SUMMARY: Two-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases.

Summary: 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) σ factors – the largest group of alternative σ 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‐σ factors, they represent a highly modular design that primarily facilitates transmembrane signal transduction. A comprehensive analysis of the ECF σ factor protein family identified more than 40 distinct major groups of ECF σ factors. The functional relevance of this classification is supported by the sequence similarity and domain architecture of cognate anti‐σ 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 σ factor‐dependent gene regulation.

Summary: 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 σ factors (σ^B and σ^M) 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 σ^X and σ^M, 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.

Soil bacteria are among the most prodigious producers of antibiotics. The Bacillus subtilis LiaRS (formerly YvqCE) two-component system is one of several antibiotic-sensing systems that coordinate the genetic response to cell wall-active antibiotics. Upon the addition of vancomycin or bacitracin, LiaRS autoregulates the liaIHGFSR operon. We have characterized the promoter of the lia operon and defined the cis -acting sequences necessary for antibiotic-inducible gene expression. A survey for compounds that act as inducers of the lia promoter revealed that it responds strongly to a subset of cell wall-active antibiotics that interfere with the lipid II cycle in the cytoplasmic membrane (bacitracin, nisin, ramoplanin, and vancomycin). Chemicals that perturb the cytoplasmic membrane, such as organic solvents, are also weak inducers. Thus, the reporter derived from P _liaI (the liaI promoter) provides a tool for the detection and classification of antimicrobial compounds.

Background: Standardized and well-characterized genetic building blocks are a prerequisite for the convenient and reproducible assembly of novel genetic modules and devices. While numerous standardized parts exist for Escherichia coli, such tools are still missing for the Gram-positive model organism Bacillus subtilis. The goal of this study was to develop and thoroughly evaluate such a genetic toolbox. Results: We developed five BioBrick-compatible integrative B. subtilis vectors by deleting unnecessary parts and removing forbidden restriction sites to allow cloning in BioBrick (RFC10) standard. Three empty backbone vectors with compatible resistance markers and integration sites were generated, allowing the stable chromosomal integration and combination of up to three different devices in one strain. In addition, two integrative reporter vectors, based on the lacZ and luxABCDE cassettes, were BioBrick-adjusted, to enable β-galactosidase and luciferase reporter assays, respectively. Four constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of more than three orders of magnitude in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. Finally, the Bacillus BioBrick Box also provides five widely used epitope tags (FLAG, His₁₀, cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice. Conclusion: Our genetic toolbox contains three compatible empty integration vectors, two reporter vectors and a set of six promoters, two of them inducible. Furthermore, five different epitope tags offer convenient protein handling and detection. All parts adhere to the BioBrick standard and hence enable standardized work with B. subtilis. We believe that our well-documented and carefully evaluated Bacillus BioBrick Box represents a very useful genetic tool kit, not only for the iGEM competition but any other BioBrick-based project in B. subtilis.