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The endospore of Bacillus subtilis is formed intracellularly upon nutrient starvation and is encased by proteinaceous shells. The outermost layer, the crust, is a postulated glycoprotein layer that is composed of six proteins: CotV, W, X, Y, Z and CgeA. Despite some insight into protein interactions and the identification of players in glycosylation, a clear picture of its architecture is still missing. Here, we report a comprehensive mutational analysis that confirms CotZ as the anchor of the crust, while the crust structure is provided by CotV, CotX and CotY. CotY seems to be the major structural component, while CotV and CotX are polar and co-depend on each other and partially on CotW. CotW is independent of other crust proteins, instead depending on outer coat proteins, indicating a role at the interface of crust and coat. CgeA is co-expressed with putative glycosyltransferases (CgeB and CgeD) and implicated in crust glycosylation. In accordance, we provide evidence that CgeB, CgeCDE, SpsA-L, SpsM and SpsNOPQR (formerly YfnHGFED) contribute to the glycosylation state of the spore. The crust polysaccharide layer consists of functionally linked rhamnose- and galactose-related variants and could contain rare sugars. It may therefore protect the crust against biological degradation and scavenging.

The σ subunit of the bacterial RNA polymerase determines promoter specificity. The extracytoplasmic function σ factors (ECFs) represent the most abundant and diverse group of alternative σ factors and are present in the vast majority of bacterial genomes. Typically, ECFs are regulated by anti-σ factors that sequester their cognate ECFs, thereby preventing their interaction with the RNA polymerase. Beyond these ECF paradigms, a number of distinct modes of regulation have been proposed and experimentally investigated. Regulatory extensions represent one such alternative mechanism of ECF regulation that can be found in 18 phylogenetically distinct ECF groups. Here, the σ factors contain additional domains that are fused to the ECF core domains and are involved in stimulus perception and modulation of σ factor activity. We will summarize the current state of knowledge on regulating ECF activity by C-terminal extensions. We will also discuss newly identified ECF groups containing either N- or C-terminal extensions and propose possible mechanisms by which these extensions have been generated and affect ECF σ factor activity. Based on their modular architecture and the resulting physical connection between stimulus perception and transcriptional output, these ECFs are analogous to one-component systems, the primary mechanism of bacterial signal transduction.

Summary The extracytoplasmic function (ECF) σ factor, σE, is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation‐sequencing (ChIP‐seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. Seventeen novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE‐binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin‐binding proteins, two L,D‐transpeptidases, a LytR‐CpsA‐Psr‐family protein predicted to be involved in cell wall teichoic acid deposition and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE‐mediated cell envelope stress response in the genus Streptomyces.

Summary When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two-component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino-terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two-component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non-stress conditions. The resulting EsrISR three-component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI-esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane-associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322-3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI-esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress-responsive system whose components are highly conserved in the phylum Actinobacteria. This article is protected by copyright. All rights reserved.

Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well-characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle-inhibiting AMP bacitracin. The underlying regulatory network orchestrates the production of the ABC transporter BceAB, the UPP phosphatase BcrC and the phage-shock proteins LiaIH. Our systems-level analysis reveals a clear hierarchy, allowing us to discriminate between primary (BceAB) and secondary (BcrC and LiaIH) layers of bacitracin resistance. Deleting the primary layer provokes an enhanced induction of the secondary layer to partially compensate for this loss. This study reveals a direct role of LiaIH in bacitracin resistance, provides novel insights into the feedback regulation of the Lia system, and demonstrates a pivotal role of BcrC in maintaining cell wall homeostasis. The compensatory regulation within the bacitracin network can also explain how gene expression noise propagates between resistance layers. We suggest that this active redundancy in the bacitracin resistance network of B. subtilis is a general principle to be found in many bacterial antibiotic resistance networks.

When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two-component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino-terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two-component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non-stress conditions. The resulting EsrISR three-component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI-esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane-associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322-3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI-esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress-responsive system whose components are highly conserved in the phylum Actinobacteria.

The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage.

The response regulator/histidine kinase pair LiaRS of Bacillus subtilis, together with its membrane-bound inhibitor protein LiaF, constitutes an envelope stress-sensing module that is conserved in Firmicutes bacteria. LiaR positively autoregulates the expression of the liaIH-liaGFSR operon from a strictly LiaR-dependent promoter (P(liaI) ). A comprehensive perturbation analysis revealed that the functionality of the LiaFSR system is very susceptible to alterations of its protein composition and amounts. A genetic analysis indicates a LiaF:LiaS:LiaR ratio of 18:4:1. An excess of LiaS over LiaR was subsequently verified by quantitative Western analysis. This stoichiometry, which is crucial to maintain a functional Lia system, differs from any other two-component system studied to date, in which the response regulator is present in excess over the histidine kinase. Moreover, we demonstrate that LiaS is a bifunctional histidine kinase that acts as a phosphatase on LiaR in the absence of a suitable stimulus. An increased amount of LiaR - both in the presence and in the absence of LiaS - leads to a strong induction of P(liaI) activity due to phosphorylation of the response regulator by acetyl phosphate. Our data demonstrate that LiaRS, in contrast to other two-component systems, is non-robust with regard to perturbations of its stoichiometry.

BceRS and PsdRS are paralogous two-component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, P

Investigations into the resistance mechanisms of Firmicutes bacteria against antimicrobial peptides have revealed unique resistance modules comprised of an unusual type of ATP-binding cassette (ABC) transporter, paired with a two-component regulatory system. In these systems, the ABC-transporter is not only involved in detoxification of the peptides, but also in their detection and resulting regulation of gene expression. The manuscript by Hiron et al. (2011) published in this issue describes an intriguing complexity of regulatory circuits and division of labour between the three paralogous modules in Staphylococcus aureus, providing important mechanistic insights and new perspectives for future investigations of these unique systems.

Summary In addition to stress-specific responses, most bacteria can mount a general stress response (GSR), which protects the cells against a wide range of unspecific stress conditions. The best-understood examples of GSR are the σB-cascade of Bacillus subtilis and the RpoS response in Escherichia coli. While the latter is conserved in many other proteobacteria of the β-, γ- and δ-clades, RpoS homologues are absent in α-proteobacteria and their GSR has long been a mystery. Recent publications finally unraveled the core of the GSR in this proteobacterial class, which is mediated by EcfG-like σ-factors. EcfG activity is controlled by NepR-like anti-σ factors and PhyR-like proteins that act as anti-anti-σ factors. These unusual hybrid proteins contain an N-terminal EcfG-like domain that acts as a docking interface for NepR, and a C-terminal receiver domain typical for bacterial response regulators. Upon phosphorylation, PhyR titrates NepR away from EcfG, thereby releasing the σ-factor to recruit RNA polymerase and initiate transcription of its target genes. In this issue of Molecular Microbiology, Herrou et al. describe the function and three-dimensional structure of PhyR from Caulobacter crescentus. This structure is key to understanding the mechanism of the reversible, phosphorylation-dependent partner switching module that orchestrates the GSR in α-proteobacteria.

The ability of a bacterial cell to monitor and adaptively respond to its environment is crucial for survival. After one- and two-component systems, extracytoplasmic function (ECF) sigma factors - the largest group of alternative sigma factors - represent the third fundamental mechanism of bacterial signal transduction, with about six such regulators on average per bacterial genome. Together with their cognate anti-sigma factors, they represent a highly modular design that primarily facilitates transmembrane signal transduction. A comprehensive analysis of the ECF sigma factor protein family identified more than 40 distinct major groups of ECF sigma factors. The functional relevance of this classification is supported by the sequence similarity and domain architecture of cognate anti-sigma factors, genomic context conservation, and potential target promoter motifs. Moreover, this phylogenetic analysis revealed unique features indicating novel mechanisms of ECF-mediated signal transduction. This classification, together with the web tool ECFfinder and the information stored in the Microbial Signal Transduction (MiST) database, provides a comprehensive resource for the analysis of ECF sigma factor-dependent gene regulation.

The extracellular presence of antibiotics is a common threat in microbial life. Their sensitive detection and subsequent induction of appropriate resistance mechanisms is therefore a prerequisite for survival. The bacitracin stress response network of Bacillus subtilis consists of four signal-transducing systems, the two-component systems (TCS) BceRS, YvcPQ and LiaRS, and the extracytoplasmic function (ECF) sigma factor sigma(M). Here, we investigated the mechanism of bacitracin perception and the response hierarchy within this network. The BceRS-BceAB TCS/ABC transporter module is the most sensitive and efficient bacitracin resistance determinant. The ABC transporter BceAB not only acts as a bacitracin detoxification pump, but is also crucial for bacitracin sensing, indicative of a novel mechanism of stimulus perception, conserved in Firmicutes bacteria. The Bce system seems to respond to bacitracin directly (drug sensing), whereas the LiaRS TCS and sigma(M) respond only at higher concentrations and indirectly to bacitracin action (damage sensing). The YvcPQ-YvcRS system is subject to cross-activation via the paralogous Bce system, and is therefore only indirectly induced by bacitracin. The bacitracin stress response network is optimized to respond to antibiotic gradients in a way that maximizes the gain and minimizes the costs of this stress response.

In response to sublethal concentrations of antibiotics, bacteria often induce an adaptive response that can contribute to antibiotic resistance. We report the response of Bacillus subtilis to bacitracin, an inhibitor of cell wall biosynthesis found in its natural environment. Analysis of the global transcriptional profile of bacitracin-treated cells reveals a response orchestrated by two alternative sigma factors (sigmaB and sigmaM) and three two-component systems (YvqEC, YvcPQ and BceRS). All three two-component systems are located next to target genes that are strongly induced by bacitracin, and the corresponding histidine kinases share an unusual topology: they lack about 100 amino acids in their extracellular sensing domain, which is almost entirely buried in the cytoplasmic membrane. Sequence analysis indicates that this novel N-terminal sensing domain is a characteristic feature of a subfamily of histidine kinases, found almost entirely in Gram-positive bacteria and frequently linked to ABC transporters. A systematic mutational analysis of bacitracin-induced genes led to the identification of a new bacitracin-resistance determinant, bceAB, encoding a putative ABC transporter. The bcrC bacitracin resistance gene, which is under the dual control of sigmaX and sigmaM, was also induced by bacitracin. By comparing the bacitracin and the vancomycin stimulons, we can differentiate between loci induced specifically by bacitracin and those that are induced by multiple cell wall-active antibiotics.

Summary Bacterial cell wall homeostasis is an intricately coordinated process that ensures that envelope integrity is maintained during cell growth and division, but can also adequately respond to growth-limiting conditions such as phosphate starvation. In Bacillus subtilis, biosynthesis of the two major cell wall components, peptidoglycan and anionic polymers, is controlled by a pair of paralogous two-component systems, WalRK and PhoPR respectively. Favorable growth conditions allow for a fast rate of cell wall biosynthesis (WalRK-ON) and the incorporation of the phosphate-containing anionic polymer teichoic acids (PhoPR-OFF). In contrast, growth-restricted cells under phosphate-limiting conditions reduce the incorporation of peptidoglycan building blocks (WalRK-OFF) and switch from the phosphate-containing teichoic acids to the phosphate-free anionic polymer teichuronic acid (PhoPR-ON). Botella et al. (2014) deepen our knowledge on the PhoPR system by identifying one signal that is perceived by its histidine kinase PhoR. In fast-growing cells, intracellular intermediates of teichoic acid biosynthesis are sensed by the cytoplasmic Per-Arnt-Sim domain as an indicator of favorable conditions, thereby inhibiting the autokinase activity of PhoR and keeping the system inactive. Depletion of teichoic acid building blocks under phosphate-limiting conditions relieves this inhibition, activates PhoPR-dependent signal transduction and hence the switch to teichuronic acid biosynthesis.

The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage.

The response regulator/histidine kinase pair LiaRS of Bacillus subtilis, together with its membrane-bound inhibitor protein LiaF, constitutes an envelope stress-sensing module that is conserved in Firmicutes bacteria. LiaR positively autoregulates the expression of the liaIH-liaGFSR operon from a strictly LiaR-dependent promoter (P(liaI) ). A comprehensive perturbation analysis revealed that the functionality of the LiaFSR system is very susceptible to alterations of its protein composition and amounts. A genetic analysis indicates a LiaF:LiaS:LiaR ratio of 18:4:1. An excess of LiaS over LiaR was subsequently verified by quantitative Western analysis. This stoichiometry, which is crucial to maintain a functional Lia system, differs from any other two-component system studied to date, in which the response regulator is present in excess over the histidine kinase. Moreover, we demonstrate that LiaS is a bifunctional histidine kinase that acts as a phosphatase on LiaR in the absence of a suitable stimulus. An increased amount of LiaR - both in the presence and in the absence of LiaS - leads to a strong induction of P(liaI) activity due to phosphorylation of the response regulator by acetyl phosphate. Our data demonstrate that LiaRS, in contrast to other two-component systems, is non-robust with regard to perturbations of its stoichiometry.

Investigations into the resistance mechanisms of Firmicutes bacteria against antimicrobial peptides have revealed unique resistance modules comprised of an unusual type of ATP-binding cassette (ABC) transporter, paired with a two-component regulatory system. In these systems, the ABC-transporter is not only involved in detoxification of the peptides, but also in their detection and resulting regulation of gene expression. The manuscript by Hiron et al. (2011) published in this issue describes an intriguing complexity of regulatory circuits and division of labour between the three paralogous modules in Staphylococcus aureus, providing important mechanistic insights and new perspectives for future investigations of these unique systems.

In addition to stress-specific responses, most bacteria can mount a general stress response (GSR), which protects the cells against a wide range of unspecific stress conditions. The best-understood examples of GSR are the σ(B)-cascade of Bacillus subtilis and the RpoS response in Escherichia coli. While the latter is conserved in many other proteobacteria of the ß-, γ- and δ-clades, RpoS homologues are absent in α-proteobacteria and their GSR has long been a mystery. Recent publications finally unraveled the core of the GSR in this proteobacterial class, which is mediated by EcfG-like σ-factors. EcfG activity is controlled by NepR-like anti-σ factors and PhyR-like proteins that act as anti-anti-σ factors. These unusual hybrid proteins contain an N-terminal EcfG-like domain that acts as a docking interface for NepR, and a C-terminal receiver domain typical for bacterial response regulators. Upon phosphorylation, PhyR titrates NepR away from EcfG, thereby releasing the σ-factor to recruit RNA polymerase and initiate transcription of its target genes. In this issue of Molecular Microbiology, Herrou et al. describe the function and three-dimensional structure of PhyR from Caulobacter crescentus. This structure is key to understanding the mechanism of the reversible, phosphorylation-dependent partner switching module that orchestrates the GSR in α-proteobacteria.