Activation of the Extracytoplasmic Function σ factor σ^Pby β-lactams inBacillus thuringiensisrequires the site-2 protease RasP

Abstract

30Bacteria can utilize alternative σ factors to regulate sets of genes in response to 31 changes in the environment. The largest and most diverse group of alternative σ factors are the 32 Extracytoplasmic Function (ECF) σ factors. σ P is an ECF σ factor found in Bacillus anthracis, B. 33 cereus, and B. thuringiensis. Previous work showed σ P is induced by ampicillin, a β-lactam 34 antibiotic, and required for resistance to ampicillin. However, it was not known how activation of 35 σ P is controlled or what other antibiotics may activate σ P . Here we report that activation of σ P is 36 specific to a subset of β-lactams and σ P is required for resistance to these β-lactams. We 37 demonstrate that activation of σ P is controlled by the proteolytic destruction of the anti-σ factor, 38RsiP, and that degradation of RsiP requires multiple proteases. Upon exposure to β-lactams, 39 the extracellular domain of RsiP is cleaved by an unknown protease, which we predict cleaves 40 at site-1. Following cleavage by the unknown protease, the N-terminus of RsiP is further 41 degraded by the site-2 intramembrane protease, RasP. Our data indicate that RasP cleavage of 42RsiP is not the rate-limiting step in σ P activation. This proteolytic cascade leads to activation of 43 σ P which induces resistance to β-lactams likely via increased expression of β-lactamases. 44 45 Importance 46