Bacillus anthracis possesses two paralogs of the transcriptional regulator, Spx. SpxA1 and SpxA2 interact with RNA polymerase (RNAP) to activate the transcription of genes implicated in the prevention and alleviation of oxidative protein damage. The spxA2 gene is highly upregulated in infected macrophages, but how this is achieved is unknown. Previous studies have shown that the spxA2 gene was under negative control by the Rrf2 family repressor protein, SaiR, whose activity is sensitive to oxidative stress. These studies also suggested that spxA2 was under positive autoregulation. In the present study, we show by in vivo and in vitro analyses that spxA2 is under direct autoregulation but is also dependent on the SpxA1 paralogous protein. The deletion of either spxA1 or spxA2 reduced the diamide-inducible expression of an spxA2-lacZ construct. In vitro transcription reactions using purified B. anthracis RNAP showed that SpxA1 and SpxA2 protein stimulates transcription from a DNA fragment containing the spxA2 promoter. Ectopically positioned spxA2-lacZ fusion requires both SpxA1 and SpxA2 for expression, but the requirement for SpxA1 is partially overcome when saiR is deleted. Electrophoretic mobility shift assays showed that SpxA1 and SpxA2 enhance the affinity of RNAP for spxA2 promoter DNA and that this activity is sensitive to reductant. We hypothesize that the previously observed upregulation of spxA2 in the oxidative environment of the macrophage is at least partly due to SpxA1-mediated SaiR repressor inactivation and the positive autoregulation of spxA2 transcription. T ranscription factors of pathogenic bacteria can be important virulence determinants, since they control genes that specify toxins, proteins functioning in nutrient acquisition within the hostile host environment, and products that operate by subverting the host innate immunity (1-4). For example, in Bacillus anthracis, the causative agent of the zoonotic infectious disease, anthrax, the plasmid-borne atxA gene encodes a transcription factor required for virulence, since it is necessary for the activation of genes for toxin and protective capsule production (5, 6). Other Grampositive bacteria possess genes encoding one or more forms of the ArsC (arsenate reductase) family protein, Spx, which is an RNA polymerase (RNAP)-binding protein directing the expression of large regulons that include genes necessary for pathogenesis (7-11). Studies of streptococci, Listeria monocytogenes, Staphylococcus aureus, and Enterococcus spp. have demonstrated the requirement for Spx proteins for virulence and viability (7,8,(12)(13)(14)(15). IMPORTANCE Regulators of transcription initiationSpx was detected in studies of B. subtilis, in which it is encoded by a gene that is under complex transcriptional control involving four RNAP forms and two repressors (16)(17)(18)(19). The complex control of spx transcription operates in response to various stress conditions, such as oxidative and cell envelope stress. Spx protein levels increase under conditions caus...

Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP ␣ subunit (␣CTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in ␣CTD severely impaired Spx-activated transcription. Attempts to substitute alanine for ␣CTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoA Y263C mutant, where "Y263C" indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound ␣CTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoA E255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli ␣D259, which has been implicated in ␣CTD-70 interaction ( 70 R603, corresponding to R362 of B. subtilis A ). However, the combined rpoA E255A and sigA R362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while ␣CTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-␣CTD complex.IMPORTANCE Though extensively studied in Escherichia coli, the role of ␣CTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal ␣CTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-␣CTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several ␣CTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.KEYWORDS alpha subunit, RNA polymerase, Spx, transcription, sporulation R egulation of gene expression at the level of transcription initiation is required for bacteria to maintain fitness and survival in a dynamic environment (e.g., perturbations in temperature, nutrient availability, exposure to toxic agents, etc.). Global control