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Effect of Adaptation to Ethanol on Cytoplasmic and Membrane Protein Profiles of Oenococcus oeni. M. Graça Silveira, 2004.The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism . Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated . Ethanol triggers alterations in protein patterns of O . oeni cells stressed with 12% ethanol for 1 h and those of cells grown in the presence of 8% ethanol . Levels of inosine-5'-monophosphate dehydrogenase and phosphogluconate dehydrogenase, which generate reduced nicotinamide nucleotides, were decreased during growth in the presence of ethanol, while glutathione reductase, which consumes NADPH, was induced, suggesting that maintenance of the redox balance plays an important role in ethanol adaptation . Phosphoenolpyruvate:mannose phosphotransferase system (PTS) components of mannose PTS, including the phosphocarrier protein HPr and EIIMan, were lacking in ethanol-adapted cells, providing strong evidence that mannose PTS is absent in ethanol-adapted cells, and this represents a metabolic advantage to O . oeni cells during malolactic fermentation . In cells grown in the presence of ethanol, a large increase in the number of membrane-associated proteins was observed . Interestingly, two of these proteins, dTDT-glucose-4,6-dehydratase and D-alanine:D-alanine ligase, are known to be involved in cell wall biosynthesis . Using a proteomic approach, we provide evidence for an active ethanol adaptation response of O . oeni at the cytoplasmic and membrane protein levels . A ToxR Homolog from Vibrio anguillarum Serotype O1 Regulates Its Own Production, Bile Resistance, and Biofilm Formation. Su-Yan Wang, 2002.ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli . To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V . anguillarum serotype O1 were cloned . The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively . Deletion mutations were made in each gene and functional analyses were done . Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors . The toxR mutant but not the toxS mutant was 20% less motile than the wild type . Like many regulatory proteins, ToxR was shown to negatively regulate its own expression . Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP . The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V . cholerae, and which has an amino terminus 75% identical to that of OmpU . ToxR and to a lesser extent ToxS enhanced resistance to bile . Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR . Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli . In Situ Activation of the Quorum-Sensing Transcription Factor TraR by Cognate and Noncognate Acyl-Homoserine Lactone Ligands: Kinetics and Consequences. Zhao-Qing Luo, 2003.Conjugal transfer of Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of the transcriptional activator TraR and its acyl-homoserine lactone quormone N-(3-oxo-octanoyl)-L-homoserine lactone (3-oxo-C8-HSL) . The population density dependence of quorum-sensing systems can often be circumvented by addition of the quormone to cultures at low cell numbers . However, the quorum-dependent activation of Ti plasmid conjugal transfer exhibited a lag of almost 8 h when the quormone was added to donor cells at low population densities (Piper and Farrand, J . Bacteriol . 182:1080-1088, 2000) . As measured by activation of a TraR-dependent traG::lacZ reporter fusion, TraR in cells exposed to the cognate signal for 5 min showed detectable activity, while exposure for 15 min resulted in full activity . Thus, the lag in activation is not due to some intrinsic property of TraR . Cells exposed to the agonistic analog N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) exhibited similar induction kinetics . However, activation of the reporter in cells exposed to the poorly effective alkanoyl acyl-HSL N-hexanoyl-L-homoserine lactone (C6-HSL) required the continued presence of the signal . As measured by an in vivo repressor assay, TraR activated by 3-oxo-C6-HSL or by 3-oxo-C8-HSL remained active for as long as 8 h after removal of exogenous signal . However, TraR activated by the alkanoyl quormone C6-HSL rapidly lost activity following removal of the signal . In quormone retention assays, which measure signal binding by TraR, cells grown with either of the two 3-oxo-acyl-HSL quormones retained the ligand after washing, while cells grown with C6-HSL lost the alkanoyl-HSL concomitant with the rapid loss of TraR activity . We conclude that TraR rapidly binds its quormone and that, once bound, the cognate signal and its close homologs are tightly retained . Moreover, in the absence of other regulatory factors, activated TraR remains functional after removal of the signal . On the other hand, poorly active signals are not tightly bound, and their removal by washing leads to rapid loss of TraR activity . Oligonucleotide Probes That Detect Quantitatively Significant Groups of Butyrate-Producing Bacteria in Human Feces. Georgina L. Hold, 2003.16S rRNA-targeted oligonucleotide probes were designed for butyrate-producing bacteria from human feces . Three new cluster-specific probes detected bacteria related to Roseburia intestinalis, Faecalibacterium prausnitzii, and Eubacterium hallii at mean populations of 2.3, 3.8, and 0.6%, respectively, in samples from 10 individuals . Additional species-level probes accounted for no more than 1%, with a mean of 7.7%, of the total human fecal microbiota identified as butyrate producers in this study . Bacteria related to E . hallii and the genera Roseburia and Faecalibacterium are therefore among the most abundant known butyrate-producing bacteria in human feces .
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