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Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo. Graham S. Timmins, 2004.Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species . Here we investigated the requirements and the pathway of nitric oxide (NO·) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro . We also provide in vivo evidence that INH-derived NO· can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH . YopD and LcrH Regulate Expression of Yersinia enterocolitica YopQ by a Posttranscriptional Mechanism and Bind to yopQ RNA. Deborah M. Anderson, 2002.Pathogenic yersiniae secrete 14 Yop proteins via the type III pathway . Synthesis of YopQ occurs when the type III machinery is activated by a low-calcium signal, but not when the calcium concentration is above 100 µM . To characterize the mechanism that regulates the expression of yopQ, mutants that permit synthesis of YopQ in the presence of calcium were isolated . Yersiniae bearing deletion mutations in yopN, tyeA, sycN, or yscB synthesized and secreted YopQ in both the presence and the absence of calcium . In contrast, yersiniae with a deletion in yopD or lcrH synthesized YopQ in the presence of calcium but did not secrete the polypeptide . These variants displayed no defect in YopQ secretion under low-calcium conditions, revealing that yopD and lcrH are required for the regulation of yopQ expression . Experiments with transcriptional and translational fusions to the npt reporter gene suggest that yopD and lcrH regulate yopQ expression at a posttranscriptional step . YopD and LcrH form a complex in the bacterial cytosol and bind yopQ mRNA . Models that can account for posttranscriptional regulatory mechanisms of yop expression are discussed . Bacillus subtilis Diacylglycerol Kinase (DgkA) Enhances Efficient Sporulation. Samuel Amiteye, 2003.The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis . After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores . Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration . In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed . We also found that dgkA is expressed mainly during the vegetative phase . It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function . The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the traJ Promoter of the Escherichia coli Conjugative Plasmid pRK100. Marjanca Star, 2003.The TraJ protein is a central activator of F-like plasmid conjugal transfer . In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system . A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100 . The defect in the cya mutant was partially restored by addition of exogenous cAMP . Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription . Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region . DNase I footprint experiments mapped the CRP binding site around position -67.5 upstream of the putative traJ promoter . Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site . Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100 .
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