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The BH1999 Protein of Bacillus halodurans C-125 Is Gentisyl-Coenzyme A Thioesterase. Zhihao Zhuang, 2004.In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a kcat/Km of 1.6 x 106 M-1 s-1 and the hydrolysis of 3-hydroxybenzoyl-CoA with a kcat/Km of 3.0 x 105 M-1 s-1 . All other acyl-CoA thioesters tested had low or no substrate activity . The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation . It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase . Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily . A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity . Relative Neurotoxin Gene Expression in Clostridium botulinum Type B, Determined Using Quantitative Reverse Transcription-PCR. Maria Lövenklev, 2004.A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum . The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA . The patterns and relative expression of cntB were different in the different strains . Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase . In the proteolytic strain C . botulinum ATCC 7949, the level of cntB mRNA was four- to fivefold higher than the corresponding levels in the other strains . This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay . When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number . Instead, the level of cntB mRNA remained the same . When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred . In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations . This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in the cntB mRNA level was observed in all growth phases . Characterization of Amino Acid Substitutions in KdpA, the K+-Binding and -Translocating Subunit of the KdpFABC Complex of Escherichia coli. Martin van der Laan, 2002.When grown under K+ limitation, Escherichia coli induces the K+-translocating KdpFABC complex . The stimulation of ATPase activity by NH4+ ions was shown for the first time . Substitutions in KdpA, which is responsible for K+ binding and translocation, revealed that enzyme complexes KdpA:G232A and KdpA:G232S have completely lost their cation selectivity . Protective Role of tolC in Efflux of the Electron Shuttle Anthraquinone-2,6-Disulfonate. J. Bruce H. Shyu, 2002.Extracellular electron transfer can play an important role in microbial respiration on insoluble minerals . The humic acid analog anthraquinone-2,6-disulfonate (AQDS) is commonly used as an electron shuttle during studies of extracellular electron transfer . Here we provide genetic evidence that AQDS enters Shewanella oneidensis strain MR-1 and causes cell death if it accumulates past a critical concentration . A tolC homolog protects the cell from toxicity by mediating the efflux of AQDS . Electron transfer to AQDS appears to be independent of the tolC pathway, however, and requires the outer membrane protein encoded by mtrB . We suggest that there may be structural and functional relationships between quinone-containing electron shuttles and antibiotics . Enterotoxigenicity and Genetic Relatedness of Clostridium perfringens Isolates from Retail Foods in the United States. Yuan-Tong Lin, 2003.Clostridium perfringens is a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high . For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness . Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic . It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness . We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States . Forty isolates were obtained by using the iron milk method at 45°C, with confirmation by use of motility nitrate and lactose gelatin media . The presence of the C . perfringens enterotoxin (cpe) and alpha toxin (cpa) genes was determined by PCR using previously published primer sequences . All isolates possessed cpa . None of the isolates were identified as carrying the cpe gene by this method or by another method using a digoxigenin-labeled gene probe . Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination . Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates . About 5% of the isolates were considered to be closely related (2- to 3-band difference) . The others were considered to be unrelated to one another . The results demonstrate the rarity of cpe+ strains in retail foods and the genetic diversity among nonoutbreak strains . Preslaughter Holding Environment in Pork Plants Is Highly Contaminated with Salmonella enterica. M. H. Rostagno, 2003.The objective of this study was to determine whether abattoir pens can provide a Salmonella enterica infection source during the 2 to 4 h of preharvest holding . Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens . For 24 groups of pigs studied (  150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded . Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period (2.5 h) . After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group . All samples were cultured for the isolation and identification of S . enterica by primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification . The study pens were highly contaminated with S . enterica; all holding pens sampled had at least one positive sample . Additionally, 33% (8 of 24) of drinking water samples were positive for S . enterica . All 24 groups of pigs had S . enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples . From pigs, trailers, and pens, 586 isolates representing 36 different Salmonella serovars were isolated . Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers . Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source . This study demonstrates highly contaminated abattoir holding pens and watering sources . It also demonstrates that holding pens can serve as an infection source . This study identifies the abattoir holding pens as a significant hazard and a potential control point for Salmonella contamination in the preharvest pork production chain .
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