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The classical TCS system in bacterial signal transduction is composed of two proteins—a histidine kinase and its cognate response regulator. More and more studies have revealed the presence of accessory proteins that can modulate the histidine kinase activity and affect signal transduction, but their mechanisms remain largely elusive. This study unveils a previously unrecognized mechanism by which bacterial accessory lipoproteins mediate TCS activation. We provide compelling evidence that QseG directly interacts with QseE through an evolutionarily conserved structural interface, readily and sufficiently activating QseE’s autokinase activity and downstream signaling. Given the essential role of QseEF in bacterial virulence and stress adaptation, our findings pave the way for the development of antimicrobial strategies targeting this conserved lipoprotein-mediated activation mechanism.

There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two‐component systems from marine Shewanella species, and validate them in laboratory Escherichia coli . Then, we port these sensors into a gut‐adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways. Synopsis A sensor bacterium that uses a novel two‐component signaling system is engineered to detect thiosulfate and colon inflammation. This work suggests thiosulfate as a novel biomarker of colon inflammation and demonstrates the potential of engineered bacteria in disease diagnostics. Novel two‐component system sensors of thiosulfate and tetrathionate from marine Shewanella species are identified computationally. Both sensors are characterized in laboratory Escherichia coli and then ported to the gut‐adapted probiotic strain Nissle 1917. A flow cytometry protocol is developed for identifying the engineered bacteria in the colon contents or feces of mice with intact microbiota. The thiosulfate sensor has elevated output in inflamed mice, suggesting thiosulfate as a novel biomarker of inflammation. Graphical Abstract A sensor bacterium that uses a novel two‐component signaling system is engineered to detect thiosulfate and colon inflammation. This work suggests thiosulfate as a novel biomarker of colon inflammation and demonstrates the potential of engineered bacteria in disease diagnostics.

Bacteria constantly sense and respond to their surroundings through two-component systems. In Gram-negative bacteria, phosphate sensing is mediated by the PhoB/PhoR two-component system with additional components, the PstSCAB phosphate transporter and the PhoU protein. Bacteria utilize two-component regulatory systems to sense and respond to their surroundings. Unlike other two-component systems that directly sense through a sensory domain in the histidine kinase (HK), the PhoB/PhoR two-component system requires additional proteins, including the PstSCAB phosphate transporter and the PhoU protein, to sense phosphate levels. Although PhoU is involved in phosphate signaling by connecting the PstSCAB transporter and PhoR histidine kinase, the mechanism by which PhoU controls expression of pho regulon genes has not yet been clearly understood. Here, we identified PhoU residues required for interacting with PhoR histidine kinase from the intracellular pathogen Salmonella enterica serovar Typhimurium. The PhoU Ala147 residue interacts with the PhoR PAS domain and is involved in repressing pho expression in high phosphate. Unexpectedly, the PhoU Arg184 residue interacts with the PhoR histidine kinase domain and is required for activating pho expression in low Mg 2+ by increasing PhoR autophosphorylation, revealing its new function. The substitution of the Arg184 to Gly codon decreased Salmonella virulence both in macrophages and in mice, suggesting that PhoU’s role in promoting PhoR autophosphorylation is required during Salmonella infection. IMPORTANCE Bacteria constantly sense and respond to their surroundings through two-component systems. In Gram-negative bacteria, phosphate sensing is mediated by the PhoB/PhoR two-component system with additional components, the PstSCAB phosphate transporter and the PhoU protein. PhoU, a regulatory protein that connects the PstSCAB phosphate transporter to the PhoB/PhoR two-component system, is believed to function as a negative regulator in phosphate signaling because the phoU deletion mutant loses the ability to repress pho expression in high phosphate. Using single amino acid substitutions in the intracellular pathogen Salmonella enterica serovar Typhimurium, PhoU turns out to control PhoR histidine kinase differently, depending on the conditions. The PhoU-PhoR PAS domain interaction is involved in repressing pho expression in high phosphate, whereas the PhoU-PhoR HK domain interaction is involved in activating autophosphorylation of PhoR histidine kinase in low Mg 2+ and thus promotes Salmonella virulence. Therefore, PhoU appears to modulate phosphate signaling exquisitely according to external conditions.

Bacteria employ two‐component systems (TCSs) to rapidly sense and respond to their surroundings often and during plant infection. Poplar canker caused by Lonsdalea populi is an emerging woody bacterial disease that leads to high mortality and poplar plantation losses in China. Nonetheless, the information about the underlying mechanism of pathogenesis remains scarce. Therefore, in this study, we reported the role of a TCS pair CpxA/CpxR in regulating virulence and stress responses in L. populi. The CpxA/R system is essential during infection, flagellum formation, and oxidative stress response. Specifically, the Cpx system affected flagellum formation by controlling the expression of flagellum‐related genes. CpxR, which was activated by phosphorylation in the presence of CpxA, participated in the transcriptional regulation of a chaperone sctU and the type III secretion system (T3SS)‐related genes, thereby influencing T3SS functions during L. populi infection. Phosphorylated CpxR directly manipulated the transcription of a membrane protein‐coding gene yccA and the deletion of yccA resulted in reduced virulence and increased sensitivity to H2O2. Furthermore, we mutated the conserved phosphorylation site of CpxR and found that CpxRD51A could no longer bind to the yccA promoter but could still bind to the sctU promoter. Together, our findings elucidate the roles of the Cpx system in regulating virulence and reactive oxygen species resistance and provide further evidence that the TCS is crucial during infection and stress response. Lonsdalea populi CpxA/CpxR manipulates pathogenesis and stress response by influencing flagella formation and function, regulating the type III secretion system and modulating transcription of membrane protein‐coding gene yccA.

Plant pathogenic bacteria of the genus Xanthomonas cause a variety of diseases in economically important monocotyledonous and dicotyledonous crop plants worldwide. Successful infection and bacterial multiplication in the host tissue often depend on the virulence factors secreted including adhesins, polysaccharides, LPS and degradative enzymes. One of the key pathogenicity factors is the type III secretion system, which injects effector proteins into the host cell cytosol to manipulate plant cellular processes such as basal defense to the benefit of the pathogen. The coordinated expression of bacterial virulence factors is orchestrated by quorum-sensing pathways, multiple two-component systems and transcriptional regulators such as Clp, Zur, FhrR, HrpX and HpaR. Furthermore, virulence gene expression is post-transcriptionally controlled by the RNA-binding protein RsmA. In this review, we summarize the current knowledge on the infection strategies and regulatory networks controlling secreted virulence factors from Xanthomonas species.

Summary The critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.), led to the formation of pseudo‐nodules even in the absence of rhizobia. Hence, a genome‐wide analysis of the cytokinin signalling, two‐component system (TCS) genes, was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out, which revealed that histidine kinases (HKs) were highly conserved, whereas there was diversification leading to neofunctionalization at the level of response regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1Δ mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule‐deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13, was functionally characterized. Its localization in the nucleus and its Y1H assay‐based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signalling molecule regulating early events of nodule organogenesis in chickpea.

Vancomycin (VAN)‐intermediate Staphylococcus aureus (VISA) is a critical cause of VAN treatment failure worldwide. Multiple genetic changes are reportedly associated with VISA formation, whereas VISA strains often present common phenotypes, such as reduced autolysis and thickened cell wall. However, how mutated genes lead to VISA common phenotypes remains unclear. Here, we show a metabolism regulatory cascade (CcpA‐GlmS), whereby mutated two‐component systems (TCSs) link to the common phenotypes of VISA. We found that ccpA deletion decreased VAN resistance in VISA strains with diverse genetic backgrounds. Metabolic alteration in VISA was associated with ccpA upregulation, which was directly controlled by TCSs WalKR and GraSR. RNA‐sequencing revealed the crucial roles of CcpA in changing the carbon flow and nitrogen flux of VISA to promote VAN resistance. A gate enzyme (GlmS) that drives carbon flow to the cell wall precursor biosynthesis was upregulated in VISA. CcpA directly controlled glmS expression. Blocking CcpA sensitized VISA strains to VAN treatment in vitro and in vivo. Overall, this work uncovers a link between the formation of VISA phenotypes and commonly mutated genes. Inhibition of CcpA‐GlmS cascade is a promising strategy to restore the therapeutic efficiency of VAN against VISA infections. The metabolic alteration in vancomycin‐intermediate Staphylococcus aureus (VISA) is associated with ccpA upregulation. WalKR and GraSR TCSs control the expression of CcpA, which orchestrates GlmS to form a CcpA‐GlmS cascade to drive carbon and nitrogen flow for the biosynthesis of cell wall precursor in VISA. Inhibition of CcpA‐GlmS cascade is a promise for the therapeutic efficiency of vancomycin against VISA infections.

The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function σ factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.

VemR is a response regulator of the two‐component signalling systems (TCSs). It consists solely of a receiver domain. Previous studies have shown that VemR plays an important role in influencing the production of exopolysaccharides and exoenzymes, cell motility, and virulence of Xanthomonas campestris pv. campestris (Xcc). However, whether VemR is involved in the essential pathogenicity determinant type III secretion system (T3SS) is unclear. In this work, we found by transcriptome analysis that VemR modulates about 10% of Xcc genes, which are involved in various cellular processes including the T3SS. Further experiments revealed that VemR physically interacts with numerous proteins, including the TCS sensor kinases HpaS and RavA, and the TCS response regulator HrpG, which directly activates the transcription of HrpX, a key regulator controlling T3SS expression. It has been demonstrated previously that HpaS composes a TCS with HrpG or VemR to control the expression of T3SS or swimming motility, while RavA and VemR form a TCS to control the expression of flagellar genes. Mutation analysis and in vitro transcription assay revealed that phosphorylation might be essential for the function of VemR and phosphorylated VemR could significantly enhance the activation of hrpX transcription by HrpG. We infer that the binding of VemR to HrpG can modulate the activity of HrpG to the hrpX promoter, thereby enhancing hrpX transcription. Although further studies are required to validate this inference and explore the detailed functional mechanism of VemR, our findings provide some insights into the complex regulatory cascade of the HpaS/RavA‐VemR/HrpG‐HrpX signal transduction system in the control of T3SS. VemR, a single‐domain response regulator, modulates the expression of type III secretion system via binding to the key regulator HrpG in Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) can cause black rot in cruciferous plants worldwide. Two‐component systems (TCSs) are key for bacterial adaptation to various environments, including hosts. VemR is a TCS response regulator and crucial for Xcc motility and virulence. Here, we report that RavA is the cognate histidine kinase (HK) of VemR and elucidate the signalling pathway by which VemR regulates Xcc motility and virulence. Genetic analysis showed that VemR is epistatic to RavA. Using bacterial two‐hybrid experiments and pull‐down and phosphorylation assays, we found that RavA can interact with and phosphorylate VemR, suggesting that RavA is the cognate HK of VemR. In addition, we found that RpoN2 and FleQ are epistatic to VemR in regulating bacterial motility and virulence. In vivo and in vitro experiments demonstrated that VemR interacts with FleQ but not with RpoN2. RavA/VemR regulates the expression of the flagellin‐encoding gene fliC by activating the transcription of the rpoN2‐vemR‐fleQ and flhF‐fleN‐fliA operons. In summary, our data show that the RavA/VemR TCS regulates FleQ activity and thus influences the expression of motility‐related genes, thereby affecting Xcc motility and virulence. The identification of this novel signalling pathway will deepen our understanding of Xcc–plant interactions. RavA phosphorylates VemR and might affect the interaction of VemR with FleQ, which regulates Xanthomonas campestris pv. campestris motility and virulence via FliA and Clp.