Novel pathways of regulation of the transcription factor Spx in Bacillus subtilis

Abstract

Spx is a transcription factor present in low G+C Gram-positive bacteria, including Bacillus subtilis and several human pathogens. By direct binding to the αCTD domain of the RNA polymerase, Spx modulates the expression of a large set of genes in B. subtilis. The Spx regulon is active in growing cells, and its expression is further induced in response to some stresses. Spx activation has been typically studied using diamide to generate disulfide stress. By using molecular biology, genetic, and biochemistry techniques, I show that the Spx regulon is also induced in response to cell wall stress, and that Spx is critical for survival of B. subtilis upon treatment with cell wall antibiotics. I further show the molecular mechanisms that lead to activation of Spx. Unlike disulfide stress, induction of the Spx regulon in response to cell wall stress requires transcriptional induction of the spx gene. This induction is mediated by the alternative extracytoplasmic sigma factor σM, and occurs at a promoter upstream of the yjbC-spx operon (i.e. PM1). Interestingly, activation of the Spx regulon in response to cell wall stress also requires stabilization; however, unlike disulfide stress, this process is mediated by a small protein called YirB. YirB is an anti-adaptor protein that binds with high affinity to YjbH, the adaptor protein required for ClpXP-mediated proteolysis, and therefore reduces the rate of Spx degradation. Transcriptional induction and post-translational stabilization are thus required for activation of the Spx regulon in response to antibiotics that inhibit peptidoglycan biosynthesis. Then, I show that yirB is also induced in response to cell wall stress, and that its activation requires the coordinated action of the YuxN repressor and the CssRS two-component system. Finally, I show that Spx is not only degraded through ClpXP, but also by ClpCP. The adaptor that mediates this degradation in unstressed cells appears to be MecA, and the evidence comes from the Spx-dependent synthetic lethality of ClpX and MecA. The McsB arginine kinase, which also acts as a ClpCP adaptor, as well as the YwlE arginine phosphatase are also shown to play an important role in Spx regulation. Overall, this work expands the regulatory mechanisms that control the activity of a pleiotropic transcription factor in B. subtilis.