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PubMed1983

Reversion by vitamin K of aflatoxin B1 (AFB)-induced inhibition of oxygen uptake in three AFB-susceptible bacteria.

The effect of Vitamin K, on the inhibition of oxygen uptake in Bacillus brevis (2611), Bacillus megaterium (1368) and Flavobacterium aurantiacum by aflatoxin B1 (AFB) has been investigated. At 1.5 mM, vitamin K completely reversed oxygen inhibition by 10 micrograms/ml AFB in the three bacteria and by 50 micrograms/ml AFB in B. brevis, the most susceptible of the three bacteria to AFB. Vitamin K did not reverse the inhibitory effect of 100 micrograms/ml AFB in any of the three bacteria except B. megaterium, neither did 1.5 mM 2,4-dinitrophenol (DNP) reverse the inhibition at 10 micrograms/ml and 100 micrograms/ml levels of AFB in any of the three bacteria.

2,4-Dinitrophenol↗

PubMed1996

Ribotyping of Chryseobacterium meningosepticum: its use as an epidemiological tool and its correlation with serovars.

Chryseobacterium meningosepticum (basonym, Flavobacterium meningosepticum King 1959) is associated with neonatal meningitis and is isolated from normal and immunocompromised adults. AAF-labelled Escherichia coli 16 + 23S rRNA was used as a probe for ribotype analysis of 92 clinical isolates from tracheal exsudate, blood culture, cerebrospinal fluid, urine and pus. The 92 isolates belonged to the 15 described serovars of C. meningosepticum, and included 21 strains isolated during an outbreak in an intensive care unit, all belonging to serovar G. Three restriction endonucleases, EcoRI, HindIII and PstI, were selected for use in ribotyping after preliminary experiments. Epidemiologically unrelated isolates were discriminated by ribotyping and could be classified into 48 ribotypes according to the hybridization banding patterns obtained after restriction with the three enzymes. Strains which were not discriminated by combined ribotype analysis belonged to the same serovar, and were of identical geographic origin. In one case, analysis with an additional enzyme, PvuII, was necessary for separating strains from two different serovars. However, three strains from different serovars (two isolated from the same place and one elsewhere within eight years) showed the same combined ribotype. Analysis of the rRNA gene patterns revealed 6 different patterns for clinical isolates of the outbreak, suggesting unrelated sources of infection. In three patients, isolation of C. meningosepticum with different combined ribotypes suggested superinfection. Ribotyping enabled differentiation between isolates belonging to the same serovar as well as between isolates of different serovars and provided a useful molecular epidemiological tool for the study of C. meningosepticum. Combined ribotype analysis with several restriction endonucleases increased the discriminating power of the method. However, there was only a partial correlation between serovars and the extent of DNA relatedness.

Adult↗

PubMed2004

The synthesis of a novel thio-linked disaccharide of chondroitin as a potential inhibitor of polysaccharide lyases.

A thio-linked disaccharide based on the structure of the glycosaminoglycan chondroitin was synthesized as a potential inhibitor of chondroitin AC lyase from Flavobacterium heparinum for structural analysis of the active site. Instead it was found to be a slow substrate, thereby demonstrating that lyases, in contrast to glycosidases, can cleave thioglycoside links between sugars.

Carbohydrate Sequence↗

PubMed2005

Non-specific biodegradation of the organophosphorus pesticides, cadusafos and ethoprophos, by two bacterial isolates.

An enrichment culture technique was used for the isolation of microorganisms responsible for the enhanced biodegradation of the nematicide cadusafos in soils from a potato monoculture area in Northern Greece. Mineral salts medium supplemented with nitrogen (MSMN), where cadusafos (10 mg l(-1)) was the sole carbon source, and soil extract medium (SEM) were used for the isolation of cadusafos-degrading bacteria. Two pure bacterial cultures, named CadI and CadII, were isolated and subsequently characterized by sequencing of 16S rRNA genes. Isolate CadI showed 97.4% similarity to the 16S rRNA gene of a Flavobacterium strain, unlike CadII which showed 99.7% similarity to the 16S rRNA gene of a Sphingomonas paucimobilis. Both isolates rapidly metabolized cadusafos in MSMN and SEM within 48 h with concurrent population growth. This is the first report for the isolation and characterization of soil bacteria with the ability to degrade rapidly cadusafos and use it as a carbon source. Degradation of cadusafos by both isolates was accelerated when MSMN was supplemented with glucose. In contrast, addition of succinate in MSMN marginally reduced the degradation of cadusafos. Both isolates were also able to degrade completely ethoprophos, a nematicide chemical analog of cadusafos, but did not degrade the other organophosphorus nematicides tested such as isazofos and isofenphos. Inoculation of a soil freshly treated with cadusafos or ethoprophos (10 mg l(-1)) with high inoculum densities (4.3 x 10(8) cells g(-1)) of Sphingomonas paucimobilis resulted in the rapid degradation of both nematicides. These results indicate the potential of this bacterium to be used in the clean-up of contaminated pesticide waste in the environment.

Antinematodal Agents↗

PubMed2025

TamL is a Key Player of the Outer Membrane Homeostasis in Bacteroidota.

In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidota, TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the function of TamL in Flavobacterium johnsoniae, an environmental Bacteroidota. Unlike its homologs in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we show that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe an overall decrease in the levels of β-barrel outer membrane proteins, nor substantial alterations in outer membrane lipid composition. By pull-down assays, we found TamL co-purifying with TamB in F. johnsoniae, suggesting an interaction. Furthermore, we found that while TamL and TamB monocistronic genes are conserved among Bacteroidota, only some species encode multiple TamL, TamB and TamA proteins. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria.

Bacterial Outer Membrane Proteins↗

PubMed2006

Stationary phase culture supernatant containing membrane vesicles induced immunity to rainbow trout Oncorhynchus mykiss fry syndrome.

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout (Oncorhynchus mykiss) fry syndrome (RTFS). Logarithmic phase formalin-killed cells (FKC) of F. psychrophilum induced immunity to BCWD in ayu (Plecoglossus altivelis) by using an oral administration. In this study, we investigated the effective antigens of logarithmic phase cells in rainbow trout. Rainbow trout fry immunized with logarithmic phase FKC resulted in near complete protection, but the vaccine effect was low in fry immunized with stationary phase FKC. Scanning electron microscopy showed that logarithmic phase cells had many membrane vesicles (MVs) on the surface of F. psychrophilum cells. The MVs were released into medium at the stationary phase. MVs rich supernatant was collected from the stationary phase culture supernatant by using an ammonium precipitation method. Immunization with MVs rich supernatant combined with stationary phase FKC resulted in a relative percentage survival (RPS) of 94-100%, but immunization with MVs rich supernatant only resulted in no protection against F. psychrophilum infection. These data show that MVs have an adjuvant efficacy and suggest that combination of MVs and cells is necessary to obtain efficient protection.

Administration, Oral↗

PubMed2002

Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates.

Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.

Catalysis↗

PubMed1981

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound AH-Sepharose 4B.

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono("phospho")-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S), as well as delta 4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (delta UA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxylapatite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and delta 4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (delta UA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxylapatite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.

Chromatography, Affinity↗

PubMed1979

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B.

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.

Chondroitinases and Chondroitin Lyases↗

PubMed2003

Chondroitin O-methyl ester: an unusual substrate for chondroitin AC lyase.

Chondroitin O-methyl ester was depolymerized by chondroitin AC lyase (EC 4.2.2.5) from Flavobacterium heparinum. The major product isolated from the depolymerization reaction was found to be methyl alpha-L-threo-hex-4-enopyranosyluronate-(1-->4)-2-acetamido-2-deoxy-alpha,beta-D-galactopyranoside.

Carbohydrate Conformation↗

PubMed1998

Particle clearance from the gills of rainbow trout (Oncorhynchus mykiss).

The rate of particle clearance from the gills was assessed in healthy rainbow trout (Oncorhynchus mykiss), challenged with the formalin-killed bacterium Flavobacterium branchiophilum, as well as in fish with altered ventilation levels produced by varying the concentrations of dissolved oxygen (DO) in the water. The clearance of F. branchiophilum from the gills was quantified by means of an enzyme-linked immunosorbent assay. Fish held under normoxic conditions (DO = 9.5 mg/l) showed an initial rapid reduction in bacterial antigen, with 50% of the bacteria being cleared in the first 12 h after exposure, followed by slower clearance for the remaining bacteria; total elimination was achieved by 40 h. Fish with reduced ventilation rates (hyperoxia; DO = 25 mg/l) and elevated ventilation rates (hypoxia; DO = 4.5 mg/l) had significantly impaired particle clearance (r < 0.05), achieving only 60 and 20% reduction, respectively, at 72 h after challenge.

Animals↗

PubMed2000

Heparinase I acts on a synthetic heparin pentasaccharide corresponding to the antithrombin III binding site.

A synthetic pentasaccharide, containing an intact antithrombin III (ATIII) binding site that is in clinical studies a specific antifactor Xa agent, serves as a substrate for a heparin lyase (heparinase I, EC 4.2.2.7) from Flavobacterium heparinum. Heparinase I, currently being assessed as a heparin reversal agent, also reverses the antifactor Xa activity of this synthetic pentasaccharide by breaking it down to inactive disaccharide and trisaccharide products.

Antithrombin III↗

PubMed1985

Septicaemia in a teaching hospital in Kuwait--I: Incidence and aetiology.

During a period of 18 months of study, blood cultures were performed on 3845 patients in hospital with clinical signs of infection. Among these, 214 (5.6%) episodes of septicaemia were diagnosed which correspond to 10.9/1000 hospital admissions. About 80% of the episodes were due to Gram-negative organisms, most common of which were Escherichia coli (19.6%), Salmonella spp (16.5%) and Klebsiella spp (15.1%). Gram-positive organisms implicated in 20% of episodes were mainly Staphylococcus aureus (9.3%) and enterococci (4.9%). Of all the septicaemias 62.0% were community-acquired with Salmonella spp. being the organism most commonly implicated. Hospital-acquired infections were mainly due to Serratia spp, Pseudomonas spp and Flavobacterium spp. The antibiotic resistance pattern of the organisms showed that hospital-acquired organisms had relatively high resistance to most antibiotics as compared with community-acquired organisms.

Cross Infection↗

PubMed1998

Sequence analysis of the Porphyromonas gingivalis dipeptidyl peptidase IV gene.

We previously constructed a Porphyromonas gingivalis genomic library and isolated the 2.9 kb EcoRV fragment which specified glycylprolyl dipeptidyl aminopeptidase (GPase). Nucleotide sequencing of this fragment identified the single 2169 bp open reading frame which coded for a 723 amino acid protein. The amino acid sequencing of the NH2-terminal domain of the native and recombinant mature enzymes suggested that the protease possessed a 16 amino acid residue signal peptide. The calculated mass of the precursor and mature proteases were 82,018 and 80,235 daltons, respectively. The homology search of this enzyme in registered protein sequences revealed that this enzyme was homologous to dipeptidyl peptidase (DPP) IV from the Flavobacterium meningosepticum and that from eukaryotic cells, including the human, mouse, and rat. Three amino acid residues, Ser-593, Asp-668, and His-700, were identified as a putative catalytic triad, a common feature of eukaryotic serine proteases. In addition, this enzyme showed a broad proteolytic spectrum toward synthetic substrates capable of splitting not only Gly-Pro-derivative but also Ala-Pro, Lys-Pro, and Phe-Pro-derivatives. Therefore, we conclude that this enzyme belongs to DPP IV rather than GPase.

Amino Acid Sequence↗

PubMed2003

Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences.

The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method. The occurrence of multiple melting domains and poor coupling between Tm and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities. We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus. Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers.

Base Sequence↗

PubMed1999

The taxonomy, ecology and cultivation of bacterial genera belonging to the family Flavobacteriaceae.

The group known as the 'flavobacteria' has previously been regarded as synonymous with the genus Flavobacterium. Today, however, flavobacteria refers to the family Flavobacteriaceae comprising 10 genera. This review deals with the rapid changes in the taxonomy of these bacteria, especially over the last decade. It also briefly reviews the ecology of the genera in this family and describes the media that have been utilized in the general and selective cultivation of these organisms.

Culture Media↗

PubMed1984

Differentiation of gram negative rods other than Enterobacteriaceae and Vibrionaceae by a micromethod for determination of carbon substrate assimilation.

The assimilation techniques described for taxonomic study are unsuitable for routine diagnosis because of the need for purification and standardization of substrates, the great quantity of medium consumed and difficulties in interpreting the results. A standardized micromethod (API Strip) for the study of carbon substrate assimilation by bacteria has recently been described (Zbl. Bakt. Hyg., I. Abt. Orig. A 255 (1983) 479-488). The prototype gallery used consisted of two strips of 32 microtubes each containing dehydrated carbon substrate. Each strip contained 30 tests plus positive and negative controls. The suspension medium was a synthetic semi-gel. A total of 1046 strains of Gram-negative rods representing 41 species belonging to the genera Pseudomonas, Alcaligenes, Moraxella, Acinetobacter, Flavobacterium and to the EF4 group were tested (Tables 1 to 6). Automatic reading was performed after incubation at 32 degrees C for one day, or two days for slow growing bacteria, using an ATB 1500 reader (API System) linked to a HP 85 microcomputer (Hewlett-Packard). Most species showed typical carbon substrate assimilation patterns allowing their differentiation from other species within each genus. The results obtained with the micromethod agreed in large measure with the nutritional patterns reported by other workers. It should be possible to use these results to construct a set of tests suitable for identifying species of Gram-negative rods other than Enterobacteriaceae and Vibrionaceae.

Acinetobacter↗

PubMed1988

Growth dependent enzymatic profiles of some gram-negative nonfermentative bacteria of clinical significance.

A total of 734 strains of gram-negative nonfermentative bacteria (46 species and biochemically defined groups) of the genera Pseudomonas, Alcaligenes, Bordetella, Moraxella, Acinetobacter, Agrobacterium, and Flavobacterium were investigated for their ability to hydrolyze 25 different chromogenic substrates. All tests were carried out in growth-stimulating media. Results were read photometrically and evaluated automatically following a 24-h incubation. Many of the 46 different species and biochemical groups exhibited uniform patterns of enzyme production. Some of the enzyme tests may serve as additional valuable tools for differential diagnosis of the organisms investigated. In combination with other biochemical tests, qualitative enzyme demonstration tests can facilitate the identification of gram-negative nonfermentative bacteria.

Acinetobacter↗