Activation of the Extracytoplasmic Function σ factor σP by β-lactams in Bacillus thuringiensis requires the site-2 protease RasP

Abstract

Bacteria can utilize alternative {sigma} factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative {sigma} factors are the Extracytoplasmic Function (ECF) {sigma} factors. {sigma}P is an ECF {sigma} factor found in Bacillus anthracis, B. cereus, and B. thuringiensis. Previous work showed {sigma}P is induced by ampicillin, a {beta}-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of {sigma}P is controlled or what other antibiotics may activate {sigma}P. Here we report that activation of {sigma}P is specific to a subset of {beta}-lactams and {sigma}P is required for resistance to these {beta}-lactams. We demonstrate that activation of {sigma}P is controlled by the proteolytic destruction of the anti-{sigma} factor, RsiP, and that degradation of RsiP requires multiple proteases. Upon exposure to {beta}-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 {sigma}P activation. This proteolytic cascade leads to activation of {sigma}P which induces resistance to {beta}-lactams likely via increased expression of {beta}-lactamases.\n\nImportanceThe 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 {beta}-lactam antibiotics. Resistance to {beta}-lactams is often dependent upon {beta}-lactamases. In B. cereus, B. thuringiensis and some B. anthracis strains the expression of some {beta}-lactamases is inducible. This inducible {beta}-lactamase expression is controlled by activation of an alternative {sigma} factor called {sigma}P. Here we show that {beta}-lactam antibiotics induce {sigma}P activation by degradation of the anti-{sigma} factor RsiP.