Showing papers in "Fems Microbiology Letters in 2002"

The microbiology of butyrate formation in the human colon.

Abstract: Butyrate arising from microbial fermentation is important for the energy metabolism and normal development of colonic epithelial cells and has a mainly protective role in relation to colonic disease. While certain dietary substrates such as resistant starch appear to be butyrogenic in the colon, it is not known to what extent these stimulate butyrate production directly, e.g. by promoting amylolytic species, or indirectly, e.g. through cross-feeding of fermentation products. Cultural and molecular studies indicate that the most numerous butyrate-producing bacteria found in human faeces are highly oxygen-sensitive anaerobes belonging to the Clostridial clusters IV and XIVa. These include many previously undescribed species related to Eubacterium, Roseburia, Faecalibacterium and Coprococcus whose distribution and metabolic characteristics are under investigation. A better understanding of the microbial ecology of colonic butyrate-producing bacteria will help to explain the influence of diet upon butyrate supply, and to suggest new approaches for optimising microbial activity in the large intestine.

Role of membranes in the activities of antimicrobial cationic peptides

Abstract: Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.

Carbon catabolite repression in bacteria : choice of the carbon source and autoregulatory limitation of sugar utilization

Abstract: Carbon catabolite repression (CCR) in bacteria is generally regarded as a regulatory mechanism to ensure sequential utilization of carbohydrates. Selection of the carbon sources is mainly made at the level of carbohydrate-specific induction. Since virtually all carbohydrate catabolic genes or operons are regulated by specific control proteins and require inducers for high level expression, direct control of the activity of regulators or control of inducer formation is an efficient measure to keep them silent. By these mechanisms, bacteria are able to establish a hierarchy of sugar utilization. In addition to the control of induction processes by CCR, bacteria have developed global transcriptional regulation circuits, in which pleiotropic regulators are activated. These global control proteins, the catabolite gene activator protein (CAP), also known as cAMP receptor protein, in Escherichia coli or the catabolite control protein (CcpA) in Gram-positive bacteria with low GC content, act upon a large number of catabolic genes/operons. Since practically any carbon source is able to trigger global transcriptional control, expression of sugar utilization genes is restricted even in the sole presence of their cognate substrates. Consequently, CAP- or CcpA-dependent catabolite repression serves as an autoregulatory device to keep sugar utilization at a certain level rather than to establish preferential utilization of certain carbon sources. Together with other autoregulatory mechanisms that are not acting at the gene expression level, CCR helps bacteria to adjust sugar utilization to their metabolic capacities. Therefore, catabolic/metabolic balance would perhaps better describe the physiological role of this regulatory network than the term catabolite repression.

Diversity of CTX‐M β‐lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals

Abstract: Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M β-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) β-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M β-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some blaCTX-M genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata β-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.

The filamentation pathway controlled by the Efg1 regulator protein is required for normal biofilm formation and development in Candida albicans.

Abstract: Candida albicans biofilms are structured microbial communities composed of a mixture of yeast cells and hyphal elements, suggesting a pivotal role for the dimorphic switch in the development of biofilms. We have used C. albicans mutants defective in genes involved in filamentation (Δcph1, Δefg1, Δhst7, and Δcst20) and compared these mutants to wild-type strains to determine whether filamentation is an integral factor for biofilm formation. Scanning electron microscopy revealed that Δcph1, Δhst7 and Δcst20 mutants were able to filament and form structured biofilms displaying three-dimensional architecture similar to those formed by wild-type strains. However, Δefg1 and Δcph1/Δefg1 mutants were unable to filament and did not form biofilms, but rather sparse monolayers of loosely attached elongated, rod-like, cells. Antimicrobial susceptibility testing showed intrinsic resistance of all mutant strains to fluconazole and amphotericin B when attached to the surface of biomaterials. These results suggest that hyphal formation is pivotal for biofilm development in C. albicans. However, the sessile lifestyle associated with adherent cells confers antifungal resistance, regardless of coherent biofilm formation.

Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery

Abstract: Airborne bacteria isolated from a tannery air environment were screened for the property of solubilization of insoluble zinc oxide and zinc phosphate. Out of 10 strains tested, a strain of Pseudomonas aeruginosa (CMG 823) showed the best solubilization and solubilized both zinc oxide and zinc phosphate. Colonies of the bacterium produced clear haloes on solid medium which contained these insoluble metal compounds, but only when glucose was provided as a carbon source. Solubilization of zinc oxide and phosphate was accompanied by an increase in the H+ concentration of the medium, probably a consequence of the production of 2-ketogluconic acid.

A PCR-based method for identification of lactobacilli at the genus level

Abstract: We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level. One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains. Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species. Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

Diversity and phylogenetic analysis of bacteria in the mucosa of chicken ceca and comparison with bacteria in the cecal lumen

Abstract: We reported the first attempt to describe mucosa-associated bacterial populations in the chicken ceca by molecular analysis of 16S rRNA genes. Bacteria in the mucosa were highly diverse but mainly Gram-positive with low G+C. Fusobacterium prausnitzii and butyrate-producing bacteria comprised the largest groups among 116 cloned sequences. Twenty five percent of the clones had less than 95% homology to database sequences. Many sequences were related to those of uncultured bacteria identified in human feces or the bovine rumen. Terminal restriction fragment length polymorphism (T-RFLP) analysis revealed some differences between bacterial populations present in the mucosa and lumen of ceca. Greater resolution of bacterial population was obtained using a culture-independent approach rather than a culture-based approach.

Current status of defensins and their role in innate and adaptive immunity.

Abstract: Naturally occurring antimicrobial cationic polypeptides play a major role in innate and adaptive immunity. These polypeptides are found to be either linear and unstructured or structured through disulfide bonds. Among the structured antimicrobial polypeptides, defensins comprise a family of cysteine-rich cationic polypeptides that contribute significantly to host defense against the invasion of microorganisms in animals, humans, insects and plants. Their wide-spread occurrence in various tissues of these diverse organisms, and their importance in innate and adaptive immunity have led to their identification, isolation and characterization. A large volume of literature is available on defensins’ occurrence, structural characterization, gene expression and regulation under normal and pathological conditions. Much has also been published regarding their antimicrobial, antiviral and chemoattractive properties, and their molecular and cellular interactions. In this review, we describe the current status of our knowledge of defensins with respect to their molecular, cellular and structural biology, their role in host defense, future research paradigms and the possibility of their utilization as a new class of non-toxic antimicrobial agents and immuno-modulators.

Copper and cadmium increase laccase activity in Pleurotus ostreatus.

Abstract: Addition of copper (0.5–5 mM) or cadmium (1–5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and H2O2 decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05–50 mM) increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy metals.

Degradation of contrasting pesticides by white rot fungi and its relationship with ligninolytic potential.

Abstract: The capacity of nine species of white rot fungus from a variety of basidiomycete orders to degrade contrasting mono-aromatic pesticides was investigated. There was no relationship between degradation of the dye Poly R-478, a presumptive test for ligninolytic potential, and degradation of the highly available pesticides diuron, metalaxyl, atrazine or terbuthylazine in liquid culture. However, there were significant positive correlations between the rates of degradation of the different pesticides. Greatest degradation of all the pesticides was achieved by Coriolus versicolor, Hypholoma fasciculare and Stereum hirsutum. After 42 days, maximum degradation of diuron, atrazine and terbuthylazine was above 86%, but for metalaxyl less than 44%. When grown in the organic matrix of an on-farm ‘biobed’ pesticide remediation system, relative degradation rates of the highly available pesticides by C. versicolor, H. fasciculare and S. hirsutum showed some differences to those in liquid culture. While H. fasciculare and C. versicolor were able to degrade about a third of the poorly available compound chlorpyrifos in biobed matrix after 42 days, S. hirsutum, which was the most effective degrader of the available pesticides, showed little capacity to degrade the compound. 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis.

Abstract: The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.

Antibacterial properties of cationic steroid antibiotics.

Abstract: Cationic steroid antibiotics have been developed that display broad-spectrum antibacterial activity. These compounds are comprised of steroids appended with amine groups arranged to yield facially amphiphilic morphology. Examples of these antibiotics are highly bactericidal, while related compounds effectively permeabilize the outer membranes of Gram-negative bacteria sensitizing these organisms to hydrophobic antibiotics. Cationic steroid antibiotics exhibit various levels of eukaryote vs. prokaryote cell selectivity, and cell selectivity can be increased via charge recognition of prokaryotic cells. Studies of the mechanism of action of these antibiotics suggest that they share mechanistic aspects with cationic peptide antibiotics.

Protein kinases and protein phosphatases in prokaryotes: a genomic perspective.

Abstract: For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain. Over time, however, incidents of apparent domain trespass involving the presence of ‘eukaryotic’ protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency. Today, genomics has provided the means to examine the phylogenetic distribution of ‘eukaryotic’ protein kinases and protein phosphatases in a comprehensive and systematic manner. The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism.

The identification of five genetic loci of Francisella novicida associated with intracellular growth

Abstract: Five transposon mutants of Francisella novicida were isolated that are compromised in their ability to grow in mouse macrophages in vitro. Sequence analysis of the DNA flanking the transposon insertions identified the genes that were interrupted in these mutants. One of the inactivated loci corresponds to the Francisella tularensis gene that encodes a 23-kDa protein that is the most prominently induced protein following macrophage infection. Another insertion was localised to approximately 2 kb upstream of the gene encoding the 23-kDa protein. By analysis of the incomplete Francisella genome sequence it was surmised that these two insertions disrupt different portions of a putative operon that encodes four proteins, none of which have discernible functions. Three other interrupted loci associated with poor intramacrophage growth showed similarity at the deduced amino acid level to alanine racemase, the ClpB heat-shock protease, and the purine biosynthetic enzyme, glutamine phosphoribosylpyrophosphate amidotransferases.

A bacterial cell to cell signal in the lungs of cystic fibrosis patients.

Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.

Identification and molecular characterisation of CmeB, a Campylobacter jejuni multidrug efflux pump.

Abstract: A multidrug efflux pump gene ( cmeB ) was identified from the published Campylobacter jejuni genome sequence. Secondary structural analysis showed that the gene encoded a protein belonging to the resistance nodulation cell division (RND) family of efflux transporters. The gene was inactivated by insertional mutagenesis. Compared with the wild-type strain (NCTC 11168), the resultant knockout strain (NCTC 11168- cmeB::kan r) displayed increased susceptibility to a range of antibiotics including β-lactams, fluoroquinolones, macrolides, chloramphenicol, tetracycline, ethidium bromide, the dye acridine orange and the detergent sodium dodecyl sulfate. Accumulation of ciprofloxacin was increased in the knockout mutant, but carbonyl cyanide m -chlorophenyl hydrazone, a proton motive force inhibitor, had less effect upon ciprofloxacin accumulation in the knockout mutant compared with NCTC 11168. These data show that the identified gene encodes an RND-type multi-substrate efflux transporter, which contributes to intrinsic resistance to a range of structurally unrelated compounds in C. jejuni . This efflux pump has been named CmeB (for Campylobacter multidrug efflux).

Energetics and kinetics of lactate fermentation to acetate and propionate via methylmalonyl-CoA or acrylyl-CoA

Abstract: Fermentation balances and growth yields were determined with various bacteria fermenting lactate to acetate plus propionate either via methylmalonyl-CoA or via acrylyl-CoA. All strains fermented lactate to acetate plus propionate at approximately a 1:2 ratio. Growth yields of Propionibacterium freudenreichii were more than twice as high as those of Clostridium homopropionicum or Veillonella parvula. Hydrogen was formed as a side product to a significant extent only by V. parvula and Pelobacter propionicus; the latter formed hydrogen preferentially when using ethanol as substrate. Acrylyl-CoA reductase of C. homopropionicum and Clostridium neopropionicum was found nearly exclusively in the cytoplasm thus confirming that this reduction step is unlikely to be involved in energy conservation. C. homopropionicum exhibited higher KS and higher μmax values, as well as higher specific substrate turnover rates than P. freudenreichii. The results allow us to conclude that C. homopropionicum using the acrylyl-CoA pathway with low growth yield obtains its specific competitive advantage compared to P. freudenreichii not through higher substrate affinity or metabolic shift toward enhanced acetate-plus-hydrogen formation but through faster specific substrate turnover.

Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects

Abstract: Polysaccharides were extracted from fruiting bodies and cultured mycelia from five Antrodia camphorata strains Polysaccharide profiles of the five strains, as determined by high-performance anion-exchange chromatography, showed varying yields and composition of neutral sugars A camphorata fruiting bodies also had different polysaccharide patterns compared to the cultured mycelium Analysis of 26-day-old mycelia showed that the neutral sugars galactose, glucose, mannose, and galactosamine were predominant All mycelia polysaccharide preparations exhibited anti-hepatitis B virus activity Polysaccharides from strain B86 at a concentration of 50 μg ml−1 showed the highest level of anti-hepatitis B surface antigen effect, which was higher than α-interferon at a dosage of 1000 U ml−1 Only strains B86 and 35398 had substantial anti-hepatitis B e antigen activities None of the polysaccharides exhibited cytotoxic effects

Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR.

Abstract: Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2–3 h after the material has arrived in the laboratory.

Altamira cave Paleolithic paintings harbor partly unknown bacterial communities

Abstract: Since it has been reported that microorganisms can affect painting pigments, Paleolithic painting microbiology deserves attention. The present study is the first report on the bacterial colonization of the valuable Paleolithic paintings in the famous Altamira cave (Spain). One sample taken from a painting area in the Polychromes Hall was analyzed culture-independently. This was the first time microbiologists were allowed to take sample material directly from Altamira paintings. Identification methods included PCR amplification of 16S rRNA genes (16S rDNA) and community fingerprinting by denaturing gradient gel electrophoresis (DGGE). The applied approach gave insight into a great bacterial taxonomic diversity, and allowed the detection of unexpected and unknown bacteria with potential effects on the conservation of the painting. Regarding the number of 29 visible DGGE bands in the community fingerprint, the numbers of analyzed clones described about 72% of the phylogenetic diversity present in the sample. Thirty-eight percent of the sequences analyzed were phylogenetically most closely related to cultivated bacteria, while the majority (62%) were most closely related to environmental 16S rDNA clones. Bacteria identified in Altamira were related with sequence similarities between 84.8 and 99.4% to members of the cosmopolitan Proteobacteria (52.3%), to members of the Acidobacterium division (23.8%), Cytophaga/Flexibacter/Bacteroides phylum (9.5%), green non-sulfur bacteria (4.8%), Planctomycetales (4.8%) and Actinobacteria (4.8%). The high number of clones most closely related to environmental 16S rDNA clones showed the broad spectrum of unknown and yet to be cultivated bacteria in Altamira cave.

Design and evaluation of oligonucleotide-microarray method for the detection of human intestinal bacteria in fecal samples

Abstract: An oligonucleotide-microarray method was developed for the detection of intestinal bacteria in fecal samples collected from human subjects. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to glass slides. Cyanine5 (CY5)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or bacterial DNA using two universal primers and were hybridized to the oligo-microarray. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides fragilis, Bacteroides distasonis, Clostridium clostridiiforme, Clostridium leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus albus, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis, Eubacterium biforme, Eubacterium aerofaciens, Lactobacillus acidophilus, Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the oligo-microarray method developed in this study is a reliable method for the detection of predominant human intestinal bacteria in the fecal samples.

Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum

Abstract: Pseudomonas aeruginosa and Burkholderia cepacia cause destructive lung disease in cystic fibrosis (CF) patients. Both pathogens employ 'quorum sensing', i.e. cell-to-cell communication, via diffusible N-acyl-L-homoserine lactone (AHL) signal molecules, to regulate the production of a number of virulence determinants in vitro. However, to date, evidence that quorum sensing systems are functional and play a role in vivo is lacking. This study presents the first direct evidence for the presence of AHLs in CF sputum. A total of 42 samples from 25 CF patients were analysed using lux-based Escherichia coli AHL biosensors. AHLs were detected in sputum from patients colonised by P. aeruginosa or B. cepacia but not Staphylococcus aureus. Furthermore, using liquid chromatography-mass spectrometry and thin layer chromatography, we confirmed the presence of N-hexanoylhomoserine lactone and N-(3-oxododecanoyl)homoserine lactone respectively in sputum samples from patients colonised by P. aeruginosa.

Cloning, expression and characterization of l-arabinose isomerase from Thermotoga neapolitana: bioconversion of d-galactose to d-tagatose using the enzyme

Abstract: Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.

Identification and characterization of toxigenic Bacillus cereus isolates responsible for two food-poisoning outbreaks

Abstract: The epidemiology of Bacillus cereus strains responsible for food poisoning is scantly known, mostly because the genotypic and toxigenic properties of the B. cereus strains isolated during food-poisoning outbreaks have been never catalogued. The occurrence of two simultaneous food-poisoning outbreaks gave us the opportunity to wonder whether (i) the identity of individual strains isolated from clinical, environmental, and food samples could be established by random amplified polymorphic DNA (RAPD)-PCR and multiplex RAPD-PCR, and (ii) the toxigenic potential of the isolates could be determined by testing their ability to secrete hemolysin BL, phosphatidylcholine-specific phospholipase C, and cereulide, as well as by determining the presence of the genes encoding enterotoxins NHE, T, and FM/S, cytotoxin K, sphingomyelinase, and phosphatidylinositol-specific phospholipase C. This is the first report demonstrating that the combination of several phenotypic and genotypic traits provides a powerful tool for tracing the source of infection of toxigenic B. cereus strains relevant for epidemiological survey.

Anaerobic C-ring cleavage of genistein and daidzein by Eubacterium ramulus

Abstract: Eubacterium ramulus, a flavonoid-degrading anaerobic bacterium from the human gastrointestinal tract, was tested for its ability to transform the isoflavonoids genistein-7-O-glucoside (genistin), genistein and daidzein. Genistein was completely degraded by E. ramulus via 6'-hydroxy-O-desmethylangolensin to 2-(4-hydroxyphenyl)-propionic acid. Dihydrogenistein was neither observed as an intermediate in this transformation nor converted itself by growing cells or cell-free extracts of E. ramulus. Genistein-7-O-glucoside was partially transformed by way of genistein to the product 2-(4-hydroxyphenyl)-propionic acid. Daidzein was in part degraded to O-desmethylangolensin, the corresponding metabolite to 6'-hydroxy-O-desmethylangolensin. The hydroxyl group in position 6' of O-desmethylangolensin is crucial for further degradation.

Plasmid profiling and antibiotic resistance of Vibrio strains isolated from cultured penaeid shrimp

Abstract: Resistance to different antibiotics was found in 26 of the 30 strains analyzed, more than 70% of the strains analyzed were resistant to carbenicillin and ampicillin and a significant correlation was found between the resistance to both antibiotics. Plasmids were found in 80% of the strains analyzed, and 11 different plasmid profiles were observed. The most common profile obtained had only a 21.2-kbp plasmid, a significant correlation was found between the presence of this plasmid and resistance to carbenicillin, although some exceptions could be detected. Plasmids were cured from a cephalothin resistant strain and reintroduced into the plasmid-free cell and into Escherichia coli DH5α, both strains gained resistance to this antibiotic.

Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto‐Inland Sea, Japan

Abstract: Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.

Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni

Abstract: Malolactic fermentation (MLF) is carried out by Oenococcus oeni under very harsh conditions. This paper shows that stress compounds in wine such as SO2, fatty acids and copper have an inhibitory effect on cell growth and MLF duration, and relates this effect to an inhibition of ATPase activity. Of the stress compounds, SO2 and dodecanoic acid had the strongest effect, decreasing the ATPase specific activity to 37% and 58%, respectively. It can be concluded that ATPase is a good indicator of the physiological state of the cells and their ability to lead MLF.

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum

Abstract: A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum.