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PubMed2003

Determination of urinary myo-inositol concentration by an improved enzymatic cycling method using myo-inositol dehydrogenase from Flavobacterium sp.

BACKGROUND: To determine myo-inositol more accurately, we improved the enzymatic cycling method. METHODS: We screened myo-inositol dehydrogenase (MIDH; EC.1.1.1.18) from Flavobacterium sp., which was highly specific to myo-inositol. We measured urinary myo-inositol/creatinine ratio 2 h after 75-g oral glucose tolerance test (2 h MI) of 71 volunteers, and investigated the relationship between diabetes and urinary myo-inositol concentration. RESULTS: The calibration curve was linear (r = 1.00) up to 2000 micromol/l, and the detection limit was 10 micromol/l. Within-run and between-run CVs were 0.5-1.1% and 0.4-1.3%, respectively. The 2 h MI of impaired fasting glycemia (IFG; 65.1 +/- 46.6 mg/g Cr, P < 0.005), impaired glucose tolerance (IGT; 85.0 +/- 73.7 mg/g Cr, P < 0.001) and diabetes (163.4 +/- 73.7 mg/g Cr, P < 0.0001) increased significantly compared with that of normal glucose tolerance (NGT; 24.0 +/- 14.4 mg/g Cr). From receiver operating characteristic analyses on 2 h MI, with 50 mg/g Cr as a tentative cutoff value to detect diabetes, the sensitivity and specificity were 100% and 77%, respectively. With 40 mg/g Cr as a tentative cutoff value to detect NGT, the sensitivity and specificity were 74% and 85%, respectively. CONCLUSIONS: The myo-inositol measurement method demonstrated high specificity and yielded accurate results. The results of clinical trials suggested that 2 h MI could not only determine diabetes but also distinguish IFG and IGT from NGT.

Adult↗

PubMed1985

Flavobacterium odoratum ventriculitis treated with intraventricular cefotaxime.

A 6-week-old infant admitted to the University Hospital of the West Indies with hydrocephalus later developed ventriculitis. A heavy growth of Flavobacterium odoratum susceptible to gentamicin and cefotaxime was recovered from the ventricular fluid. Since intraventricular therapy was envisaged, a Pudenz reservoir was installed and ventricular fluid aspirated every 24 h to monitor treatment. Initial therapy consisted of intravenous cefotaxime, 50 mg/kg q.i.d. for 4 days. No significant reduction in the number of organisms in the ventricular fluid was achieved with this regimen. Intravenous therapy was therefore discontinued. On day 5 intraventricular therapy began with 5 mg cefotaxime 24 h for 6 days, followed by 1 mg/24 h for 4 days. Daily monitoring of intraventricular fluid indicated a high degree of antibacterial activity with rapid elimination of bacteria. Ventricular fluid remained sterile 10 days after therapy stopped. The Pudenz reservoir was removed, a ventriculoperitoneal shunt installed, and the patient discharged from hospital 4 days later without noticeable sequelae.

Bacterial Infections↗

PubMed2002

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum.

Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through beta-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords approximately 10-fold higher K(m) values but no significant change in V(max), consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage.

Amino Acid Substitution↗

PubMed1999

Novel bacterial peroxidase without catalase activity from Flavobacterium meningosepticum: purification and characterization.

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¿ omitted¿000 Da, and that of its subunit was 54¿ omitted¿000 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.

Amino Acid Sequence↗

PubMed2003

Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare.

A 16-S rRNA gene from the chromosomal DNA of the fish-pathogenic bacterium Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set. The primer set amplified a specific 1193-bp DNA fragment from F. columnare strains but not from related bacteria, F. psychrophilum, F. aquatile, F. branchiophilum, or other bacterial pathogens of fish, Flexibacter maritimus, Cytophaga johnsonae, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, and Streptococcus iniae or from the non-fish pathogen Escherichia coli. The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if the more sensitive nested PCR is used). DNA was extracted by a boiled-extraction method or by commercial column purification. The PCR product was detected at DNA concentrations below 0.1 ng and from as few as 100 bacterial cells. Nested PCR using universal eubacterial primers increased the sensitivity five-fold, allowing detection of F. columnare strains at DNA concentrations below 0.05 ng and from as few as 10 bacterial cells in apparently healthy, asymptomatic fish. The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al. [Fish Pathol. 29 (1994) 271.] and that of Bader and Shotts [J. Aquat. Anim. Health 10 (1998) 311.]. The new primer set is as good or better than the previously published primer sets for detecting F. columnare in all samples and under all conditions tested.

Animals↗

PubMed1986

Isolation and some properties of an arylamidase from Flavobacterium IIb.

A constitutive L-leucylarylamidase (EC 3.4.11) hydrolase able to cleave L-aminoacyl-beta naphthylamide and L-aminoacyl-4 nitroanilide substrates, was isolated from sonicated cells of Flavobacterium IIb and partially purified with a 0.9% yield and a 159-fold recovery. Its molecular weight was estimated to be about 170,000 +/- 10%. This arylamidase exhibited optimum activity at pH 7.0 and 28 degrees C for the hydrolysis of L-leucine-4NA and is inhibited strongly by metal chelating agents, and to a weaker extent, by some sulfhydryl and reducing agents. Heavy metal ions: Cd2+, Zn2+, Cu2+, Hg2+ and Co2+, markedly inhibit it, and Zn2+ is a competitive inhibitor. This metalloenzyme, free of carboxypeptidase, proteinase and L-leucine aminopeptidase (L-leucylglycine substrate) activities, hydrolyzes aminoacyl-beta NA, aminoacyl-4NA and some dipeptides with unsubstituted amino groups of the L-configuration. The lowest Km values are associated with substrates having neutral or basic residues, with large side chains.

Alkaline Phosphatase↗

PubMed2003

Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum.

To go further into the characterization of the proteolysis exocellular system of the salmonid pathogen Flavobacterium psychrophilum, the purification and characterization of a novel protease designated Fpp2 (F. psychrophilum protease 2) was undertaken. A protease (Fpp2) hydrolyzing azocasein was purified. The Fpp2 can be defined as a metalloprotease, it had an estimated molecular mass of 62 kDa with calcium playing an important role in the thermostability of the enzyme. Proteolytic activity was optimal at pH 6.0-7.0 and 24 degrees C and activation energy for the hydrolysis of azocasein was determined to be 5.4 kcal mol(-1), being inactive at temperatures above 42 degrees C. All these results are characteristic of 'cold adapted enzymes'. Fpp2 proved to be a broad range hydrolytic enzyme because in optimal conditions it was able to hydrolyze matrix and muscular proteins. It can be concluded that the Fpp1, a previously characterized 55 kDa metalloprotease, and the Fpp2 protease were produced under different physiological conditions and were immunologically as well as biochemically different.

Calcium↗

PubMed1998

Cloning, functional expression and purification of endo-beta-galactosidase from Flavobacterium keratolyticus.

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

Amino Acid Sequence↗

PubMed1998

Characterization of four Flavobacterium columnare (Flexibacter columnaris) strains isolated from tropical fish.

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

Agglutination Tests↗

PubMed1999

The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops).

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

Animals↗

PubMed2000

16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides.

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.

Animals↗

PubMedDate not supplied

[Isolation and description of strains of Flavobacterium multivorum of telluric origin].

Twenty encapsulated strains of Flavobacterium multivorum were isolated from soil by elective culture in minimal medium containing inulin as the sole source of carbon and energy. These strains were compared with the type strain and with 5 other strains (including 4 clinical strains) of F. multivorum. Of 168 substrates tested, 18 carbohydrates were used as the sole carbon and energy source by all 26 strains. A few amino acids were used by some strains. The yellow pigment produced was found to be a carotene and its production was photo-inducible. The presence of a cytochrome c oxidase of the aa3 type was suggested by absorption spectra. The major cell lipids were phosphatidylethanolamine and sphingolipids; the major fatty acids were 13-methyltetradecanoic and 2-hydroxy-13-methyltetradecanoic acids. Soil and clinical strains of F. multivorum showed roughly similar patterns of antibiotic multiresistance. The average G + C content of the DNA of 11 strains was 40.8 +/- 1.5 mol%.

Anti-Bacterial Agents↗

PubMed1999

Production of viable cultures of Flavobacterium psychrophilum: approach and control.

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

Animals↗

PubMed1998

The specificity of prolyl endopeptidase from Flavobacterium meningoseptum: mapping the S' subsites by positional scanning via acyl transfer.

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

Acylation↗

PubMed2003

Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro.

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum

Animals↗

PubMed2002

Adherence of Flavobacterium psychrophilum on the body surface of the ayu Plecoglossus altivelis.

Bacterial cold water disease in the ayu Plecoglossus altivelis caused by Flavobacterium psychrophilum is a serious problem in the Japanese freshwater culture industry. The distribution and activity of this bacterium on the body surface of the ayu in the infection process was investigated. The survival of F. psychrophilum in tap water showed that this bacterium might sustain its infectivity for 24 h. In an experimental infection, juvenile ayu were immersed in water containing 10(8.9) CFU/ml F. psychrophilum, and the progressing infection was followed by scanning electron microscopy during a 24-h period. This bacterium was observed in the ayu for 24 h adhering to the lower jaw and caudal peduncle, where the epidermis tissue was collapsed. This study showed that bacterial suspension in water sustains the activity of this bacterium. F. psychrophilum attaches especially to the jaw and caudal peduncle, growing at these sites, collapsing the dermal structure and invading the tissues.

Animals↗

PubMed1995

Heparinase I from Flavobacterium heparinum: the role of the cysteine residue in catalysis as probed by chemical modification and site-directed mutagenesis.

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

Base Sequence↗

PubMed2002

Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity.

The soil bacterium Flavobacterium heparinum produces several enzymes that degrade heparan sulfate glycosaminoglycans (HSGAGs) in a sequence-specific manner. Among others, these enzymes include the heparinases and an unusual glycuronidase that hydrolyzes the unsaturated Delta4,5 uronic acid at the nonreducing end of oligosaccharides resulting from prior heparinase eliminative cleavage. We report here the molecular cloning of the Delta4,5 glycuronidase gene from the flavobacterial genome and its recombinant expression in Escherichia coli as a highly active enzyme. We also report the biochemical and kinetic characterization of this enzyme, including an analysis of its substrate specificity. We find that the Delta4,5 glycuronidase discriminates on the basis of both the glycosidic linkage and the sulfation pattern within its saccharide substrate. In particular, we find that the glycuronidase displays a strong preference for 1-->4 linkages, making this enzyme specific to heparin/heparan sulfate rather than 1-->3 linked glycosaminoglycans such as chondroitin/dermatan sulfate or hyaluronan. Finally, we demonstrate the utility of this enzyme in the sequencing of heparinase-derived HSGAG oligosaccharides.

Amino Acid Sequence↗