Linda J. Kenney

Co‐regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ

Analysis of the transcriptome of slyA mutant Salmonella enterica serovar Typhimurium revealed that many SlyA‐dependent genes, including pagC, pagD, ugtL, mig‐14, virK, phoN, pgtE, pipB2, sopD2, pagJ and pagK, are also controlled by the PhoP/PhoQ regulatory system. Many SlyA‐ and PhoP/PhoQ‐co‐regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicrobial peptides. Purified His‐tagged SlyA binds to the pagC and mig‐14 promoters in regions homologous to a previously proposed ‘SlyA‐box’. The pagC promoter lacks a consensus PhoP binding site and does not bind PhoP in vitro, suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low‐magnesium PhoP‐inducing conditions in which pagC expression is profoundly elevated, however, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti‐repressor to allow PhoP activation. The absence of almost all SlyA‐activated genes from the Escherichia coli K12 genome suggests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmonella evolved its distinctive pathogenic lifestyle.

A divalent switch drives H-NS/DNA-binding conformations between stiffening and bridging modes

Heat-stable nucleoid structuring protein (H-NS) is an abundant prokaryotic protein that plays important roles in organizing chromosomal DNA and gene silencing. Two controversial binding modes were identified. H-NS binding stimulating DNA bridging has become the accepted mechanism, whereas H-NS binding causing DNA stiffening has been largely ignored. Here, we report that both modes exist, and that changes in divalent cations drive a switch between them. The stiffening form is present under physiological conditions, and directly responds to pH and temperature in vitro. Our findings have broad implications and require a reinterpretation of the mechanism by which H-NS regulates genes.

Dual regulation by phospho-OmpR of ssrA/B gene expression in Salmonella pathogenicity island 2

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 the spiC–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.

Structure/function relationships in OmpR and other winged-helix transcription factors.

Response regulators are the output component of two-component regulatory systems, the predominant form of signal transduction systems utilized by prokaryotes. The majority of response regulators function as transcription factors, yet detailed descriptions of their mechanisms of DNA binding and its consequences are lacking. Versatility in the modes of DNA binding is evident with winged helix-turn-helix proteins, raising doubts that mechanisms of DNA binding will be generalizable among members of the family. The current focus of some of the research efforts aimed at understanding activation and DNA binding by response regulators is highlighted in this review.

The Escherichia coli CpxA-CpxR Envelope Stress Response System Regulates Expression of the Porins OmpF and OmpC

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.

The response regulator SsrB activates expression of diverse Salmonella pathogenicity island 2 promoters and counters silencing by the nucleoid-associated protein H-NS.

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.

The response regulator SsrB activates transcription and binds to a region overlapping OmpR binding sites at Salmonella pathogenicity island 2

OmpR activates expression of the two‐component regulatory system located on Salmonella pathogenicity 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 of ssrA and vice versa. This result was suggested from our previous studies, in which two promoters at ssrA/B were identified. The isolated C‐terminus of SsrB binds to DNA and protects regions upstream of ssrA, ssrB and srfH from DNase I digestion. Furthermore, the C‐terminus of SsrB alone is capable of activating transcription in the absence of the N‐terminus. Results from β‐galactosidase assays indicate that the N‐terminal phosphorylation domain inhibits the C‐terminal effector domain. A previous study from our laboratory reported that ssrA–lacZ and ssrB–lacZ transcriptional fusions were substantially reduced in an ssrB null strain. Results from DNase I protection assays provide direct evidence that SsrB binds at ssrA and ssrB, 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 to Salmonella virulence, also affects ssrA/B gene expression.

The inner membrane histidine kinase EnvZ senses osmolality via helix‐coil transitions in the cytoplasm

Two‐component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZc). Increasing osmolality decreased deuterium exchange localized to the four‐helix bundle containing the autophosphorylation site (His243). EnvZc exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His243, suggesting that these two events are linked. In‐vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H‐bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli.

MzrA: a novel modulator of the EnvZ/OmpR two‐component regulon

Analysis of suppressors that alleviate the acute envelope stress phenotype of a ΔbamBΔdegP 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 EnvZs 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.

Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR

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.