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The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA, from Clostridium perfringens. Jackie K. Cheung, 2004.The transcriptional regulation of toxin production in the gram-positive anaerobe Clostridium perfringens involves a two-component signal transduction system that comprises the VirS sensor histidine kinase and its cognate response regulator, VirR . Previous studies showed that VirR binds independently to a pair of imperfect direct repeats, now designated VirR box 1 and VirR box 2, located immediately upstream of the promoter of the pfoA gene, which encodes the cholesterol-dependent cytolysin, perfringolysin O . For this study, we introduced mutated VirR boxes into a C . perfringens pfoA mutant and found that both VirR boxes are essential for transcriptional activation . Furthermore, the spacing between the VirR boxes and the distance between the VirR boxes and the –35 region are shown to be critical for perfringolysin O production . Other VirR boxes that were previously identified from the strain 13 genome sequence were also analyzed, with perfringolysin O production used as a reporter system . The results showed that placement of the different VirR boxes at the same position upstream of the pfoA promoter yields different levels of perfringolysin O activity . In all of these constructs, VirR was still capable of binding to the target DNA, indicating that DNA binding alone is not sufficient for transcriptional activation . Finally, we show that the C . perfringens RNA polymerase binds more efficiently to the pfoA promoter in the presence of VirR, indicating that interactions must occur between these proteins . We propose that these interactions are required for VirR-mediated transcriptional activation . Biochemical Characterization of Streptococcus pneumoniae Penicillin-Binding Protein 2b and Its Implication in ß-Lactam Resistance. Estelle Pagliero, 2004.Extensive use of ß-lactam antibiotics has led to the selection of pathogenic streptococci resistant to ß-lactams due to modifications of the penicillin-binding proteins (PBPs) . PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan . The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 ß-lactam-sensitive strain . We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain . The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S443SN) . The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein . A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S . pneumoniae strain . Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations . Degradation of a Nonylphenol Single Isomer by Sphingomonas sp . Strain TTNP3 Leads to a Hydroxylation-Induced Migration Product. P. F. X. Corvini, 2004. Microautoradiographic Study of Rhodocyclus-Related Polyphosphate-Accumulating Bacteria in Full-Scale Enhanced Biological Phosphorus Removal Plants. Yunhong Kong, 2004.The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization . The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass . The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures . Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate . They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously . Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources . Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed . Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation . The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor . Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor . This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants .
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