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Abstract
The investigation of complex regulatory networks through in vitro transcription assays is critical to expand our understanding of genome expression and metabolism regulation in prokaryotes. However, the execution of such a project is dependent on the availability of the RNA polymerase holoenzyme (RNApholo), an essential element of transcription. Ideally, the RNApholo applied to structural and/or biochemical assays should be obtained from the species being studied or, alternatively, from the closest available organism. Acinetobacter baylyi ADP1 is a non-pathogenic, easily cultured and genetically- manipulated bacterium with a vast repertoire of metabolic enzymes that make it a compelling model organism for the study of bacterial metabolism. Before this dissertation, no RNApholo from A. baylyi ADP1 or from any other Acinetobacter species was recombinantly expressed and purified, thus limiting transcription studies. Here, we developed a method for the expression and purification of A. baylyi ADP1 RNApholo that provided high yields of a highly pure and active enzyme that can be applied to regulatory studies. In this regard, the A. baylyi ADP1 RNApholo was essential for conducting experiments that characterized the biological function of ACIAD0746, a LysR-type transcriptional regulator (LTTR) from A. baylyi, and for identifying a second class of transcriptional regulators involved in the assimilation of sulfur. Aerobic bacteria rely on the assimilation of sulfur to make cysteine, which serves as a reservoir of bioavailable sulfur to be used in the synthesis of critical S-containing cell components, such as iron-sulfur clusters, siderophores, and co-factors. The transcriptional regulation of sulfur metabolism in prokaryotes has long been associated with the LTTRs CysB and Cbl, two proteins that are mainly found in Betaproteobacteria and Gammaproteobacteria. In this dissertation, the combination of X-ray crystallographic, transcriptional assays, and other supportive experiments revealed the existence of another set of proteins that participate in the metabolism of sulfur and are more diversely distributed in bacteria.