Activation of the Extracytoplasmic Function σ Factor σ(P) by β-Lactams in Bacillus thuringiensis Requires the Site-2 Protease RasP

Bacteria can utilize alternative σ factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative σ factors are the extracytoplasmic function (ECF) σ factors. σ(P) is an ECF σ factor found in Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Previous work showed that σ(P) is induced by ampicillin, a β-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of σ(P) is controlled or what other antibiotics may activate σ(P). Here, we report that activation of σ(P) is specific to a subset of β-lactams and that σ(P) is required for resistance to these β-lactams. We demonstrate that activation of σ(P) is controlled by the proteolytic destruction of the anti-σ factor RsiP and that degradation of RsiP requires multiple proteases. Upon exposure to β-lactams, the extracellular domain of RsiP is cleaved by an unknown protease, which we predict cleaves at site-1. Following cleavage by the unknown protease, the N terminus of RsiP is further degraded by the site-2 intramembrane protease RasP. Our data indicate that RasP cleavage of RsiP is not the rate-limiting step in σ(P) activation. This proteolytic cascade leads to activation of σ(P), which induces resistance to β-lactams likely via increased expression of β-lactamases. IMPORTANCE The discovery of antibiotics to treat bacterial infections has had a dramatic and positive impact on human health. However, shortly after the introduction of a new antibiotic, bacteria often develop resistance. The bacterial cell envelope is essential for cell viability and is the target of many of the most commonly used antibiotics, including β-lactam antibiotics. Resistance to β-lactams is often dependent upon β-lactamases. In B. cereus, B. thuringiensis, and some B. anthracis strains, the expression of some β-lactamases is inducible. This inducible β-lactamase expression is controlled by activation of an alternative σ factor called σ(P). Here, we show that β-lactam antibiotics induce σ(P) activation by degradation of the anti-σ factor RsiP.