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Electron Microscopic Analysis of Membrane Assemblies Formed by the Bacterial Chemotaxis Receptor Tsr. Robert M. Weis, 2003.The serine receptor (Tsr) from Escherichia coli is representative of a large family of transmembrane receptor proteins that mediate bacterial chemotaxis by influencing cell motility through signal transduction pathways . Tsr and other chemotaxis receptors form patches in the inner membrane that are often localized at the poles of the bacteria . In an effort to understand the structural constraints that dictate the packing of receptors in the plane of the membrane, we have used electron microscopy to examine ordered assemblies of Tsr in membrane extracts isolated from cells engineered to overproduce the receptor . Three types of assemblies were observed: ring-like "micelles" with a radial arrangement of receptor subunits, two-dimensional crystalline arrays with approximate hexagonal symmetry, and "zippers," which are receptor bilayers that result from the antiparallel interdigitation of cytoplasmic domains . The registration among Tsr molecules in the micelle and zipper assemblies was sufficient for identification of the receptor domains and for determination of their contributions to the total receptor length . The overall result of this analysis is compatible with an atomic model of the receptor dimer that was constructed primarily from the X-ray crystal structures of the periplasmic and cytoplasmic domains . Significantly, the micelle and zipper structures were also observed in fixed, cryosectioned cells expressing the Tsr receptor at high abundance, suggesting that the modes of Tsr assembly found in vitro are relevant to the situation in the cell . MgrA, an Orthologue of Mga, Acts as a Transcriptional Repressor of the Genes within the rlrA Pathogenicity Islet in Streptococcus pneumoniae. Carolyn Hemsley, 2003.Streptococcus pneumoniae normally resides in the human nasopharynx in a nondisease state . In response to unknown triggers this organism can descend to the lower respiratory tract and/or invade the bloodstream . Regulation and activation of virulence genes play essential roles in this process of disease development . Characterization of S . pneumoniae regulatory networks has been a recent area of interest, but despite inroads little is known about regulation of virulence genes in this pathogen . A putative transcriptional regulator in S . pneumoniae, mgrA, which exhibits homology to the virulence gene activator mga of group A streptococcus, was previously identified as a regulator that is required for development of pneumonia in a murine model . In this study we confirmed that mgrA plays a role in both nasopharyngeal carriage and pneumonia . Transcriptional profiling by microarray technology was used to show that mgrA acts as a repressor of the previously characterized rlrA pathogenicity islet . This is manifested phenotypically by a decrease in adherence to epithelial cells in tissue culture since the rlrA pathogenicity islet contains genes mediating adherence . Contribution of the RpoA C-Terminal Domain to Stimulation of the Salmonella enterica hilA Promoter by HilC and HilD. Igor N. Olekhnovich, 2004.Expression of invasion genes in Salmonella pathogenicity island 1 (SPI-1) is mainly driven by the transcriptional activator HilA . Transcription of hilA is subject to complex control and is stimulated by the SPI-1-encoded HilC and HilD proteins . The C-terminal domain of RpoA contributes to hilA activation by HilC/D under certain inducing conditions . Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution. Yolanda Nebra, 2003.A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described . The procedure is based on the detection of certain Bifidobacterium species in the samples . Two 16S rRNA gene-targeted probes are described . One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin . The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins . The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization . DNA-DNA hybridization with the BDE probe showed it to be specific for B . dentium, whereas that with the BAN probe showed it to be specific for B . animalis, B . asteroides, B . coryneforme, B . cuniculi, B . globosum, B . magnum, B . minimum, and B . subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces) . DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats . Thus, this probe is not suitable for the identification of any animal fecal pollution . Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did . Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin . Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor. Peter R. Girguis, 2003.Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture . Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments . To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea . We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system . Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments . Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners . Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia . Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments . The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia .
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