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Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter. Yang Wang, 2003.The Escherichia coli transcription factor sigma 32 binds to core RNA polymerase to form the holoenzyme responsible for transcription initiation at heat shock promoters, utilized upon exposure of the cell to higher temperatures . We have developed two ways to assay sigma 32-dependent RNA synthesis in E . coli . The plasmid-borne reporter gene for both is lacZ (ß-galactosidase), driven by the groE promoter . In one application, the cells are exposed to a temperature of 42°C in order to induce accumulation of endogenous sigma 32 . The other involves isopropylthiogalactopyranoside (IPTG)-induced synthesis of sigma 32 at 30°C from a gene contained on a second plasmid . The latter employs DnaK- cells, which additionally contained a second mutation, inactivating the endogenous sigma 32 gene (Bukau and Walker, EMBO J . 9:4027-4036, 1990) . These assays were used to delineate the sequences CTTGA (-37 to -33) and GNCCCCATNT (-18 to -9) as important for sigma 32 promoter activity . At each of the specified base pairs, substitutions were found which reduced promoter activity by greater than 75% . Activity was also dependent upon the number of base pairs separating the two regions . 2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum. Mary M. Watrous, 2003.The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated . An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis . The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a Km of 152 µM . Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction . Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates . The level of hydrogenase activity in a cell system correlated (R2 = 0.89) with the organism's ability to reduce TNT . A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains . Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression . The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C . acetobutylicum in whole-cell systems . A mechanism for the reaction is proposed . Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities .
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