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Two Biosynthetic Pathways for Aromatic Amino Acids in the Archaeon Methanococcus maripaludis. Iris Porat, 2004.Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon . Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR) . In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted . This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 ± 0.2 mU mg–1 in the wild-type extract . The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate . The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-13C]phenylacetate. M . maripaludis has two IOR homologs . A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type . Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth . Genetic complementation of the IOR mutant also restored much of the wild-type phenotype . Thus, aryl acids appear to regulate the expression or activity of the de novo pathway . The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes . Efflux Pump-Mediated Intrinsic Drug Resistance in Mycobacterium smegmatis. Xian-Zhi Li, 2004.The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps . Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism . We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc2155 . Null mutants each carrying an in-frame deletion of these genes were then constructed in M . smegmatis . The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs) . The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs) . Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin . The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR . The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs . Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells . Finally, use of isogenic ß-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain ß-lactams in M . smegmatis . A Special Fondness for Lactobacilli. Gerald W. Tannock, 2004. Transcription-Defective soxR Mutants of Escherichia coli: Isolation and In Vivo Characterization. Monica Chander, 2003.The soxRS regulon protects Escherichia coli from superoxide and nitric oxide stress . SoxR protein, a transcription factor that senses oxidative stress via its [2Fe-2S] centers, transduces the signal to the soxS promoter to stimulate RNA polymerase . Here we describe 29 mutant alleles of soxR that cause defects in the activation of soxS transcription in response to paraquat, a superoxide stress agent . Owing to the selection and screen used in their isolation, most of these mutant alleles encode proteins that retained specific binding activity for the soxS promoter in vivo . The mutations were found throughout the SoxR polypeptide, although those closer to the N terminus typically exhibited greater defects in DNA binding . The degree of the defect in the transcriptional response to superoxide caused by each mutation was closely paralleled by its impaired response to nitric oxide . This work begins the general identification of the residues in the SoxR polypeptide that are critical for transducing oxidative stress signals into gene activation .
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