Comprehensive and Comparative Transcriptional Profiling of the Cell Wall Stress Response in Bacillus subtilis

The bacterial cell wall is an essential protective barrier, the frontline of cellular interactions with the environment and also a target for numerous antimicrobial agents. Accordingly, its integrity and homeostasis are closely monitored, and rapid adaptive responses by transcriptional reprogramming induce appropriate counter-measures against perturbations. Here, we report a comprehensive and comparative transcriptional profiling of the cell envelope stress response (CESR) in the Gram-positive model bacterium Bacillus subtilis, by integrating RNAseq and high-resolution tiling array data. We exposed growing cells to a range of antimicrobial compounds that interfere with cytoplasmic, membrane-coupled or extracellular steps of peptidoglycan (PG) biosynthesis. Transcriptomic studies revealed the complexity of the resulting cell wall stress responses of B. subtilis and unraveled the contribution of extracytoplasmic function (ECF) sigma factors and two-component signal transduction systems (TCSs). While membrane-anchored steps are tightly controlled, early cytoplasmic and late extracellular steps of PG biosynthesis are hardly monitored at all. The ECF σ factors σ^W and σ^M both respond to several cell wall synthesis inhibitors and protect the cytoplasmic membrane and cell wall, respectively, while σ^V is almost exclusively induced by lysozyme, against which it provides specific resistance. Remarkably, σ^X was slightly repressed by most antibiotics, pointing towards a role in envelope homeostasis rather than cell wall stress. It shares this role with the essential TCS WalRK, which balances cell wall growth with controlled autolysis. In contrast, all remaining TCSs are envelope stress-inducible systems. LiaRS is induced by a wide range of PG synthesis inhibitors, while the three paralogous systems BceAB, PsdRS, and ApeRS are more compound-specific detoxification modules. Induction of the CssRS TCS by all antibiotics interfering with membrane-anchored steps of PG biosynthesis points toward a physiological link between CESR and secretion stress. Based on the expression signatures, a series of CESR-specific B. subtilis whole-cell biosensors were developed and carefully evaluated. Overall, this comprehensive transcriptomic study establishes a reference framework for future studies of Gram-positive stress responses triggered by cell wall-targeting antibiotics.