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