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Regulation of Hypercompetence in Legionella pneumophila. Jessica A. Sexton, 2004.Although many bacteria are known to be naturally competent for DNA uptake, this ability varies dramatically between species and even within a single species, some isolates display high levels of competence while others seem to be completely nontransformable . Surprisingly, many nontransformable bacterial strains appear to encode components necessary for DNA uptake . We believe that many such strains are actually competent but that this ability has been overlooked because standard laboratory conditions are inappropriate for competence induction . For example, most strains of the gram-negative bacterium Legionella pneumophila are not competent under normal laboratory conditions of aerobic growth at 37°C . However, it was previously reported that microaerophilic growth at 37°C allows L . pneumophila serogroup 1 strain AA100 to be naturally transformed . Here we report that another L . pneumophila serogroup 1 strain, Lp02, can also be transformed under these conditions . Moreover, Lp02 can be induced to high levels of competence by a second set of conditions, aerobic growth at 30°C . In contrast to Lp02, AA100 is only minimally transformable at 30°C, indicating that Lp02 is hypercompetent under these conditions . To identify potential causes of hypercompetence, we isolated mutants of AA100 that exhibited enhanced DNA uptake . Characterization of these mutants revealed two genes, proQ and comR, that are involved in regulating competence in L . pneumophila . This approach, involving the isolation of hypercompetent mutants, shows great promise as a method for identifying natural transformation in bacterial species previously thought to be nontransformable . Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis. Zhongtang Yu, 2004.Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples . We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used . When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions . The temperature of the lowest-melting-temperature (Tm(L)) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to Tm(L) . The average Tm(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared . The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products . The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons . Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles . Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted . Mutant Analysis Shows that Alanine Racemases from Pseudomonas aeruginosa and Escherichia coli Are Dimeric. Ulrich Strych, 2002.Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine . We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers . Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E . coli suppresses bacterial growth in a dominant negative fashion . Integration of Global Regulation of Two Aromatic-Responsive  54-Dependent Systems: a Common Phenotype by Different Mechanisms.Chun Chau Sze, 2002.Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related 54-dependent activators that control transcription of genes involved in catabolism of aromatic compounds . The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters . The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding
54 RNA polymerase, the integration host factor, and the regulator . These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors . In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media . Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation . Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case . The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of
54-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host . The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties . The mechanistic implications of how each of the host factors exerts its action are discussed .
Use of Bacterial  -Glutamyltranspeptidase for Enzymatic Synthesis of
-D-Glutamyl Compounds.Hideyuki Suzuki, 2003.An enzymatic method for synthesizing various -D-glutamyl compounds efficiently and stereospecifically involving bacterial
-glutamyltranspeptidase (EC 2.3.2.2) with D-glutamine as a
-glutamyl donor was developed . With D-glutamine as a
-glutamyl donor instead of L-glutamine in
-glutamyltaurine synthesis, by-products such as
-glutamylglutamine and
-glutamyl--glutamyltaurine were not synthesized and the yield of
-glutamyltaurine dramatically increased from 25 to 71% . It was also shown that the purification could be simplified without these
-glutamyl by-products . The possibility of synthesizing various
-D-glutamyl compounds was also shown .
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