The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function sigma factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.

The regulatory network of the cell envelope stress response in Bacillus subtilis involves both extracytoplasmic function -factors and two-component signal transducing systems. One such system, LiaRS, responds to cell wall antibiotics that interfere with the undecaprenol cycle and to perturbation of the cytoplasmic membrane. It is encoded by the last two genes of the liaIHGFSR locus. Here, we analyzed the expression of two LiaRdependent operons, liaIHGFSR and yhcYZ-yhdA, and characterized a palindromic sequence required for LiaR-dependent activation. Since induction of the strong liaI promoter leads to both liaIH and liaIHGFRS transcripts, LiaR is positively autoregulated. Systematic deletion analysis of the liaI operon revealed that LiaF is a potent negative regulator of LiaR-dependent gene expression: a nonpolar liaF deletion led to constitutive activation of both characterized LiaR-dependent promoters. The liaF gene is conserved in both sequence and genomic context in the Firmicutes group of gram-positive bacteria, located directly upstream of liaSR orthologs. LiaH, a homolog of Escherichia coli phage shock protein A, also plays a more subtle role in negatively modulating the bacitracin-inducible expression from LiaR-dependent promoters. Our results support a model in which the LiaFRS module integrates both positive and negative feedback loops to transduce cell envelope stress signals.

The Lia system, a cell envelope stress response module of Bacillus subtilis, is comprised of the LiaRS two-component system and a membrane-anchored inhibitor protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all orthologs investigated so far are activated by cell wall antibiotics. In response to envelope stress, the systems in Firmicutes cocci induce the expression of a number of genes that are involved in conferring resistance against its inducers. In contrast, a complete picture of the LiaR regulon of B. subtilis is still missing and no phenotypes could be associated with mutants lacking LiaRS. Here, we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a comprehensive picture of the Lia response of Bacillus subtilis. In addition to the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene affected by LiaR-dependent regulation. The results of detailed follow-up gene expression studies, together with proteomic analysis, demonstrate that the liaIH operon represents the only relevant LiaR target locus in vivo. It encodes a small membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms large oligomeric rings reminiscent of those described for Escherichia coli PspA or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies demonstrated that the gene products of the liaIH operon are involved in protecting the cell against oxidative stress and some cell wall antibiotics. Our data suggest that the LiaFSR system of B. subtilis and, presumably, other Firmicutes bacilli coordinates a phage shock protein-like response.

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