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PubMed1993

Cloning and expression of heparinase I gene from Flavobacterium heparinum.

Heparinases, enzymes that cleave heparin and heparin sulfate, are implicated in physiological and pathological functions ranging from wound healing to tumor metastasis and are useful in deheparinization therapies. We report the cloning of the heparinase I (EC 4.2.2.7) gene from Flavobacterium heparinum using PCR. Two degenerate oligonucleotides, based on the amino acid sequences derived from tryptic peptides of purified heparinase, were used to generate a 600-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. This probe was used to screen a Flavobacterium genomic DNA library in pUC18. The open reading frame of heparinase I is 1152 bp in length, encoding a precursor protein of 43.8 kDa. Eleven of the tryptic peptides (approximately 35% of the total amino acids) mapped onto the open reading frame. The amino acid sequence reveals a consensus heparin binding domain and a 21-residue leader peptide with a characteristic Ala-(Xaa)-Ala cleavage site. Recombinant heparinase was expressed in Escherichia coli as a soluble protein, using the T7 polymerase pET expression system. The recombinant heparinase cleavage of heparin was identical to that of native heparinase.

Amino Acid Sequence↗

PubMed1998

Crystal structures of Flavobacterium glycosylasparaginase. An N-terminal nucleophile hydrolase activated by intramolecular proteolysis.

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

Amino Acid Sequence↗

PubMed1993

Unusually biased nucleotide sequences on sense strands of Flavobacterium sp. genes produce nonstop frames on the corresponding antisense strands.

From investigation of eight Flavobacterium sp. genes encoding enzyme proteins, it was found that six genes had nonstop frames (NSFs) on the antisense strands, and base sequences of the genes are mainly composed of repeating triplet sequence(s), 5'-GNC-3' (where G and C are guanine and cytosine, and N is either of the four bases), in the reading frames. Thus, we concluded that the biased nucleotide sequences on the sense strands produce NSFs on the corresponding antisense strands. Furthermore, from the precise alignments of both nucleotide and amino acid sequences of two related Flavobacterium sp. genes, nyIB and nyIB', it was found that base replacements might have occurred symmetrically in the codons. That is, transversions between G and C were observed at high frequencies at the first and third positions of codons, but not at the second positions. At the first position, AG base transitions were observed much more than similar CT transitions, whereas CT transitions were found at the third positions at a relatively high frequency. These suggest that symmetrical base replacements in codons might be the main contribution to evolution in Flavobacterium sp. genes.

Amidohydrolases↗

PubMed2001

Flavobacterium frigidarium sp. nov., an aerobic, psychrophilic, xylanolytic and laminarinolytic bacterium from Antarctica.

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

Aerobiosis↗

PubMed2005

Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa.

Earthworms emit nitrous oxide (N(2)O) via the activity of bacteria in their gut. Four N(2)O-producing facultative aerobes, ED1(T), ED5(T), MH21(T) and MH72, were isolated from the gut of the earthworm Aporrectodea caliginosa. The isolates produced N(2)O under conditions that simulated the microenvironment of the earthworm gut. ED1(T) and ED5(T) were Gram-negative, motile rods that carried out complete denitrification (i.e. the reduction of nitrate to N(2)) and contained membranous c-type cytochromes. ED1(T) grew optimally at 30 degrees C and pH 7. ED1(T) oxidized organic acids and reduced (per)chlorate, sulfate, nitrate and nitrite. The closest phylogenetic relative of ED1(T) was Dechloromonas agitata. ED5(T) grew optimally at 25 degrees C and pH 7. ED5(T) grew mainly on sugars, and nitrate and nitrite were used as alternative electron acceptors. The closest phylogenetic relatives of ED5(T) were Flavobacterium johnsoniae and Flavobacterium flevense. MH21(T) and MH72 were motile, spore-forming, rod-shaped bacteria with a three-layered cell wall. Sugars supported the growth of MH21(T) and MH72. Cells of MH21(T) grew in chains, were linked by connecting filaments and contained membranous b-type cytochromes. MH21(T) grew optimally at 30-35 degrees C and pH 7.7, grew by fermentation and reduced low amounts of nitrite to N(2)O. The closest phylogenetic relatives of MH21(T) were Paenibacillus borealis and Paenibacillus chibensis. Based on morphological, physiological and phylogenetic characteristics, ED1(T) (= DSM 15892(T) = ATCC BAA-841(T)), ED5(T) (= DSM 15936(T) = ATCC BAA-842(T)) and MH21(T) (=DSM 15890(T) = ATCC BAA-844(T)) are proposed as type strains of the novel species Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov. and Paenibacillus anaericanus sp. nov., respectively. MH72 is considered a new strain of Paenibacillus terrae.

Aerobiosis↗

PubMed2006

Flavobacterium saliperosum sp. nov., isolated from freshwater lake sediment.

An aerobic, yellow-pigmented, Gram-negative bacterium, designated strain S13T, was isolated from freshwater sediment of Taihu Lake in central China. The taxonomy of strain S13T was studied by using phenotypic and phylogenetic methods. Cells of strain S13T were rod-shaped, non-motile and with a size range of 0.35-0.55x1.5-2.5 microm. The nearly complete 16S rRNA gene of strain S13T was amplified and sequenced. A blast search and phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain S13T was related to members of the genus Flavobacterium, with the highest sequence similarity of 93.8% to Flavobacterium columnare (ATCC 23463T). Cells contained menaquinone-6 (MK-6) as the major respiratory quinone and the genomic DNA G+C content was 41 mol%. The major fatty acids were iso-C15:0 (28.2%) and iso-C17:1omega9c (19.0%). It is proposed that S13T (=CGMCC 1.3801T=JCM 13331T) represents the type strain of a novel species, Flavobacterium saliperosum sp. nov.

Bacterial Typing Techniques↗

PubMed2005

Identification and expression of a host-recognized antigen, FspA, from Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome, an economically important disease of immature salmonid fish for which there is no vaccine. Convalescent serum from the host, rainbow trout (Oncorhynchus mykiss), reacted strongly with a approximately 20 kDa, Flavobacterium-specific protein antigen (subsequently named FspA) from F. psychrophilum. Protein-enriched, detergent-partitioned samples were separated by two-dimensional gel electrophoresis and the protein target was excised, proteolytically cleaved and the resulting peptides analysed by MS. Quadrupole-time-of-flight MS was used to generate a fragmented peptide spectrum. The resulting peptide sequences were then used to design degenerate PCR primers to amplify the gene (fspA) of interest: 612 bp encoding 203 aa, including a putative 19 aa N-terminal signal sequence which predicted a processed 19 303.6 Da protein. FspA proved to be unique and only homologous to two unspecified sequences reported from Flavobacterium johnsoniae, although weakly homologous to a Yersinia pseudotuberculosis adhesin. An amplified gene fragment (537 bp, encoding 179 aa) was further cloned into an expression vector, expressed as a approximately 30 kDa N-terminal fusion protein and found to retain its strong reactivity with host serum antibodies. These results suggest that the surface-localized FspA may be an important subunit vaccine candidate antigen against F. psychrophilum.

Animals↗

PubMed1991

Complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp.

The complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp. has been determined by analysis of peptides after cleavage with lysyl endopeptidase, pepsin and chymotrypsin. The protein consists of a single polypeptide chain consisting of 267 amino acid residues and a molecular mass of 27972 Da. The sequence of Flavobacterium endo-beta-N-acetylglucosaminidase is very close to that of the Streptomyces enzyme (endo-H), having 60% similarity and very similar hydropathy profiles. Similarities were also found between Flavobacterium endo-beta-N-acetylglucosaminidase and chitinases from Bacillus circulans, Serratia marcescens and Phaseolus vulgaris.

Amino Acid Sequence↗

PubMed1991

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.

Amino Acid Sequence↗

PubMed1992

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.

Biodegradation, Environmental↗

PubMed1996

Development of techniques to genetically manipulate members of the genera Cytophaga, Flavobacterium, Flexibacter, and Sporocytophaga.

The Bacteroides-Cytophaga-Flavobacterium branch of the eubacterial phylogenetic tree contains a diverse group of bacterial species. Techniques for the genetic manipulation of Bacteroides spp. are well developed (A. A. Salyers, N. B. Shoemaker, and E. P. Guthrie, Crit. Rev. Microbiol. 14:49-71, 1987). Recently we developed techniques to genetically manipulate the gliding bacterium Cytophaga johnsonae (M. J. McBride and M. J. Kempf, J. Bacteriol. 178:583-590, 1996). We now demonstrate that some of these techniques allow genetic manipulation of a number of environmentally or medically significant bacteria in this group. The Bacteroides transposon Tn4351 was introduced into Cytophaga hutchinsonii, Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadensis, Flexibacter sp. strain FS1, and Sporocytophaga myxococcoides by conjugation. Tn4351 integrated itself into the host chromosomes and conferred erythromycin resistance. We isolated several auxotrophic mutants of Flavobacterium meningosepticum following Tn4351 mutagenesis. The C. johnsonae-Escherichia coli shuttle vector pCP11 functioned in C. succinicans but not in the other bacteria. pLYL03 did not replicate in any of these bacteria and should function as a convenient suicide vector. The identification of a system of gene transfer, a selectable marker, a suicide vector, and a transposon that functions in these diverse bacteria allows genetic manipulations to be performed.

Blotting, Southern↗

PubMed2001

Polyamine distribution profiles in newly validated genera and species within the Flavobacterium-Flexibacter-Cytophaga-Sphingobacterium complex.

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

Bacteria↗

PubMed1992

Flavobacterium indologenes infection in leopard frogs.

An investigation of an epidemic of infectious disease in a frog (Rana pipiens) colony was conducted. Six of 40 frogs in a continuous (once through) water flow housing system had weight loss, swollen abdomen, corneal edema, uveitis, subcutaneous edema, petechial hemorrhage, incoordination, and respiratory distress. The frogs had lesions consistent with bacterial septicemia. A gram-negative, nonfermenting bacillus, Flavobacterium indologenes (Flavobacterium sp biovar IIb), was isolated in pure culture from tissues and blood. The clinical isolate was used to inoculate healthy frogs sc. An isolate identical to the one isolated from the sick frogs was recovered from tissues and blood of the inoculated frogs. Inoculation of the housing water in a nonflow-through system did not result in disease, despite proliferation of the Flavobacterium spp in the water; therefore, it is likely that establishment of infection requires the presence of the organism in sufficient numbers and a portal of entry into the body.

Animals↗

PubMed2004

[Isolation and analysis of the drug resistance of the flavobacterium and its production of beta-lactamases].

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

Anti-Bacterial Agents↗

PubMed1990

5S rRNA sequences of representatives of the genera Chlorobium, Prosthecochloris, Thermomicrobium, Cytophaga, Flavobacterium, Flexibacter and Saprospira and a discussion of the evolution of eubacteria in general.

5S rRNA sequences were determined for the green sulphur bacteria Chlorobium limicola, Chlorobium phaeobacteroides and Prosthecochloris aestuarii, for Thermomicrobium roseum, which is a relative of the green non-sulphur bacteria, and for Cytophaga aquatilis, Cytophaga heparina, Cytophaga johnsonae, Flavobacterium breve, Flexibacter sp. and Saprospira grandis, organisms allotted to the phylum 'Bacteroides-Cytophaga-Flavobacterium' and relatives as determined by 16S rRNA analyses. By using a clustering algorithm a dendrogram was constructed from these sequences and from all other known eubacterial 5S RNA sequences. The dendrogram showed differences, as well as similarities, with respect to results obtained by 16S RNA analyses. The 5S RNA sequences of green sulphur bacteria were closely related to one another, and to a cluster containing 5S RNA sequences from Bacteroides and its relatives, including Cytophaga aquatilis. 5S RNA sequences of all other representatives of the 'Bacteroides-Cytophaga-Flavobacterium' phylum as distinguished by 16S RNA analysis failed to group with Bacteroides and related clusters. On the basis of 5S RNA sequences, Thermomicrobium roseum clustered with Chloroflexus aurantiacus, as was expected from 16S RNA analysis.

Base Sequence↗

PubMed1963

METABOLISM OF PHENOXYALKYL CARBOXYLIC ACIDS BY A FLAVOBACTERIUM SPECIES.

Macrae, I. C. (Cornell University, Ithaca, N.Y.), and M. Alexander. Metabolism of phenoxyalkyl carboxylic acids by a Flavobacterium species. J. Bacteriol. 86:1231-1235. 1963.-A Flavobacterium sp. isolated from soil and grown in media containing 4-(2,4-dichlorophenoxy) butyric acid metabolized omega-linked 2,4-dichlorophenoxyalkyl carboxylic acids in the series from 3-(2,4-dichlorophenoxy)propionic acid through 11-(2,4-dichlorophenoxy)undecanoic acid rapidly and without a preliminary induction phase. There was no detectable oxidation of 2,4-dichlorophenoxyacetic acid. Phenols and the fatty acids corresponding to the aliphatic side chains were liberated during the decomposition of the dichlorophenoxy alkanoates from propionate to octanoate. The data indicate that the initial step in the degradation of omega-linked 2,4-dichlorophenoxyalkyl carboxylic acids by the bacterium involves a cleavage of the ether linkage, a new mechanism for the microbial metabolism of these compounds.

Bacteria↗

PubMed2005

Volatile organic compounds from arctic bacteria of the Cytophaga-Flavobacterium-Bacteroides group: a retrobiosynthetic approach in chemotaxonomic investigations.

Volatile organic compounds emitted by different marine arctic strains of the Cytophaga-Flavobacterium-Bacteroides group were investigated by using a modified closed-loop stripping apparatus (CLSA). Seven of nine strains emitted volatiles, dominated by methyl ketones, in specific patterns. The methyl ketones were aliphatic saturated, or unsaturated, and comprised 12 to 18 C-atoms, sometimes with terminal Me branches. They were identified by GC/MS, retention-index calculations, derivatization with dimethyl disulfide for C=C bond location, and GC/FTIR to elucidate their uniform (Z)-configuration. The proposed structures of all methyl ketones were subsequently confirmed by synthesis, while the absolute configuration of chiral volatiles was elucidated by stereoselective synthesis. From retrobiosynthetic considerations, it was found that strain ARK10267 uses mainly valine, and strain ARK10063 mainly isoleucine for formation of starters for the ketone biosynthesis, which is correlated to fatty acid biosynthesis. Four strains (ARK10223, ARK10044, ARK10141, and ARK10146) use leucine. These separations are supported by phylogenetic affiliations based on 16S rRNA. Strain ARK10255b, in the course of this study found to be not a member of the Cytophaga-Flavobacterium-Bacteroides phylum, did not emit aliphatic ketones of medium chain length, but methionine-derived 4-(methylsulfanyl)butan-2-one and corresponding 4-(methylsulfanyl)butan-2-ol. Most of the compounds described have not been reported previously from nature.

Arctic Regions↗

PubMed1980

Deoxyribonucleic acid relatedness of some menaquinone-producing Flavobacterium and Cytophaga strains.

Nine menaquinone-forming strains of the Flavobacterium--Cytophaga complex with DNA base compositions between 35 and 45 moles percent guanine-plus-cytosine were investigated for genome sizes and DNA relatedness by DNA:DNA hybridization in vitro, using the optically recorded initial reassociation kinetics. Two strains representing C. hutchinsonii and C. marinoflava proved to be related on the 50 percent binding level, i.e. on a level of DNA relatedness commonly found within well-classified conventional genera of bacteria. Strains of C. johnsonae, F. heparinum, F. meningosepticum, F. odoratum, F. pectinovorum, and an unnamed Flavobacterium--Cytophaga strain were found to be interrelated, and linked to the genus Cytophaga, on the 30, or 20 percent binding levels, respectively. These findings indicate that the organisms in question are related to Cytophaga. They therefore should be transferred into the family Cytophagaceae.

Base Composition↗