The life cycle of Bacillus anthracis involves two cellular states, both of which function in establishing a productive infection. The dormant spores are able to escape immune defenses and become engulfed by macrophages, where the spores germinate to generate actively growing vegetative cells that perpetuate the disease condition. The B. anthracis genes that are induced in the macrophage upon germination include the ortholog of the global transcriptional regulator, Spx, an ArsC protein family member, which in Gram-positive bacteria activates genes that function in the oxidative stress response. Spx is highly conserved in Gram-positive bacteria, and has been implicated in the expression of virulence determinants in Listeria and Staphylococcus.. It is kept at low concentrations by proteolytic control that is exerted by the protease ClpXP and a substrate-binding adaptor protein YjbH. B. anthracis contains two paralogous (SpxA1 and SpxA2) forms of spx that are expressed at different stages of the life cycle. The objective of the proposed project is to define the regulons controlled by both Spx paralogs using microarray hybridization analysis. Mutant versions of the B. anthracis spx genes encoding protease resistant forms of SpxA1 and SpxA2 will be introduced into B. anthracis by conjugation using the B. subtilis ICEBs1 element. We will verify the microarray results in vivo by RT-PCR and by reconstructing Spx-dependent activation of transcription in vitro of genes identified in microarray analysis as requiring Spx for induction. We will determine which stage of the life cycle (vegetative growth versus sporulation) the Spx regulons are expressed. We will determine if both Spx-activated transcription and control of Spx stability is affected by changes in redox balance by treating cultures with oxidants known to induce Spx activity in B. subtilis. The phenotype of null mutations in both spx paralogs, of the spx double mutant will be examined with respect to oxidant sensitivity and Spx-controlled gene expression. Lastly, we will determine if holoenzymes bearing both paralogous Spx proteins recognize a distinct set of promoters, different from those recognized by either SpxA1 and SpxA2 holoenzyme forms. The findings will provide information pertaining to the roles of the two Spx paralogs in the B. anthracis life cycle and pathogenesis. The project will also develop and exploit a new tool in the genetic manipulation of B. anthracis, highlighted by the use of the interspecies conjugation system mediated by the B. subtilis ICEBs1 conjugative element. PUBLIC HEALTH RELEVANCE: The cause of Anthrax is the spore-forming bacterium Bacillus anthracis. Upon infection, the anthrax spore enters the human immune defense cell and begins a transformation from spore into a growing, virulent bacterium. This is accomplished through activation of the bacterial processes that promote growth and defense against the toxic oxidative attack mounted by the human immune system. The Spx protein, a known regulator of the oxidative stress response, is one of the first control factors produced by the invading bacterium, and understanding its role in Bacillus anthracis gene expression will provide clues to the virulence mechanisms that are active during infection.