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RelA Is a Component of the Nutritional Stress Activation Pathway of the Bacillus subtilis Transcription Factor  B.Shuyu Zhang, 2003.The general stress regulon of Bacillus subtilis is induced by the activation of the B transcription factor . Activation of
B occurs when one of two phosphatases (RsbU and RsbP), each responding to a unique type of stress, actuates a positive regulator of
B by dephosphorylation . Nutritional stress triggers the RsbP phosphatase . The mechanism by which RsbP becomes active is unknown; however, its activation coincides with culture conditions that are likely to reduce the cell's levels of high-energy nucleotides . We now present evidence that RelA, a (p)ppGpp synthetase and the key enzyme of the stringent response, plays a role in nutritional stress activation of
B . An insertion mutation that disrupts relA blocks the activation of
B in response to PO4 or glucose limitation and inhibits the drop in ATP/GTP levels that normally accompanies
B induction under these conditions . In contrast, the activation of
B by physical stress (e.g., ethanol treatment) is not affected by the loss of RelA . RelA's role in
B activation appears to be distinct from its participation in the stringent response . Amino acid analogs which induce the stringent response and RelA-dependent (p)ppGpp synthesis do not trigger
B activity . In addition, neither a missense mutation in relA (relA240GE) nor a null mutation in rplK (rplK54), either of which is sufficient to inhibit the stringent response and RelA-dependent (p)ppGpp synthesis, fails to block
B activation by PO4 or glucose limitation .
Global Characterization of Disulfide Stress in Bacillus subtilis. Lars Ingo Ole Leichert, 2003.We used DNA macroarray and proteome analysis to analyze the regulatory networks in Bacillus subtilis that are affected by disulfide stress . To induce disulfide stress, we used the specific thiol oxidant diamide . After addition of 1 mM diamide to an exponentially growing culture, cell growth stopped until the medium was cleared of diamide . Global analysis of the mRNA expression pattern during growth arrest revealed 350 genes that were induced by disulfide stress by greater than threefold . Strongly induced genes included known oxidative stress genes that are under the control of the global repressor PerR and heat shock genes controlled by the global repressor CtsR . Other genes that were strongly induced encode putative regulators of gene expression and proteins protecting against toxic elements and heavy metals . Many genes were substantially repressed by disulfide stress, among them most of the genes belonging to the negative stringent response . Two-dimensional gels of radioactively labeled protein extracts allowed us to visualize and quantitate the massive changes in the protein expression pattern that occurred in response to disulfide stress . The observed dramatic alteration in the protein pattern reflected the changes found in the transcriptome experiments . The response to disulfide stress seems to be a complex combination of different regulatory networks, indicating that redox-sensing cysteines play a key role in different signaling pathways sensing oxidative stress, heat stress, toxic element stress, and growth inhibition . Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization. Tamás Bakonyi, 2003.PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures . Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared . The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P . larvae cultures . The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey . The PCR assays also amplified these related bacteria, but at lower sensitivity . In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries . Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P . larvae spores . The most sensitive primer pair detected P . larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood . Honey specimens containing saprophyte bacilli and paenibacilli, but not P . larvae, were PCR negative . Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs .
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