Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HK–RR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the α1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the broadly occurring HisKA family of sensor kinases in bacteria.

Summary In the intracellular pathogen Brucella abortus, the general stress response (GSR) signaling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signaling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK efficiently and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signaling pathway that regulates B. abortus stress physiology and infection biology.

The Alphaproteobacteria uniquely integrate features of two-component signal transduction and alternative σ factor regulation to control transcription of genes that ensure growth and survival across a range of stress conditions. Research over the past decade has led to the discovery of the key molecular players of this general stress response (GSR) system, including the sigma factor σEcfG, its anti-σ factor NepR, and the anti-anti-σ factor PhyR. The central molecular event of GSR activation entails aspartyl phosphorylation of PhyR, which promotes its binding to NepR and thereby releases σEcfG to associate with RNAP and direct transcription. Recent studies are providing a new understanding of complex, multilayered sensory networks that activate and repress this central protein partner switch. This review synthesizes our structural and functional understanding of the core GSR regulatory proteins and highlights emerging data that are defining the systems that regulate GSR transcription in a variety of species.

Summary Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight‐stranded β‐barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O‐polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O‐polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O‐polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.