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Bacteria respond to physical forces perceived as mechanical stress as part of their comprehensive environmental sensing strategy. Histidine kinases can then funnel diverse environmental stimuli into changes in gene expression through a series of phosphorelay reactions. Because histidine kinases are most often embedded in the inner membrane, they can be sensitive to changes in membrane tension that occurs, for example, in response to osmotic stress, or when deformation of the cell body occurs upon encountering a surface before forming biofilms, or inside the host in response to shear stress in the kidney, intestine, lungs, or blood stream. A summary of our recent work that links the histidine kinase EnvZ to mechanical changes in the inner membrane is provided and placed in a context of other bacterial systems that respond to mechanical stress. [ABSTRACT FROM AUTHOR]

After uptake by epithelial cells or engulfment by macrophages, Salmonella resides in an acidic vacuole. Salmonella senses this acidic compartment through the action of the EnvZ/OmpR two-component regulatory system. OmpR, in turn, represses the cadC/BA system, preventing neutralization of the bacterial cytoplasm. New, single cell techniques now enable us to observe that in response to acid stress, the pH is low in bacterial cells and acidification is critical for infection. Instead of recovering from acid stress, Salmonella uses acid pH as a signal to drive pathogenesis. The relevant molecular mechanisms employed by Salmonella to couple acid stress with the expression of virulence genes that promote intracellular survival are explored. [ABSTRACT FROM AUTHOR]

Analysis of suppressors that alleviate the acute envelope stress phenotype of a Δ bamBΔ degP strain of Escherichia coli identified a novel protein MzrA and pleiotropic envZ mutations. Genetic evidence shows that overexpression of MzrA – formerly known as YqjB and EcfM – modulates the activity of EnvZ/OmpR similarly to pleiotropic EnvZ mutants and alter porin expression. However, porin expression in strains devoid of MzrA or overexpressing it is still sensitive to medium osmolarity, pH and procaine, all of which modulate EnvZ/OmpR activities. Thus, MzrA appears to alter the output of the EnvZ/OmpR system but not its ability to receive and respond to various environmental signals. Localization and topology experiments indicate that MzrA is a type II membrane protein, with its N-terminus exposed in the cytoplasm and C-terminus in the periplasm. Bacterial two-hybrid experiments determined that MzrA specifically interacts with EnvZ but not with OmpR or the related membrane sensor kinase, CpxA. This and additional genetic and biochemical evidence suggest that the interaction of MzrA with EnvZ would either enhance EnvZ's kinase activity or reduce its phosphatase activity, thus elevating the steady state levels of OmpR∼P. Furthermore, our data show that MzrA links the two-component envelope stress response regulators, CpxA/CpxR and EnvZ/OmpR. [ABSTRACT FROM AUTHOR]

All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation. [ABSTRACT FROM AUTHOR]

The two-component system SsrA–SsrB activates expression of a type III secretion system required for replication in macrophages and systemic infection in mice. Here we characterize the SsrB-dependent regulation of genes within Salmonella pathogenicity island 2 (SPI-2). Primer extension and DNase I footprinting identified multiple SsrB-regulated promoters within SPI-2 located upstream of ssaB, sseA, ssaG and ssaM. We previously demonstrated that ssrA and ssrB transcription is uncoupled. Overexpression of SsrB in the absence of its cognate kinase, SsrA, is sufficient to activate SPI-2 transcription. Because SsrB requires phosphorylation to relieve inhibitory contacts that occlude its DNA-binding domain, additional components must phosphorylate SsrB. SPI-2 promoters examined in single copy were highly SsrB-dependent, activated during growth in macrophages and induced by acidic pH. The nucleoid structuring protein H-NS represses horizontally acquired genes; we confirmed that H-NS is a negative regulator of SPI-2 gene expression. In the absence of H-NS, the requirement for SsrB in activating SPI-2 genes is substantially reduced, suggesting a role for SsrB in countering H-NS silencing. SsrB activates transcription of multiple operons within SPI-2 by binding to degenerate DNA targets at diversely organized promoters. SsrB appears to possess dual activities to promote SPI-2 gene expression: activation of transcription and relief of H-NS-mediated repression. [ABSTRACT FROM AUTHOR]

OmpR activates expression of the two-component regulatory system located onSalmonellapathogenicity island 2 (SPI-2) that controls the expression of a type III secretion system, as well as many other genes required for systemic infection in mice. Measurements of SsrA and SsrB protein levels under different growth conditions indicate that expression of these two components is uncoupled, i.e. SsrB is produced in the absence ofssrAand vice versa. This result was suggested from our previous studies, in which two promoters atssrA/Bwere identified. The isolated C-terminus of SsrB binds to DNA and protects regions upstream ofssrA,ssrBandsrfHfrom DNase I digestion. Furthermore, the C-terminus of SsrB alone is capable of activating transcription in the absence of the N-terminus. Results fromβ-galactosidase assays indicate that the N-terminal phosphorylation domain inhibits the C-terminal effector domain. A previous study from our laboratory reported thatssrA–lacZandssrB–lacZtranscriptional fusions were substantially reduced in anssrBnull strain. Results from DNase I protection assays provide direct evidence that SsrB binds atssrAandssrB, although the binding sites lie within the transcribed regions. Additional regulators clearly affect gene expression at this important locus, and here we provide evidence that SlyA, a transcription factor that contributes toSalmonellavirulence, also affectsssrA/Bgene expression. [ABSTRACT FROM AUTHOR]

Expression of genes located on Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection in mice. This region encodes a type III secretion system, secreted effectors and the two-component regulatory system SsrA/B (also referred to as SpiR), as well as additional uncharacterized genes. In the present work, we demonstrate that phospho-OmpR (OmpR-P) functions as an activator at thespiC-ssrA/ B locus. There are two promoters at spiR; one is upstream of ssrA and the other upstream of ssrB. Our results indicate that, in contrast to many two-component regulatory systems, regulation of the sensor kinase SsrA appears to be uncoupled and distinct from regulation of the response regulator SsrB. OmpR regulation of ssrA/B is one of only a few examples known in which a two-component response regulator directly regulates the expression of another two-component regulatory system. [ABSTRACT FROM AUTHOR]

We performed transposon mutagenesis of a two-color fluorescent reporter strain to identify new regulators of the porin genes ompF and ompC in Escherichia coli. Screening of colonies by fluorescence microscopy revealed numerous mutants that exhibited interesting patterns of porin expression. One mutant harbored an insertion in the gene encoding the histidine kinase CpxA, the sensor for a two-component signaling system that responds to envelope stress. The cpxA mutant exhibited increased transcription of ompC and a very strong decrease in transcription of ompF under conditions in which acetyl phosphate levels were high. Subsequent genetic analysis revealed that this phenotype is dependent on phosphorylation of the response regulator CpxR and that activation of CpxA in wild-type cells results in similar regulation of porin expression. Using DNase I footprinting, we demonstrated that CpxR binds upstream of both the ompF and ompC promoters. It thus appears that two distinct two-component systems, CpxA-CpxR and EnvZ-OmpR, converge at the porin promoters. Within the context of envelope stress, outer membrane beta-barrel proteins have generally been associated with the sigma E pathway. However, at least for the classical porins OmpF and OmpC, our results show that the Cpx envelope stress response system plays a role in regulating their expression. [ABSTRACT FROM AUTHOR]

The response regulator SsrB activates expression of genes encoded within and outside of a pathogenicity island (SPI-2), which is required for systemic infection of Salmonella. SsrB binds upstream of the sifA, siJB, and sseJ effector genes and directly regulates transcription. SsrB also relieves gene silencing by the nucleoid protein H-NS. Single molecule experiments with magnetic tweezers demonstrated that SsrB displaces H-NS from DNA only when it is bound in a polymerization (stiffening) mode and not when H-NS is bound to DNA in the bridging mode. Thus, in contrast to previous views, the polymerization binding mode of H-NS is the relevant form for counter-silencing by SsrB. Our results reveal that response regulators can directly activate transcription and also relieve H-NS silencing. This study adds to the repertoire of mechanisms by which NarL/FixJ subfamily members regulate transcription. Because SsrB-dependent promoters are diversely organized, additional mechanisms of transcriptional activation at other loci are likely. [ABSTRACT FROM AUTHOR]

In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB (SsrBc) by NMR and analyzed the effect of amino acid substitutions on function. We identified residues in the DNA recognition helix (Lys179, Met186) and the dimerization interface (Val197, Leu201) that are important for SsrB transcriptional activation and DNA binding. An essential cysteine residue in the N-terminal receiver domain was also identified (Cys45), and the effect of Cys203 on dimerization was evaluated. Our results suggest that although disulfide bond formation is not required for dimerization, dimerization occurs upon DNA binding and is required for subsequent activation of transcription. Disruption of the dimer interface by a C203E substitution reduces SsrB activity. Modification of Cys203 or Cys45 may be an important mode of SsrB inactivation inside the host. [ABSTRACT FROM AUTHOR]