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

Abstract

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. σP is an ECF σ factor found in Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Previous work showed that σP is induced by ampicillin, a β-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of σP is controlled or what other antibiotics may activate σP. Here, we report that activation of σP is specific to a subset of β-lactams and that σP is required for resistance to these β-lactams. We demonstrate that activation of σP 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 σP activation. This proteolytic cascade leads to activation of σP, 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 σP. Here, we show that β-lactam antibiotics induce σP activation by degradation of the anti-σ factor RsiP.

The Penicillin-Binding Protein PbpP Is a Sensor of β-Lactams and Is Required for Activation of the Extracytoplasmic Function σ Factor σ P in Bacillus thuringiensis

“…In Bacillus anthracis , Bacillus cereus , and Bacillus thuringiensis , resistance to penicillin and other β-lactam antibiotics is dependent upon σ P , an ECF σ factor ( 33 , 34 ). σ P was originally classified as a member of the ECF01 group of ECF σ factors but was recently reclassified to the ECF265 group, the members of which are primarily found in Firmicutes ( 14 , 15 ).…”

“…The activation of σ P results in the expression of at least two genes that encode β-lactamases and are involved in resistance to penicillin, ampicillin, and other β-lactam antibiotics. σ P also activates the expression of its operon, thus controlling the expression of sigP and rsiP ( 33 , 34 ). We previously demonstrated that σ P is activated in the presence of a subset of β-lactams, ampicillin, methicillin, cefoxitin, cephalothin, and cefmetazole, but not other cell envelope stresses ( 34 ).…”

“…σ P also activates the expression of its operon, thus controlling the expression of sigP and rsiP ( 33 , 34 ). We previously demonstrated that σ P is activated in the presence of a subset of β-lactams, ampicillin, methicillin, cefoxitin, cephalothin, and cefmetazole, but not other cell envelope stresses ( 34 ). We also identified a subset of β-lactams that do not activate σ P : piperacillin, cefsulodin, and cefoperazone ( 34 ).…”

“…We previously demonstrated that σ P is activated in the presence of a subset of β-lactams, ampicillin, methicillin, cefoxitin, cephalothin, and cefmetazole, but not other cell envelope stresses ( 34 ). We also identified a subset of β-lactams that do not activate σ P : piperacillin, cefsulodin, and cefoperazone ( 34 ). In response to the activating β-lactams, RsiP is destroyed by a cascade of proteases, resulting in σ P activation ( 34 ).…”

“…We also identified a subset of β-lactams that do not activate σ P : piperacillin, cefsulodin, and cefoperazone ( 34 ). In response to the activating β-lactams, RsiP is destroyed by a cascade of proteases, resulting in σ P activation ( 34 ). An unidentified site 1 protease initiates the proteolytic cascade by cleaving RsiP at site 1, which is then followed by cleavage at site 2 by RasP, the highly conserved site 2 protease ( 34 ) ( Fig.…”