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PubMed2005

Iron acquisition mechanisms of Flavobacterium psychrophilum.

Forty strains of Flavobacterium psychrophilum were tested for the production of siderophores using the universal Chrome Azurol S (CAS) assay. The majority of the strains (85%) were CAS positive (CAS+) and some (15%) were CAS negative (CAS-). The cryptic plasmid pCP1 was carried by all positive strains and was lacking from negative strains. While a weak catechol reaction was detectable in CAS+ culture supernatants, the CAS reaction was, to some extent, heat sensitive, questioning whether the positive reaction was caused only by siderophores. The ability to grow in vitro under iron-restricted conditions did not correlate with the CAS reactivity, as growth of both CAS+ and CAS- strains was similarly impaired under iron restriction induced by 2,2 dipyridyl. Suppressed growth under these conditions was restored by addition of FeCl3, haemoglobin and transferrin for both CAS+ and CAS- strains.

2,2'-Dipyridyl↗

PubMed2005

Detection and identification of Flavobacterium psychrophilum from gill washings and benthic diatoms by PCR-based sequencing analysis.

A nested polymerase chain reaction (PCR) amplification technique was used to detect Flavobacterium psychrophilum from washings of fish gill surfaces and benthic diatoms as environmental samples. Gill washing samples were prepared from kawamutsu, Zacco temminckii (Temminck & Schlegel) and oikawa, Z. platypus (Temminck & Schlegel). Benthic diatom samples were collected from stone surfaces. All samples were collected from rivers in Wakayama Prefecture, Japan from November 2003 to January 2004. Following simple DNA extraction using a chelating resin, nested PCR techniques targeting 16S-rDNA and gyrB regions were performed, and PCR products were cloned and sequenced. With nested PCR amplification for the 16S-rDNA gene, ambiguous PCR products were detected from two of six samples, and by cloning and sequencing analysis were found not to be DNA fragments amplified from F. psychrophilum. Using nested PCR for the gyrB gene, however, five of six samples were clearly positive for F. psychrophilum in agarose gel electrophoresis, and were found to be identical with nucleotide sequences of F. psychrophilumgyrB deposited in DNA databases by sequencing analysis. Results indicate that nested PCR for the gyrB region is a useful technique to detect low levels of F. psychrophilum from environmental samples contaminated with many other organisms.

Animals↗

PubMed2006

Efficacy of injection vaccines against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum).

Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.

Animals↗

PubMed2006

Quantitative expression (Walbaum) of immunological factors in rainbow trout, Oncorhynchus mykiss (Walbaum), after infection with either Flavobacterium psychrophilum, Aeromonas salmonicida, or infectious haematopoietic necrosis virus.

To further enhance our understanding of immunological gene expression in rainbow trout, Oncorhynchus mykiss, after infection with naturally occurring pathogens, a series of probes and primers were developed for the quantification of immune factors. Separate groups of specific-pathogen-free rainbow trout were infected with either Flavobacterium psychrophilum, Aeromonas salmonicida or infectious haematopoietic necrosis virus (IHNV). Three different concentrations of each pathogen were used and samples from infected and mock-infected fish were taken at either 1 or 5 days after infection. Ten fish were sampled at each time point for individual sections of liver, spleen and head kidney. Organ specimens from five of the fish were used to re-isolate and quantify the pathogen at the time the samples were taken. Total RNA was extracted from the organs of the remaining five animals. Using real-time polymerase chain reaction with fluorescent-labelled probes, the RNA from these organs was examined for the level of expression of the following immunological factors; an interferon related protein (MX-1), interleukin-8 (IL-8), the cytotoxic T-cell marker CD-8 and complement factor C3 (C3). They were also measured for the level of beta-actin, which was used as a standardization control for cellular RNA expression. Infection with IHNV produced the greatest change in expression level for all the immunological related factors examined in this study. IHNV elicited the best dose response profile, which was typically seen at 5-days post-infection for MX-1, C3, IL-8 and CD-8. Infection with A. salmonicida and F. psychrophilum showed elevated, but variable expression levels for several of the genes tested.

Actins↗

PubMed2006

Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare.

The virulence of eight Flavobacterium columnare strains was studied to find correlations between several virulence-related factors and virulence. Virulence was tested in vivo using rainbow trout, Oncorhynchus mykiss (Walbaum). Suggested virulence-related factors such as production of the degradative enzyme chondroitin lyase, plasmid occurrence and adhesion capability were studied in vitro. Infection with the four most virulent strains resulted in 95-100% mortality within 114 h. Chondroitin lyase activity was found to be significantly related to the virulence of the strains at 25 degrees C and it was also shown to be temperature-dependent, being higher at 25 degrees C than at 20 degrees C. Virulence was not plasmid associated. The adhesion capability of the strains in vitro varied substantially when tested on crude mucus-coated slides and no statistical relationship between adhesion and virulence was found using this method.

Animals↗

PubMed1981

A bacterial glucoamylase degrading cyclodextrins. Partial purification and properties of the enzyme from a Flavobacterium species.

A Flavobacterium species has been isolated which produces a cyclodextrin-degrading glucoamylase. The inducible, cell-bound enzyme was purified about 10-fold to 75% purity in 57% yield. The action of the enzyme was studied with the main cyclodextrins (cyclohexaamylose, cycloheptaamylose and cyclooctaamylose) and with the typical glucoamylase substrates, respectively. The final degradation product with all the substrates was glucose. Small amounts of maltose, which could be detected in the course of cyclodextrin degradation, were hydrolyzed at a lower rate. V for cyclohexaamylase was found to be about 14-15 mumol glucose min-1 (mg pure protein)-1, the Km for cyclohexaamylose was 0.142 mM. Apparently the enzyme preferred shorter alpha-D-glucopyranosyl chains. Besides maltose, amylopectin and glycogen proved to be very poor substrates. Some properties of the enzyme have been described.

Cyclodextrins↗

PubMed1981

Purification and characterization of 6-aminohexanoic-acid-oligomer hydrolase of Flavobacterium sp. Ki72.

6-Aminohexanoic-oligomer hydrolase of Flavobacterium sp. KI72 was purified to homogeneity by column chromatography three times, and by preparation polyacrylamide gel electrophoresis twice. The purified enzyme had the following characteristics. 1. The molecular weight was estimated to be 84000 by Sephadex G-200 molecular-sieve chromatography. The enzyme consisted of two homologous subunits of 42000, judged from sodium dodecylsulfate/polyacrylamide gel electrophoresis. 2. The optimum pH for activity was between 8 and 9, the optimum temperature was 40 degrees C for a 1-h reaction. The Michaelis-Menten constants and turnover numbers for the 6-aminohexanoic acid dimer and trimer were 5.9 mM and 2.4 s-1, and 6.2 mM and 2.0 s-1 respectively. 3. The enzyme was inhibited by 0.37 mM diisopropylfluorophosphate and by 0.013 mM p-chloromercuribenzoate. 4. The enzyme was active on 6-aminohexanoic acid oligomers from dimer to hexamer and icosamer but not on hectamer, and the activity decreased with the increase of the polymerization number of the oligomer. The oligomers were hydrolyzed so as to remove the 6-aminohexanoic acid residue successively from the amino terminus. The enzyme could not hydrolyze other linear amides, cyclic amides, dipeptides, tripeptides or casein. 5. 6-aminohexanoic-acid-oligomer hydrolase was classified as a new member of the linear amidases (EC 3.5.1.-).

Amidohydrolases↗

PubMed1984

Flavobacterium heparinum 2-O-sulphatase for 2-O-sulphato-delta 4,5-glycuronate-terminated oligosaccharides from heparin.

The glycosulphatase which hydrolyses the 2-O-sulphate of the disaccharide, 4-deoxy-2-O-sulphato-alpha-L-threohex-4-enopyranosyl uronic acid-(1----4)-2-deoxy-2-sulphamido-6-O-sulphato-D-glucose (delta UA-2S----GlcNS-6S), has been isolated from the soluble fraction of disrupted Flavobacterium heparinum. The activity was purified 3300-fold by chromatography on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and blue-Sepharose CL-6B. From sodium dodecylsulphate/polyacrylamide gel electrophoresis, the enzyme was homogeneous and of 62000 Mr. A novel assay was devised using the de-N-sulphonated [1-3H]alditol, 4-deoxy-2-O-sulphato-alpha-L-threo-hex-4-enopyranosyl uronic acid-(1----4)-2-amino-2-deoxy-6-O-sulphato-D-[1-3H]glucitol (delta UA-2S----[1-3H]GlcNH2-ol-6S). This alditol was shown by 13C-NMR to be desulphated in the analogous manner to the original reducing trisulphated disaccharide. The purified 2-O-sulphatase was completely free of heparinase I, heparinase II (heparitinase), chondroitinases AC, chondroitinase B, the delta 4,5-glycuronidase for heparin delta 4,5-disaccharides, the 6-O-sulphatase and the 2-sulphamidase. It was optimally active over the range pH 5.5-6.5 and was practically unaffected by Na, K, Ca or Mg ions. Inorganic phosphate inhibited the activity. The Km value for the alditol substrate was 1.22 mmol dm-3. Using 13C-NMR, the 2-O-sulphatase was found to hydrolyse the analogous esters of higher delta 4,5-oligosaccharides from heparin. This contrasts with the findings of other authors [Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408-6415].

Chemical Phenomena↗

PubMed1985

Flavobacterium heparinum 3-O-sulphatase for N-substituted glucosamine 3-O-sulphate.

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-O-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose and 2-acetamido-2-deoxy-3-O-sulpho-D-glucose. The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56 000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulpho-D-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose as respective substrates. The purified 3-O-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-3 Ca2+. Inorganic phosphate and sulphate inhibited 3-O-sulphatase activity. The Km value of the N-acetylated substrate was determined to be 42 mumol dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulpho-D-glucose.

Chemical Phenomena↗

PubMed1985

Flavobacterium heparinum 6-O-sulphatase for N-substituted glucosamine 6-O-sulphate.

A specific glyco-6-O-sulphatase has been purified to homogeneity from Flavobacterium heparinum. The enzyme hydrolyses the 6-O-sulphates of 2-deoxy-2-sulphamido-6-O-sulpho-D-glucose (GlcNS-6S), 2-acetamido-2-deoxy-6-O-sulpho-D-glucose (GlcNAc-6S) and 2-amino-2-deoxy-6-O-sulphato-D-glucose (GlcN-6S). The activity was purified 2100-fold by successive chromatography on CM-Sepharose CL-6B, Sepharose CL-4B, hydroxyapatite and blue-Sepharose CL-6B. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed a protein of relative molecular mass 64000. Four novel assays were developed using 35S-labelled and 14C-labelled monosaccharides. The purified enzyme was free of all other known heparin-degrading enzymes. In particular this was the first resolution of the 6-O-sulphatase from the sulphamidase. Optimal activity was at pH 7.5. Enzyme activity was virtually unaffected by Na+ and K+ ions. Enhancements of activity of 12% and 30% were effected by Mg2+ and Ca2+ ions respectively. Inorganic phosphate and sulphate (both 0.005 mol dm-3) inhibited activity by 48% and 50% respectively. The Km value for the free amino substrate GlcN-6S was 1.35 mmol dm-3. In contrast the Km values for the GlcNAc-6S and GlcNS-6S were 54 mumol dm-3 and 16 mumol dm-3 respectively.

Flavobacterium↗

PubMed1987

Flavobacterium heparinum sulphamidase for D-glucosamine sulphamate. Purification and characterisation.

A novel assay has been developed for 2-deoxy-2-sulphamido-D-glucose (GlcNS) sulphamidase from Flavobacterium heparinum. This has enabled the 1930-fold purification of the enzyme from a soluble fraction of bacterial homogenate. From SDS/polyacrylamide gel electrophoresis the enzyme was shown to have a relative molecular mass of 81,500. Ca2+ was essential for enzyme activity. Inorganic phosphate and sulphate inhibited activity by 28% and 29% respectively at 5 mmol dm-3. The purified sulphamidase had a pH optimum of 7.0 and a Km of 8.32 mumol dm-3 for GlcNS. The degradation of 2-deoxy-2-sulphamido-6-O-sulpho-D-glucose (GlcNS-6S) was also re-investigated. The two sulphate groups were hydrolysed sequentially in a single non-bifurcate manner, in contrast to previous reports [Dietrich, C.P., Silva, M.E. and Michelacci, Y.M. (1973) J. Biol. Chem. 248, 6408-6415].

Anions↗

PubMed1991

Heparinase II from Flavobacterium heparinum. Action on chemically modified heparins.

Five chemically modified heparins were derived from native pig mucosal heparin (pig heparin Is). These were de-N-sulphated heparin (heparin IH), N-acetylheparin (heparin IA), de-N/O-sulphated heparin (heparin IVH), de-O-sulphated heparin (heparin IVs) and de-O-sulphated N-acetyl-heparin (heparin IVA). Their structures were studied by 13C-NMR spectroscopy at 90.56 MHz. Native heparin and the derivatives were incubated with Flavobacterium heparinase II at 25 degrees C. The progress of degradation was followed by the delta A235 and the final composition examined by gel filtration with Bio-Gel P-4. Native heparin (Is) was readily degraded by heparinase II and, with the exception of heparin IVH for which degradation was negligible, the chemically modified derivatives were also degraded. Approximately 90% of the saccharides from heparins Is, IA, IVs and IVA were disaccharides and tetrasaccharides. For heparin IH, which was degraded more slowly, the proportion was 65%. Heparins Is, IVs and IVA underwent initial rapid degradation. The digestion of heparin Ia proceeded rapidly after an initial lag phase. The undegraded polymers produced similar elution profiles from Bio-Gel P-4. Following the action of heparinase II on heparins Is, IA, IVs and IVA, the elution profiles revealed a major peak of disaccharides and minor peaks of higher oligomers. The profile of heparin IH revealed a greater proportion of intermediate-molecular-mass saccharides. Our results demonstrate a broad specificity for heparinase II. It is capable of lysing both N-acetylated and N-sulphated heparins independent of O-sulphation. Heparinase II will also degrade heparin derivatives that are non-N-substituted provided that they are O-sulphated.

Animals↗

PubMed1991

Purification and characterisation of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacterium species.

3-Hydroxyphenylacetate 6-hydroxylase was purified 70-fold from a Flavobacterium sp. grown upon phenylacetic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme. SDS/PAGE of purified enzyme reveals a single band with a minimum molecular mass of 63 kDa. Analytical gel-filtration, sedimentation-equilibrium and sedimentation-velocity experiments indicate that the purified enzyme exists in solution mainly as a dimer, containing 1 molecule non-covalently bound FAD/subunit. 3-Hydroxyphenylacetate 6-hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3-hydroxyphenylacetate is hydroxylated efficiently, yielding 2,5-dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4-dihydroxyphenylacetate and 4-hydroxyphenylacetate are partially hydroxylated, exclusively at the 6' (2') position. The physiological product 2,5-dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3-hydroxyphenylacetate 6-hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate. In the native state of enzyme, two sulfhydryl groups are accessible to 5,5'-dithiobis(2-nitrobenzoate). Titration with stoichiometric amounts of either 5,5'-dithiobis(2-nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3-hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol. The N-terminal amino acid sequence of the enzyme lacks the consensus sequence GXGXXG, found at the N-termini of all flavin-dependent external monooxygenases sequenced so far. The amino acid composition of 3-hydroxyphenylacetate 6-hydroxylase is also presented.

Amino Acid Sequence↗

PubMed1991

Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp.

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181----Asp (EII type) and His266----Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.

Amidohydrolases↗

PubMed2000

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.

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.

Cells, Cultured↗

PubMed1999

Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium.

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

Amino Acid Sequence↗

PubMed1999

Molecular characterization of a carbapenem-hydrolyzing beta-lactamase from Chryseobacterium (Flavobacterium) indologenes.

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

Base Sequence↗

PubMed2007

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis.

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.

Animals↗