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Superoxide dismutase in strains of the genus Flavobacterium: isolation and characterization.

Two electrophoretically different forms of superoxide dismutase, one of them containing manganese-protein and the other iron-protein, were detected in eleven different strains of the genus Flavobacterium. The activities of the different strains were similar to those described for other bacteria. The two molecular forms of the enzyme differed clearly with regard to activity, electrophoretic behaviour, sensitivity to cyanide and peroxide, and NaCl requirement. Both molecular forms were isolated from Flavobacterium halmephilum. Molecular mass absorption spectra, metal content, optimum pH, heat-sensitivity and stability were described.

Chromatography, DEAE-Cellulose↗

The carotenoids of Flavobacterium strain R1560.

The main carotenoid of Flavobacterium strain R1560 has been identified as (3R,3'R)-zeaxanthin. Also present were small amounts of 15-cis-phytoene, phytofluene, "zeta-carotene" (7,8,7',8'-tetrahydro-psi, psi-carotene plus 7,8,11,12-tetrahydro-psi, psi-carotene), neurosporene, lycopene, beta-zeacarotene, gamma-carotene, beta-carotene, beta-cryptoxanthin, rubixanthin, 3-hydroxy-beta-zeacarotene and several apo-carotenals. Zeaxanthin production was inhibited by nicotine (10 mM), and lycopene and rubixanthin accumulated. The biosynthesis of zeaxanthin is discussed in terms of pathways and also of "half-molecule" reaction sequences. The presence of zeaxanthin may be a characteristic of a group of Flavobacterium species, and may thus be useful in the taxonomic classification of these organisms.

Carotenoids↗

The nylon oligomer biodegradation system of Flavobacterium and Pseudomonas.

This review article is a compendium of the available information on the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium and Pseudomonas strains, and discusses the molecular basis for adaptation of microorganisms toward these xenobiotic compounds. Three plasmid-encoded enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endo-type 6-aminohexanoate-oligomer hydrolase (EIII) are responsible for the degradation of the oligomers. Two repeated sequences, designated RS-I and RS-II, are found on plasmid pOAD2, which is involved in 6-aminohexanoate degradation in Flavobacterium. RS-I appears 5 times on the pOAD2, and all copies have the same sequences as insertion sequence IS6100. RS-II appears twice on the plasmid. RS-IIA contains the gene encoding EII, while RS-IIB contains the gene for the analogous EII' protein. Both EII and EII' are polypeptides of 392 amino acids, which differ by 46 amino acid residues. The specific activity of the EII enzyme is 200-fold higher than that of EII'. Construction of various hybrid genes demonstrated that only the combination of two amino acid residues in the EII' enzyme can enhance the activity of the EII' to the same level as that of EII enzyme.

Amino Acid Sequence↗

Flavobacterium meningosepticum infection in a neonatal ward.

An outbreak of infections due to Flavobacterium meningosepticum type C in a neonatal intensive care unit is described. During a period of two weeks, two infants developed meningitis and a third was colonized in the respiratory tract and had transient bacteremia. The two meningitis patients were treated with clindamycin, rifampicin and cefotaxime systemically, plus rifampicin intraventricularly. Bacteriological eradication was achieved within 48 h, and both infants recovered from the meningitis without apparent neurological sequelae; however, one infant died two months later of unrelated causes. Environmental surveillance cultures failed to demonstrate a reservoir for the epidemic strain, but other Flavobacterium strains were recovered. Two clinically healthy infants were found to be colonized in the nasopharynx with strains that were extremely difficult to differentiate phenotypically from the epidemic strain. Extensive characterization of strains is necessary in order to differentiate between strains and subsequently to determine a certain source of infection.

Anti-Bacterial Agents↗

Flavobacterium meningosepticum ventriculitis: in vivo and in vitro results with the combinations rifampicin-erythromycin and mezlocillin-cefoxitin.

A case of Flavobacterium meningosepticum ventriculitis is described in which the failure of therapy with a combination of rifampicin and erythromycin is attributed to inadequate antibiotic levels in cerebrospinal fluid. The successful eradication of the organism was achieved with the use of mezlocillin and cefoxitin given by the intravenous and intraventricular route. In vitro sensitivity tests of recently isolated strains of Flavobacterium meningosepticum suggested that the combination mezlocillin and cefoxitin is more often synergistic than rifampicin and erythromycin.

Anti-Bacterial Agents↗

Biodegradation of triiodophenol by cell-free extracts of a pentachlorophenol-degrading Flavobacterium sp.

Pentachlorophenol (PCP) degrading Flavobacterium sp. ATCC 39723 was found to degrade other polyhalogenated phenolic compounds, including triiodophenol, tribromophenol, and trichlorophenol. Each compound was able to induce the degradation of the other compounds. A PCP Flavobacterium sp. mutant, F-2, was unable to degrade any of the halogenated compounds. The results suggest that all of the polyhalogenated phenols were degraded by the same enzyme system. This observation led us to exploit the sensitive leuco crystal violet assay, which measures the iodide released from triiodophenol. Cell free extracts from PCP-induced cells were able to release iodide from triiodophenol. The reaction required NADPH and oxygen.

Biodegradation, Environmental↗

Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp.

Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.

Aryldialkylphosphatase↗

Optical microbial biosensor for detection of methyl parathion pesticide using Flavobacterium sp. whole cells adsorbed on glass fiber filters as disposable biocomponent.

An optical microbial biosensor was described for the detection of methyl parathion pesticide. Whole cells of Flavobacterium sp. were immobilized by trapping in glass fiber filter and were used as biocomponent along with optic fiber system. Flavobacterium sp. has the organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into detectable product p-nitrophenol. The immobilized microbial biocomponent was disposable, cost-effective and showed high reproducibility and uniformity. The detection of methyl parathion by the use of disposable microbial biocomponent with optical biosensor was simple, single step and direct measurement of very low quantity of the sample. The home made reaction vessel was small and needed only 75 microl of sample. A lower detection limit 0.3 microM methyl parathion was estimated from the linear range (4-80 microM) of calibration plot of organophosphorus hydrolase enzymatic assay. The applicability to synthetic methyl parathion spiked samples was demonstrated.

Aryldialkylphosphatase↗

Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S-23S rDNA spacer.

Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into three genotypes. The isolates within the genotypes were further grouped based on RFLP of the 16S-23S rDNA spacer. The first genotype had five strains that were further divided into group A (4 strains) and B (1 strain) while the second genotype had 10 strains that were also further divided into group A (4 strains) and B (6 stains). The third genotype had 12 isolates with no differences in the RFLP patterns of the 16S-23S rDNA spacers. The 16S rRNA gene sequences representing the three identified genotypes were compared to the different published sequences by phylogenetic analysis and the results showed the American genotypes 1, 2 and 3 corresponding to genomovar 1, 2, and 3, respectively, reported by Triyanto and Wakabayashi [Triyanto, Wakabayashi H. Genotyping of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999; 34: 65-71]. The study demonstrates a method for RFLP and sequencing of the 16S rRNA gene and the 16-23S rDNA spacer as a useful tool in epidemiological studies of F. columnare.

DNA, Ribosomal Spacer↗

Glutathione is the reducing agent for the reductive dehalogenation of tetrachloro-p-hydroquinone by extracts from a Flavobacterium sp.

Tetrachloro-p-hydroquinone is the first intermediate during pentachlorophenol degradation by Flavobacterium sp. strain ATCC 39723, a strict aerobe. We report here that tetrachlorohydroquinone was reductively dehalogenated to 2,3,6-trichloro-p-hydroquinone and subsequently to 2,6-dichloro-p-hydroquinone under anaerobic conditions by the cell extract from Flavobacterium. The reducing agent was identified to be the reduced form of glutathione. This is the first time glutathione has been identified as the reducing agent for reductive dehalogenation.

Anaerobiosis↗

Catabolism of pentachlorophenol by a Flavobacterium sp.

The pathway employed for pentachlorophenol (PCP) degradation by an aerobic, chlorophenol-utilizing Flavobacterium sp. was initiated by conversion of PCP to tetrachloro-p-hydroquinone (TCH). 18O labelling experiments demonstrated that the first dechlorination, where a hydroxyl replaced the chlorine at PCP ring position number 4, involved a hydrolytic reaction. Then two reductive dechlorinations of TCH followed to yield firstly trichlorohydroquinone (TrCH) and then 2,6-dichlorohydroquinone (DCH). Thus, the initial steps in catabolism of PCP by the Flavobacterium were: PCP----TCH----TrCH.

Chlorine↗

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534.

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

Alkyl and Aryl Transferases↗

Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum.

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

Animals↗

Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum.

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

Amino Acids↗

The structure of the lipopolysaccharide O-antigen produced by Flavobacterium psychrophilum (259-93).

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

Animals↗

Novel, specific O-glycosylation of secreted Flavobacterium meningosepticum proteins. Asp-Ser and Asp-Thr-Thr consensus sites.

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

Amino Acid Sequence↗

Antimicrobial treatment of Flavobacterium meningosepticum infection.

A six-month-old boy in hospital developed septicaemia and meningitis due to Flavobacterium meningosepticum type A. Although the organism was susceptible to amikacin, chloramphenicol and sulphonamides when tested by the disc diffusion method, therapy with these drugs was ineffective. Intravenous therapy with cotrimoxazole gave complete recovery. Various strains of flavobacteria including F. meningosepticum type B were isolated from the environment. Antimicrobial susceptibilities of patients and environmental strains against chloramphenicol, sulphonamides and rifampicin showed no correlation between disc diffusion results and minimum inhibitory concentration (MIC). On the other hand, all the strains were found by both methods to be susceptible to amikacin, erythromycin, clindamycin and trimethoprim. Disc diffusion method should be supplemented with determination of MIC when testing antimicrobial susceptibility of Flavobacterium sp.

Anti-Bacterial Agents↗

Proline-specific dipeptidyl aminopeptidase from Flavobacterium meningosepticum.

A proline-specific dipeptidyl aminopeptidase was highly purified from cell-free extract of Flavobacterium meningosepticum by a series of column chromatographies on DEAE-Sephadex A-50, Sephadex G-150, hydroxyapatite, and a second gel filtration on Sephadex G-150. The enzyme was most active at pH 7.4-7.8 for both Gly-Pro-beta-naphthylamide (Gly-Pro-2-NNap) and Gly-Pro-p-nitroanilide (Gly-Pro-pNA) and was stable between pH 7 and 9.5. The enzyme was markedly inhibited by diisopropylphosphofluoridate (DFP) and mercury ion but not by sulfhydryl-blocking reagents and metal chelators. The molecular weight of the enzyme was about 160,000 as judged by the gel filtration method and the subunit molecular weight was estimated to be 75,000 by sodium dodecyl sulfate (SDS)-gel electrophoresis, suggesting a dimeric form of the native enzyme. The isoelectric point was at pH 9.5. The enzyme hydrolyzed peptides and peptide amides at the carboxyl side of a proline residue penultimate to the amino-terminal amino acid, as did post-proline dipeptidyl aminopeptidases from various mammals. However, antiserum raised against post-proline dipeptidyl aminopeptidase from porcine kidney did not cross-react with the Flavobacterium dipeptidyl aminopeptidase.

Animals↗