Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

A Survey of Two-Component Systems in Coxiella burnetii Reveals Redundant Regulatory Schemes and a Requirement for an Atypical PhoBR System in Mammalian Cell Infection.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever in humans. C. burnetii transitions between a replicative, metabolically active large-cell variant (LCV) and a spore-like, quiescent small-cell variant (SCV) as a likely mechanism to ensure survival between host cells and mammalian hosts. C. burnetii encodes three canonical two-component systems, four orphan hybrid histidine kinases, five orphan response regulators, and a histidine phosphotransfer protein, which have been speculated to play roles in the signaling required for C. burnetii morphogenesis and virulence. However, very few of these systems have been characterized. By employing a CRISPR interference system for genetic manipulation of C. burnetii, we created single- and multigene transcriptional knockdown strains targeting most of these signaling genes. Through this, we revealed a role for the C. burnetii PhoBR canonical two-component system in virulence, regulation of [Pi] maintenance, and Pi transport. We also outline a novel mechanism by which PhoBR function may be regulated by an atypical PhoU-like protein. We also determined that the GacA.2/GacA.3/GacA.4/GacS orphan response regulators coordinately and disparately regulate expression of SCV-associated genes in C. burnetii LCVs. These foundational results will inform future studies on the role of C. burnetii two-component systems in virulence and morphogenesis. IMPORTANCE C. burnetii is an obligate intracellular bacterium with a spore-like stability allowing it to survive long periods of time in the environment. This stability is likely due to its biphasic developmental cycle, whereby it can transition from an environmentally stable small-cell variant (SCV) to a metabolically active large-cell variant (LCV). Here, we define the role of two-component phosphorelay systems (TCS) in C. burnetii's ability to survive within the harsh environment contained in the phagolysosome of host cells. We show that the canonical PhoBR TCS has an important role in C. burnetii virulence and phosphate sensing. Further examination of the regulons controlled by orphan regulators indicated a role in modulating gene expression of SCV-associated genes, including genes essential for cell wall remodeling.

Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes.

Metal resistance of Cupriavidus metallidurans is based on determinants that were acquired in the past by horizontal gene transfer during evolution. Some of these determinants encode transmembrane metal efflux systems. Expression of most of the respective genes is controlled by two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR). Here, we investigated the interplay between the three closely related two-component regulatory systems CzcRS, CzcR2S2, and AgrRS. All three systems regulate the response regulator CzcR, while the RRs AgrR and CzcR2 were not involved in czc regulation. Target promoters were czcNp and czcPp for genes upstream and downstream of the central czc gene region. The two systems together repressed CzcRS-dependent upregulation of czcP-lacZ at low zinc concentrations in the presence of CzcS but activated this signal transmission at higher zinc concentrations. AgrRS and CzcR2S2 interacted to quench CzcRS-mediated expression of czcNp-lacZ and czcPp-lacZ. Together, cross talk between the three two-component regulatory systems enhanced the capabilities of the Czc systems by controlling expression of the additional genes czcN and czcP. IMPORTANCE Bacteria are able to acquire genes encoding resistance to metals and antibiotics by horizontal gene transfer. To bestow an evolutionary advantage on their host cell, new genes must be expressed, and their expression should be regulated so that resistance-mediating proteins are produced only when needed. Newly acquired regulators may interfere with those already present in a host cell. Such an event was studied here in the metal-resistant bacterium Cupriavidus metallidurans. The results demonstrate how regulation by the acquired genes interacts with the host's extant regulatory network. This leads to emergence of a new system level of complexity that optimizes the response of the cell to periplasmic signals.

Hyperphosphorylation of the Group A Streptococcal Control of Virulence Regulator Increases Promoter Occupancy Specifically at Virulence Factor-Encoding Genes.

The control of virulence two-component gene regulatory system (CovRS) is critical to the pathogenesis of many medically important streptococci. In emm1 group A streptococci (GAS), CovR directly binds the promoters of numerous GAS virulence factor-encoding genes. Elimination of CovS phosphatase activity increases CovR phosphorylation (CovR~P) levels and abrogates GAS virulence. Given the emm type-specific diversity of CovRS function, in this study we used chromatin immunoprecipitation sequencing (ChIP-seq) to define global CovR DNA occupancy in the wild-type emm3 strain MGAS10870 (medium CovR~P) and its CovS phosphatase-negative derivative 10870-CovS-T284A (high CovR~P). In the wild-type emm3 strain, 89% of the previously identified emm1 CovR binding sites present in the emm3 genome were also enriched; additionally, we ascertained unique CovR binding, primarily to genes in mobile genetic elements and other sites of interstrain chromosomal differences. Elimination of CovS phosphatase activity specifically increased CovR occupancy at the promoters of a broad array of CovR repressed virulence factor-encoding genes, including those encoding the key GAS regulator Mga and M protein. However, a limited number of promoters had augmented enrichment at low CovR~P levels. Differential motif searches using sequences enriched at high versus low CovR~P levels revealed two distinct binding patterns. At high CovR~P, a pseudopalindromic AT-rich consensus sequence (WTWTTATAAWAAAAWNATDA) consistent with CovR binding as a dimer was determined. Conversely, sequences specifically enriched at low CovR~P contained isolated ATTARA motifs suggesting an interaction with a monomer. These data extend understanding of global CovR DNA occupancy beyond emm1 GAS and provide a mechanism for previous observations regarding hypovirulence induced by CovS phosphatase abrogation. IMPORTANCE Given its key role in pathogenesis of Gram-positive bacteria, CovR is one of the most important members of the OmpR/PhoB family of transcriptional regulators. Herein we extend recent GAS CovR global binding analyses done in emm1 to a non-emm1 strain, which is important considering the known inter-emm-type heterogeneity in GAS CovRS function. Our data provide mechanistic understanding for variation in CovRS function between emm types and the profound hypovirulence of CovS phosphatase-negative strains in addition to indicating differential targeting by phosphorylated and nonphosphorylated CovR isoforms at specific CovR binding sites. These findings advance knowledge regarding how a key bacterial virulence regulator impacts pathogenesis and add to the growing appreciation of the function of nonphosphorylated OmpR/PhoB family members.

The Extracellular Electron Transport Pathway Reduces Copper for Sensing by the CopRS Two-Component System under Anaerobic Conditions in Listeria monocytogenes.

The renowned antimicrobial activity of copper stems in part from its ability to undergo redox cycling between Cu1+/2+ oxidation states. Bacteria counter copper toxicity with a network of sensors that often include two-component signaling systems to direct transcriptional responses. As in typical two-component systems, ligand binding by the extracellular domain of the membrane bound copper sensor component leads to phosphorylation and activation of the cognate response regulator transcription factor. In Listeria monocytogenes, the plasmid-borne CopRS two-component system upregulates both copper resistance and lipoprotein remodeling genes upon copper challenge, but the oxidation state of copper bound by CopS is unknown. Herein, we show CopS utilizes a triad of key residues (His-His-Phe) that are predicted to be at the dimerization interface and that are analogous with the Escherichia coli CusS copper sensor to specifically bind Cu1+/Ag1+ and activate CopR transcription. We demonstrate Cu2+ only induces CopRS if first reduced by electron transport systems, as strains lacking menaquinone carriers were unable to respond to Cu2+. The flavin-dependent extracellular electron transport system (EET) was the main mechanism for metal reduction, capable of either generating inducing ligand (Cu2+ to Cu1+) or removing it by precipitation (Ag1+ to Ag0). We show that EET flux is directly proportional to the rate of Cu2+ reduction and that since EET activity is low under oxygenated conditions when a competing respiratory chain is operating, CopRS signaling in turn is activated only under anaerobic conditions. EET metal reduction thus sensitizes cells to copper while providing resistance to silver under anaerobic growth. IMPORTANCE Two-component extracellular copper sensing from the periplasm of Gram-negative bacteria has been well studied, but copper detection at the cell surface of the Gram-positive L. monocytogenes is less understood. Collectively, our results show that EET is most active under anaerobic conditions and reduces Cu2+ and Ag1+ to, respectively, generate or remove the monovalent ligands that directly bind to CopS and lead to the induction of lipoprotein remodeling genes. This reducing activity regulates CopRS signaling and links the upregulation of copper resistance genes with increasing EET flux. Our studies provide insight into how a two-component copper sensing system is integrated into a model monoderm Firmicute to take cues from the electron transport chain activity.

Identification of DraRS in Clostridioides difficile, a Two-Component Regulatory System That Responds to Lipid II-Interacting Antibiotics.

Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 220,000 infections, 12,000 deaths, and upwards of $1 billion in medical costs in the United States each year. C. difficile is highly resistant to a variety of antibiotics, but we have a poor understanding of how C. difficile senses and responds to antibiotic stress and how such sensory systems affect clinical outcomes. We have identified a spontaneous C. difficile mutant that displays increased daptomycin resistance. We performed whole-genome sequencing and found a nonsense mutation, S605*, in draS, which encodes a putative sensor histidine kinase of a two-component system (TCS). The draSS605* mutant has an ~4- to 8-fold increase in the daptomycin MIC compared to the wild type (WT). We found that the expression of constitutively active DraRD54E in the WT increases daptomycin resistance 8- to 16-fold and increases bacitracin resistance ~4-fold. We found that a selection of lipid II-inhibiting compounds leads to the increased activity of the luciferase-based reporter PdraR-slucopt, including vancomycin, bacitracin, ramoplanin, and daptomycin. Using RNA sequencing (RNA-seq), we identified the DraRS regulon. Interestingly, we found that DraRS can induce the expression of the previously identified hex locus required for the synthesis of a novel glycolipid produced in C. difficile. Our data suggest that the induction of the hex locus by DraR explains some, but not all, of the DraR-induced daptomycin and bacitracin resistance. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. C. difficile encodes ~50 annotated two-component systems (TCSs); however, only a few have been studied. The function of these unstudied TCSs is not known. Here, we show that the TCS DraRS plays a role in responding to a subset of lipid II-inhibiting antibiotics and mediates resistance to daptomycin and bacitracin in part by inducing the expression of the recently identified hex locus, which encodes enzymes required for the production of a novel glycolipid in C. difficile.

Regulation of Bacterial Two-Component Systems by Cardiolipin.

The regulation of membrane protein activity for cellular functions is critically dependent on the composition of phospholipid membranes. Cardiolipin, a unique phospholipid found in bacterial membranes and mitochondrial membranes of eukaryotes, plays a crucial role in stabilizing membrane proteins and maintaining their function. In the human pathogen Staphylococcus aureus, the SaeRS two-component system (TCS) controls the expression of key virulence factors essential for the bacterium's virulence. The SaeS sensor kinase activates the SaeR response regulator via phosphoryl transfer to bind its gene target promoters. In this study, we report that cardiolipin is critical for sustaining the full activity of SaeRS and other TCSs in S. aureus. The sensor kinase protein SaeS binds directly to cardiolipin and phosphatidylglycerol, enabling SaeS activity. Elimination of cardiolipin from the membrane reduces SaeS kinase activity, indicating that bacterial cardiolipin is necessary for modulating the kinase activities of SaeS and other sensor kinases during infection. Moreover, the deletion of cardiolipin synthase genes cls1 and cls2 leads to reduced cytotoxicity to human neutrophils and lower virulence in a mouse model of infection. These findings suggest a model where cardiolipin modulates the kinase activity of SaeS and other sensor kinases after infection to adapt to the hostile environment of the host and expand our knowledge of how phospholipids contribute to membrane protein function.

The Two-Component System YesMN Promotes Pneumococcal Host-to-Host Transmission and Regulates Genes Involved in Zinc Homeostasis.

The ability to sense and respond rapidly to the dynamic environment of the upper respiratory tract (URT) makes Streptococcus pneumoniae (Spn) a highly successful human pathogen. Two-component systems (TCSs) of Spn sense and respond to multiple signals it encounters allowing Spn to adapt and thrive in various host sites. Spn TCS have been implicated in their ability to promote pneumococcal colonization of the URT and virulence. As the disease state can be a dead-end for a pathogen, we considered whether TCS would contribute to pneumococcal transmission. Herein, we determined the role of YesMN, an understudied TCS of Spn, and observe that YesMN contributes toward pneumococcal shedding and transmission but is not essential for colonization. The YesMN regulon includes genes involved in zinc homeostasis and glycan metabolism, which are upregulated during reduced zinc availability in a YesMN-dependent fashion. Thus, we identified the YesMN regulon and a potential molecular signal it senses that lead to the activation of genes involved in zinc homeostasis and glycan metabolism. Furthermore, in contrast to Spn monoinfection, we demonstrate that YesMN is critical for high pneumococcal density in the URT during influenza A virus (IAV) coinfection. We attribute reduced colonization of the yesMN mutant possibly due to increased association with and clearance by the mucus covering the URT epithelial surface. Thus, our results highlight the dynamic interactions that occur between Spn and IAV in the URT, and the role that TCSs play in modulation of these interactions.

Omptin Proteases of Enterobacterales Show Conserved Regulation by the PhoPQ Two-Component System but Exhibit Divergent Protection from Antimicrobial Host Peptides and Complement.

Bacteria that colonize eukaryotic surfaces interact with numerous antimicrobial host-produced molecules, including host defense peptides, complement, and antibodies. Bacteria have evolved numerous strategies to both detect and resist these molecules, and in the Enterobacterales order of bacteria these include alterations of the cell surface lipopolysaccharide structure and/or charge and the production of proteases that can degrade these antimicrobial molecules. Here, we show that omptin family proteases from Escherichia coli and Citrobacter rodentium are regulated by the PhoPQ system. Omptin protease activity is induced by growth in low Mg2+, and deletion of PhoP dramatically reduces omptin protease activity, transcriptional regulation, and protein levels. We identify conserved PhoP-binding sites in the promoters of the E. coli omptin genes ompT, ompP, and arlC as well as in croP of Citrobacter rodentium and show that mutation of the putative PhoP-binding site in the ompT promoter abrogates PhoP-dependent expression. Finally, we show that although regulation by PhoPQ is conserved, each of the omptin proteins has differential activity toward host defense peptides, complement components, and resistance to human serum, suggesting that each omptin confers unique survival advantages against specific host antimicrobial factors.

The VxrAB two-component system is important for the polymyxin B-dependent activation of the type VI secretion system in Vibrio cholerae O1 strain A1552.

The type VI secretion system (T6SS) is used by bacteria for virulence, resistance to grazing, and competition with other bacteria. We previously demonstrated that the role of the T6SS in interbacterial competition and in resistance to grazing is enhanced in Vibrio cholerae in the presence of subinhibitory concentrations of polymyxin B. Here, we performed a global quantitative proteomic analysis and a targeted transcriptomic analysis of the T6SS-known regulators in V. cholerae grown with and without polymyxin B. The proteome of V. cholerae is greatly modified by polymyxin B with more than 39% of the identified cellular proteins displaying a difference in their abundance, including T6SS-related proteins. We identified a regulator whose abundance and expression are increased in the presence of polymyxin B, vxrB, the response regulator of the two-component system VxrAB (VCA0565-66). In vxrAB, vxrA and vxrB deficient mutants, the expression of both hcp copies (VC1415 and VCA0017), although globally reduced, was not modified by polymyxin B. These hcp genes encode an identical protein Hcp, which is the major component of the T6SS syringe. Thus, the upregulation of the T6SS in the presence of polymyxin B appears to be, at least in part, due to the two-component system VxrAB.

Response Regulator CD1688 Is a Negative Modulator of Sporulation in Clostridioides difficile.

Two-component signal transduction systems (TCSs), consisting of a sensor histidine kinase (HK) and a response regulator (RR), sense environmental stimuli and then modulate cellular responses, typically through changes in gene expression. Our previous work identified the DNA binding motif of CD1586, an RR implicated in Clostridioides difficile strain R20291 sporulation. To determine the role of this RR in the sporulation pathway in C. difficile, we generated a deletion strain of cd1688 in the historical 630 strain, the homolog of cd1586. The C. difficile Δcd1688 strain exhibited a hypersporulation phenotype, suggesting that CD1688 negatively regulates sporulation. Complementation of the C. difficile Δcd1688 strain restored sporulation. In contrast, a nonphosphorylatable copy of cd1688 did not restore sporulation to wild-type (WT) levels, indicating that CD1688 must be phosphorylated to properly modulate sporulation. Expression of the master regulator spo0A, the sporulation-specific sigma factors sigF, sigE, sigG, and sigK, and a signaling protein encoded by spoIIR was increased in the C. difficile Δcd1688 strain compared to WT. In line with the increased spoIIR expression, we detected an increase in mature SigE at an earlier time point, which arises from SpoIIR-mediated processing of pro-SigE. Taken together, our data suggest that CD1688 is a novel negative modulator of sporulation in C. difficile and contributes to mediating progression through the spore developmental pathway. These results add to our growing understanding of the complex regulatory events involved in C. difficile sporulation, insight that could be exploited for novel therapeutic development. IMPORTANCE Clostridioides difficile causes severe gastrointestinal illness and is a leading cause of nosocomial infections in the United States. This pathogen produces metabolically dormant spores that are the major vehicle of transmission between hosts. The sporulation pathway involves an intricate regulatory network that controls a succession of morphological changes necessary to produce spores. The environmental signals inducing the sporulation pathway are not well understood in C. difficile. This work identified a response regulator, CD1688, that, when deleted, led to a hypersporulation phenotype, indicating that it typically acts to repress sporulation. Improving our understanding of the regulatory mechanisms modulating sporulation in C. difficile could provide novel strategies to eliminate or reduce spore production, thus decreasing transmission and disease relapse.

DnaJ and ClpX Are Required for HitRS and HssRS Two-Component System Signaling in Bacillus anthracis.

Bacillus anthracis is the causative agent of anthrax. This Gram-positive bacterium poses a substantial risk to human health due to high mortality rates and the potential for malicious use as a bioterror weapon. To survive within the vertebrate host, B. anthracis relies on two-component system (TCS) signaling to sense host-induced stresses and respond to alterations in the environment through changes in target gene expression. HitRS and HssRS are cross-regulating TCSs in B. anthracis that respond to cell envelope disruptions and high heme levels, respectively. In this study, an unbiased and targeted genetic selection was designed to identify gene products that are involved in HitRS and HssRS signaling. This selection led to the identification of inactivating mutations within dnaJ and clpX that disrupt HitRS- and HssRS-dependent gene expression. DnaJ and ClpX are the substrate-binding subunits of the DnaJK protein chaperone and ClpXP protease, respectively. DnaJ regulates the levels of HitR and HitS to facilitate signal transduction, while ClpX specifically regulates HitS levels. Together, these results reveal that the protein homeostasis regulators, DnaJ and ClpX, function to maintain B. anthracis signal transduction activities through TCS regulation.

Therapeutic Inhibition of Staphylococcus aureus ArlRS Two-Component Regulatory System Blocks Virulence.

Staphylococcus aureus is a common cause of severe infections, and its widespread antibiotic resistance necessitates search for alternative therapies, such as inhibition of virulence. As S. aureus produces multiple individual virulence factors, inhibition of an entire regulatory system might provide better effects than targeting each virulence factor separately. Herein, we describe two novel inhibitors of S. aureus two-component regulatory system ArlRS: 3,4'-dimethoxyflavone and homopterocarpin. Unlike other putative ArlRS inhibitors previously identified, these two compounds were effective and specific. In vitro kinase assays indicated that 3,4'-dimethoxyflavone directly inhibits ArlS autophosphorylation, while homopterocarpin did not exhibit such effect, suggesting that two inhibitors work through distinct mechanisms. Application of the inhibitors to methicillin-resistant S. aureus (MRSA) in vitro blocked ArlRS signaling, inducing an abnormal gene expression pattern that was reflected in changes at the protein level, enhanced sensitivity to oxacillin, and led to the loss of numerous cellular virulence traits, including the ability to clump, adhere to host ligands, and evade innate immunity. The pleiotropic antivirulence effect of inhibiting a single regulatory system resulted in a marked therapeutic potential, demonstrated by the ability of inhibitors to decrease severity of MRSA infection in mice. Altogether, this study demonstrated the feasibility of ArlRS inhibition as anti-S. aureus treatment, and identified new lead compounds for therapeutic development.

The ChvG-ChvI and NtrY-NtrX Two-Component Systems Coordinately Regulate Growth of Caulobacter crescentus.

Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases directly phosphorylate their cognate response regulator to affect gene expression. Bacteria typically express multiple TCSs that are insulated from one another and regulate distinct physiological processes. There are examples of cross-regulation between TCSs, but this phenomenon remains relatively unexplored. We have identified regulatory links between the ChvG-ChvI (ChvGI) and NtrY-NtrX (NtrYX) TCSs, which control important and often overlapping processes in alphaproteobacteria, including maintenance of the cell envelope. Deletion of chvG and chvI in Caulobacter crescentus limited growth in defined medium, and a selection for genetic suppressors of this growth phenotype uncovered interactions among chvGI, ntrYX, and ntrZ, which encodes a previously uncharacterized periplasmic protein. Significant overlap in the experimentally defined ChvI and NtrX transcriptional regulons provided support for the observed genetic connections between ntrYX and chvGI. Moreover, we present evidence that the growth defect of strains lacking chvGI is influenced by the phosphorylation state of NtrX and, to some extent, by levels of the TonB-dependent receptor ChvT. Measurements of NtrX phosphorylation in vivo indicated that NtrZ is an upstream regulator of NtrY and that NtrY primarily functions as an NtrX phosphatase. We propose a model in which NtrZ functions in the periplasm to inhibit NtrY phosphatase activity; regulation of phosphorylated NtrX levels by NtrZ and NtrY provides a mechanism to modulate and balance expression of the NtrX and ChvI regulons under different growth conditions. IMPORTANCE TCSs enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell envelope function in many alphaproteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. In addition, we identified NtrZ as a periplasmic inhibitor of NtrY phosphatase activity in vivo. Our work establishes C. crescentus as an excellent model to investigate multilevel regulatory connections between ChvGI and NtrYX in alphaproteobacteria.

Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments.

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes. In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M protein, consistent with the transcriptional regulation of emm, which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

Delineation of the pH-Responsive Regulon Controlled by the Helicobacter pylori ArsRS Two-Component System.

Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site-specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ΔarsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins (including sabA, alpA, alpB, hopD [labA], and horA). When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS-mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.

The Sensor Kinase QseC Regulates the Unlinked PmrA Response Regulator and Downstream Gene Expression in Francisella.

The facultative intracellular bacterial pathogen Francisella tularensis is the causative agent of tularemia in humans and animals. Gram-negative bacteria utilize two-component regulatory systems (TCS) to sense and respond to their changing environment. No classical, tandemly arranged sensor kinase and response regulator TCS genes exist in the human virulent Francisella tularensis subsp. tularensis, but orphaned members are present. PmrA is an orphan response regulator responsible for intramacrophage growth and virulence; however, the regulation of PmrA activity is not understood. We and others have shown that PmrA represses the expression of priM, described to encode an antivirulence determinant. By screening a mutant library for increased priM promoter activity, we identified the sensor kinase homolog QseC as an upstream regulator of priM expression, and this regulation is in part dependent upon the aspartate phosphorylation site of PmrA (D51). Several examined environmental signals, including epinephrine, which is reported to activate QseC in other bacteria, do not affect priM expression in a manner dependent on PmrA. Intramacrophage survival assays also question the finding that PriM is an antivirulence factor. Thus, these data suggest that the PmrA-regulated gene priM is modulated by the QseC-PmrA (QseB) TCS in FrancisellaIMPORTANCE The disease tularemia is caused by the highly infectious Gram-negative pathogen Francisella tularensis This bacterium encodes few regulatory factors (e.g., two-component systems [TCS]). PmrA, required for intramacrophage survival and virulence in the mouse model, is encoded by an orphan TCS response regulator gene. It is unclear how PmrA is responsive to environmental signals to regulate loci, including the PmrA-repressed gene priM We identify an orphan sensor kinase (QseC) that is required for priM repression and further explore both environmental signals that might regulate the QseC-PmrA TCS and the function of PriM.

The Hybrid Histidine Kinase HrmK Is an Early-Acting Factor in the Hormogonium Gene Regulatory Network.

Filamentous, heterocyst-forming cyanobacteria belonging to taxonomic subsections IV and V are developmentally complex multicellular organisms capable of differentiating an array of cell and filament types, including motile hormogonia. Hormogonia exhibit gliding motility that facilitates dispersal, phototaxis, and the establishment of nitrogen-fixing symbioses. The gene regulatory network (GRN) governing hormogonium development involves a hierarchical sigma factor cascade, but the factors governing the activation of this cascade are currently undefined. Here, using a forward genetic approach, we identified hrmK, a gene encoding a putative hybrid histidine kinase that functions upstream of the sigma factor cascade. The deletion of hrmK produced nonmotile filaments that failed to display hormogonium morphology or accumulate hormogonium-specific proteins or polysaccharide. Targeted transcriptional analyses using reverse transcription-quantitative PCR (RT-qPCR) demonstrated that hormogonium-specific genes both within and outside the sigma factor cascade are drastically downregulated in the absence of hrmK and that hrmK may be subject to indirect, positive autoregulation via sigJ and sigC Orthologs of HrmK are ubiquitous among, and exclusive to, heterocyst-forming cyanobacteria. Collectively, these results indicate that hrmK functions upstream of the sigma factor cascade to initiate hormogonium development, likely by modulating the phosphorylation state of an unknown protein that may serve as the master regulator of hormogonium development in heterocyst-forming cyanobacteria.IMPORTANCE Filamentous cyanobacteria are morphologically complex, with several representative species amenable to routine genetic manipulation, making them excellent model organisms for the study of development. Furthermore, two of the developmental alternatives, nitrogen-fixing heterocysts and motile hormogonia, are essential to establish nitrogen-fixing symbioses with plant partners. These symbioses are integral to global nitrogen cycles and could be artificially recreated with crop plants to serve as biofertilizers, but to achieve this goal, detailed understanding and manipulation of the hormogonium and heterocyst gene regulatory networks may be necessary. Here, using the model organism Nostoc punctiforme, we identify a previously uncharacterized hybrid histidine kinase that is confined to heterocyst-forming cyanobacteria as the earliest known participant in hormogonium development.

Regulation of Virulence by Two-Component Systems in Pathogenic Burkholderia.

The regulation and timely expression of bacterial genes during infection is critical for a pathogen to cause an infection. Bacteria have multiple mechanisms to regulate gene expression in response to their environment, one of which is two-component systems (TCS). TCS have two components. One component is a sensory histidine kinase (HK) that autophosphorylates when activated by a signal. The activated sensory histidine kinase then transfers the phosphoryl group to the second component, the response regulator, which activates transcription of target genes. The genus Burkholderia contains members that cause human disease and are often extensively resistant to many antibiotics. The Burkholderia cepacia complex (BCC) can cause severe lung infections in patients with cystic fibrosis (CF) or chronic granulomatous disease (CGD). BCC members have also recently been associated with several outbreaks of bacteremia from contaminated pharmaceutical products. Separate from the BCC is Burkholderia pseudomallei, which is the causative agent of melioidosis, a serious disease that occurs in the tropics, and a potential bioterrorism weapon. Bioinformatic analysis of sequenced Burkholderia isolates predicts that most strains have at least 40 TCS. The vast majority of these TCS are uncharacterized both in terms of the signals that activate them and the genes that are regulated by them. This review will highlight TCS that have been described to play a role in virulence in either the BCC or B. pseudomallei Since many of these TCS are involved in virulence, TCS are potential novel therapeutic targets, and elucidating their function is critical for understanding Burkholderia pathogenesis.