-sulfate structure in the highly sulfated region of heparin is involved in some of the biological activities of heparin.

Rev Argent Microbiol, 1995 Apr-Jun, 27(2), 99 - 105
{Growth and survival of Azospirillum in roots and maize rhizospheres with different levels of acidity}; Eory VJ et al.; An experiment was carried out in order to evaluate the effect of pH on Azospirillum sp . growth and survival in maize rhizosphere . Sterilized maize seeds were sown in a perlite substratum with addition of a nutritive medium . The pots were buffered at two different pHs: 5.8 (group one) and 7.0 (group two) . Each group was divided in two treatments: inoculated with Azospirillum sp . Az-39 and non-inoculated . Experimental pots were incubated at 20 degrees C with a 14 hour photoperiod . Growth of non-inoculated roots was negligible . Inoculated roots showed a better response at pH 5.8 than at 7.0 . Several accompanying bacteria were found . Azospirillum grew in both groups with a low penetration into roots . A set of nutritive relationships among microorganisms and maize roots was observed; Xanthomonas is a maize pathogenic bacteria, and it is a NO3- consumer, and uses this anion as hydrogen acceptor . The Gram (-) Diplococcus is a nitrate producer . Cytophaga and Flavobacterium are related with roots decomposition . It is concluded that Azospirillum improves the root growth, mainly at pH 5.8.

Appl Biochem Biotechnol, 1995 Apr, 53(1), 1 - 9
Preparation of deglycosylated egg white avidin; Bayer EA et al.; A simple procedure for the preparation of deglycosylated avidin is described . Commercially obtained avidin was treated with a mixed microbial culture . The cells were capable of growing on the oligosaccharide residues, but generally ignored the polypeptide portion of the egg white glycoprotein . The resultant deglycosylated avidin retained its biotin-binding characteristics . The major bacterial strain (strain BECH080), responsible for the deglycosylation, was isolated . On the basis of elementary biochemical tests, fatty acid, and phenotypic analyses, the isolate was identified as a strain of Flavobacterium meningosepticum . The primary enzymatic activity that caused the removal of the oligosaccharide residues of avidin appeared to be similar to endoglycosidase F.

Proc R Soc Lond B Biol Sci, 1995 Mar 22, 259(1356), 293 - 9
The establishment of intracellular symbiosis in an ancestor of cockroaches and termites; Bandi C et al.; All cockroaches examined so far have been found to harbour a bacterial endosymbiont in specialized cells of the fat body, whereas Mastotermes darwiniensis is the only termite currently known to harbour an intracellular symbiont . The localization and mode of transmission of these bacteria are surprisingly similar, but so far no data have been published on their phylogenetic relationships . To address this issue, molecular sequence data were obtained from the genes encoding the small subunit ribosomal RNA of the M . darwiniensis endosymbiont, and compared with those obtained from endosymbionts of seven species of cockroaches . Molecular phylogenetic analysis unambiguously placed all these bacteria among the flavobacteria-bacteroides, indicating that the endosymbiont of M . darwiniensis is the sister group to the cockroach endosymbionts examined . Additionally, nucleotide divergence between the endosymbionts appears to be congruent with the palaeontological data on the hosts's evolution . These results support previous claims that the original infection occurred in an ancestor common to cockroaches and termites . A loss of endosymbionts should subsequently have occurred in all termite lineages, except that which gave rise to M . darwiniensis.

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