Analysis of the role of Bacillus subtilis σ(M) in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis.

The Bacillus subtilis extracytoplasmic function (ECF) s factor sM is inducible by, and confers resistance to, several cell envelope-acting antibiotics. Here, we demonstrate that sM is responsible for intrinsic beta-lactam resistance, with sX playing a secondary role. Activation of sM upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta-lactam cefuroxime. However, sM still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of sM in beta-lactam resistance, we characterized suppressor mutations that restore cefuroxime resistance to a sigM null mutant. The most frequent suppressors inactivated gdpP (yybT) which encodes a cyclic-di-AMP phosphodiesterase (PDE). Intriguingly, sM is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to beta-lactam antibiotics. Conversely, genetic studies indicate that at least one DAC is required for growth with depletion leading to cell lysis. These findings support a model in which c-di-AMP is an essential signal molecule required for cell wall homeostasis. Other suppressors highlight the roles of ECF s factors in counteracting the deleterious effects of autolysins and reactive oxygen species in beta-lactam-treated cells.