PMID:22211522

Citation	Luo, Y and Helmann, JD (2012) Analysis of the role of Bacillus subtilis σ(M) in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis. Mol. Microbiol. 83:623-39
Abstract	The Bacillus subtilis extracytoplasmic function (ECF) σ factor σ(M) is inducible by, and confers resistance to, several cell envelope-acting antibiotics. Here, we demonstrate that σ(M) is responsible for intrinsic β-lactam resistance, with σ(X) playing a secondary role. Activation of σ(M) upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta-lactam cefuroxime. However, σ(M) still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of σ(M) in β-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, σ(M) is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to β-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 σ factors in counteracting the deleterious effects of autolysins and reactive oxygen species in β-lactam-treated cells.
Links	PubMed PMC3306796 Online version:10.1111/j.1365-2958.2011.07953.x
Keywords	3',5'-Cyclic-AMP Phosphodiesterases/metabolism; Bacillus subtilis/drug effects; Bacillus subtilis/genetics; Bacillus subtilis/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Cefuroxime/pharmacology; Cell Wall/metabolism; Cyclic AMP/metabolism; Gene Expression Regulation, Bacterial; Homeostasis; Mutagenesis, Insertional; Peptidoglycan/metabolism; Sigma Factor/genetics; Sigma Factor/metabolism; beta-Lactam Resistance/genetics
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Significance

Overall, this is a solid work. Several questions/comments arised during journal club:

1)Is Tn7 the standard for locking out genes in Bacillus sbtilies?

2)It is difficult to trust the zone inhibition assay with out the MIC data. For Fig 2, most the data are coorelated except the last 3 MIC number and last 3 bar graph.Why?

3)Fig 3 points out that sigM is important without giving more explaination

4)Figure 4 should include MIC measurement since zone inhibition assay is untrustable. Accoding to this figure, there are basial level of reaction with oxygen for WT and abh. Why is that?

5)How difficult would it be to measure c-di-AMP?

Kenneth To

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