Xanthomonas oryzae Pv. oryzicola Response Regulator VemR Is Co-opted by the Sensor Kinase CheA for Phosphorylation of Multiple Pathogenicity-Related Targets.

Two-component systems (TCSs) (cognate sensor histidine kinase/response regulator pair, HK/RR) play a crucial role in bacterial adaptation, survival, and productive colonization. An atypical orphan single-domain RR VemR was characterized by the non-vascular pathogen Xanthomonas oryzae pv. oryzicola (Xoc) is known to cause bacterial leaf streak (BLS) disease in rice. Xoc growth and pathogenicity in rice, motility, biosynthesis of extracellular polysaccharide (EPS), and the ability to trigger HR in non-host tobacco were severely compromised in the deletion mutant strain RΔvemR as compared to the wild-type strain RS105. Site-directed mutagenesis and phosphotransfer experiments revealed that the conserved aspartate (D56) residue within the stand-alone phosphoacceptor receiver (REC) domain is essential for phosphorelay and the regulatory activity of Xoc VemR. Yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) data identified CheA as the HK co-opting the RR VemR for phosphorylation. Affinity proteomics identified several downstream VemR-interacting proteins, such as 2-oxoglutarate dehydrogenase (OGDH), DNA-binding RR SirA, flagellar basal body P-ring formation protein FlgA, Type 4a pilus retraction ATPase PilT, stress-inducible sensor HK BaeS, septum site-determining protein MinD, cytoskeletal protein CcmA, and Type III and VI secretion system proteins HrpG and Hcp, respectively. Y2H and deletion mutant analyses corroborated that VemR interacted with OGDH, SirA, FlgA, and HrpG; thus, implicating multi-layered control of diverse cellular processes including carbon metabolism, motility, and pathogenicity in the rice. Physical interaction between VemR and HrpG suggested cross-talk interaction between CheA/VemR- and HpaS/HrpG-mediated signal transduction events orchestrating the hrp gene expression.

The Two-Component System DcuS-DcuR Is Involved in Virulence and Stress Tolerance in the Poplar Canker Bacterium Lonsdalea populi.

The gram-negative bacterium Lonsdalea populi causes an emerging poplar (Populus × euramericana) canker resulting in severe losses to poplar production in China and Europe. Two-component signal transduction systems play important roles in the regulation of virulence and stress responses in phytopathogenic bacteria. We identified a two-component pair (Lqp2625-Lqp2624) in L. populi, highly homologous to DcuS-DcuR of Escherichia coli. Mutants lacking DcuS or DcuR displayed normal growth while their virulence on poplar twigs was impaired. An inability to produce flagella indicated that DcuS and DcuR are involved in biofilm formation and swimming motility. Moreover, the loss of DcuS or DcuR led to increased sensitivity to oxidative stress and chloramphenicol through downregulation of genes associated with catalases and the multidrug efflux pump, suggesting that the two-component pair contributes to cellular adaptation to oxidative and antibiotic stresses. We identified key domains and putative phosphorylation sites important for virulence and stress responses. Our findings reveal the functions of DcuS-DcuR in virulence and stress responses in L. populi and provide increasing evidence that two-component systems are crucial during the infection process and stress adaptation in bacteria.

Analysis of HrpG regulons and HrpG-interacting proteins by ChIP-seq and affinity proteomics in Xanthomonas campestris.

Gamma-proteobacteria Xanthomonas spp. cause at least 350 different plant diseases among important agricultural crops, which result in serious yield losses. Xanthomonas spp. rely mainly on the type III secretion system (T3SS) to infect their hosts and induce a hypersensitive response in nonhosts. HrpG, the master regulator of the T3SS, plays the dominant role in bacterial virulence. In this study, we used chromatin immunoprecipitation followed by sequencing (ChIP-seq) and tandem affinity purification (TAP) to systematically characterize the HrpG regulon and HrpG interacting proteins in vivo. We obtained 186 candidate HrpG downstream genes from the ChIP-seq analysis, which represented the genomic-wide regulon spectrum. A consensus HrpG-binding motif was obtained and three T3SS genes, hpa2, hrcU, and hrpE, were confirmed to be directly transcriptionally activated by HrpG in the inducing medium. A total of 273 putative HrpG interacting proteins were identified from the TAP data and the DNA-binding histone-like HU protein of Xanthomonas campestris pv. campestris (HUxcc ) was proved to be involved in bacterial virulence by increasing the complexity and intelligence of the bacterial signalling pathways in the T3SS.

[MarR family transcription regulator HpaR and XC0449 coordinately regulate the virulence of Xanthomonas campestris pv. campestris].

MarR family transcription regulators are ubiquitous among bacteria and archaea. They extensively control multiple cellular processes and elaborately regulate the expression of genes involved in virulence, stress response and antibiotics at translational level. In Xanthomonas campestris pv. campestris, insertional inactivation of MarR family transcription regulator HpaR (XC2827) resulted in significantly decrease in virulence and increase in the production of the extracellular proteases. Here, we reported that the genome of Xcc 8004 encodes nine MarR family transcription regulators. The MarR family transcription regulators, HpaR (XC2827) and XC0449, were heterologous expressed and purified. In vitro MST and Pull-down assay confirmed the physical interaction between HpaR and XC0449. Phenotypical assay determined that deletion of XC0449 resulted in substantial virulence attenuation. In vitro EMSA, in vivo qRT-PCR and GUS activity assay identified that HpaR and XC0449 coordinately act as the transcriptional activator to regulate the expression of the virulence-associated gene XC0705, and eventually control the bacterial virulence and the production of extracellular proteases.

Proteolysis of histidine kinase VgrS inhibits its autophosphorylation and promotes osmostress resistance in Xanthomonas campestris.

In bacterial cells, histidine kinases (HKs) are receptors that monitor environmental and intracellular stimuli. HKs and their cognate response regulators constitute two-component signalling systems (TCSs) that modulate cellular homeostasis through reversible protein phosphorylation. Here the authors show that the plant pathogen Xanthomonas campestris pv. campestris responds to osmostress conditions by regulating the activity of a HK (VgrS) via irreversible, proteolytic modification. This regulation is mediated by a periplasmic, PDZ-domain-containing protease (Prc) that cleaves the N-terminal sensor region of VgrS. Cleavage of VgrS inhibits its autokinase activity and regulates the ability of the cognate response regulator (VgrR) to bind promoters of downstream genes, thus promoting bacterial adaptation to osmostress.

A Large-Scale Mutational Analysis of Two-Component Signaling Systems of Lonsdalea quercina Revealed that KdpD-KdpE Regulates Bacterial Virulence Against Host Poplar Trees.

Poplar, which is a dominant species in plant communities distributed in the northern hemisphere, is commonly used as a model plant in forestry studies. Poplar production can be inhibited by infections caused by bacteria, including Lonsdalea quercina subsp. populi, which is a gram-negative bacterium responsible for bark canker disease. However, the molecular basis of the pathogenesis remains uncharacterized. In this study, we annotated the two-component signal transduction systems (TCSs) encoded by the L. quercina subsp. populi N-5-1 genome and identified 18 putative histidine kinases and 24 response regulators. A large-scale mutational analysis revealed that 19 TCS genes regulated bacterial virulence against poplar trees. Additionally, the deletion of kdpE or overexpression of kdpD resulted in almost complete loss of bacterial virulence. We observed that kdpE and kdpD formed a bi-cistronic operon. KdpD exhibited autokinase activity and could bind to KdpE (Kd = 5.73 ± 0.64 µM). Furthermore, KdpE is an OmpR family response regulator. A chromatin immunoprecipitation sequencing analysis revealed that KdpE binds to an imperfect palindromic sequence within the promoters of 44 genes, including stress response genes Lqp0434, Lqp3037, and Lqp3270. A comprehensive analysis of TCS functions may help to characterize the regulation of poplar bark canker disease.

A three-component signalling system fine-tunes expression kinetics of HPPK responsible for folate synthesis by positive feedback loop during stress response of Xanthomonas campestris.

During adaptation to environments, bacteria employ two-component signal transduction systems, which contain histidine kinases and response regulators, to sense and respond to exogenous and cellular stimuli in an accurate spatio-temporal manner. Although the protein phosphorylation process between histidine kinase and response regulator has been well documented, the molecular mechanism fine-tuning phosphorylation levels of response regulators is comparatively less studied. Here we combined genetic and biochemical approaches to reveal that a hybrid histidine kinase, SreS, is involved in the SreK-SreR phosphotransfer process to control salt stress response in the bacterium Xanthomonas campestris. The N-terminal receiver domain of SreS acts as a phosphate sink by competing with the response regulator SreR to accept the phosphoryl group from the latter's cognate histidine kinase SreK. This regulatory process is critical for bacterial survival because the dephosphorylated SreR protein participates in activating one of the tandem promoters (P2) at the 5' end of the sreK-sreR-sreS-hppK operon, and then modulates a transcriptional surge of the stress-responsive gene hppK, which is required for folic acid synthesis. Therefore, our study dissects the biochemical process of a positive feedback loop in which a 'three-component' signalling system fine-tunes expression kinetics of downstream genes.

The periplasmic PDZ domain-containing protein Prc modulates full virulence, envelops stress responses, and directly interacts with dipeptidyl peptidase of Xanthomonas oryzae pv. oryzae.

PDZ domain-containing proteases, also known as HtrA family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell-envelope stress responses. These proteases have diverse functions through proteolysis- and nonproteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes revealed that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidence demonstrating that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell-envelope stress responses.