System Response Regulator Research Articles

The ArlR-MgrA regulatory cascade regulates PIA-dependent and protein-mediated biofilm formation in Rbf-dependent and Rbf-independent pathways

The two-component system response regulator ArlR and the global regulator MgrA in Staphylococcus aureus participated in numerous biological processes including biofilm formation inhibition. Previous studies have shown that these two regulators could function as a regulatory cascade. Rbf is a positive regulator of biofilm formation enhancing the production of PIA (polysaccharide intercellular adhesin). Here we have demonstrated that both ArlR and MgrA can directly bind to the promoter of rbf and repress its expression. ArlR and MgrA can also directly bind to the promoter of ica operon and enhance the expression of icaA and PIA production, revealing that the ArlR-MgrA regulatory cascade controls PIA-dependent biofilm formation. In addition, we have found that Rbf can directly bind to the aur promoter and repress the expression of aur, which encodes a protease initiating a protease cascade to inhibit protein-mediated biofilm formation. Moreover, our data indicate that the ArlR-MgrA regulatory cascade can promote the expression of aur by directly binding to its promoter and inhibit protein-mediated biofilm formation. These findings shed light on the molecular mechanisms of both PIA-dependent and protein-mediated biofilm formation modulated by the ArlR-MgrA regulatory cascade and the new role of Rbf in protein-mediated biofilm formation, and broaden our understanding of the biofilm formation regulation in S. aureus.

Phosphotyrosine-Mediated Regulation of Enterohemorrhagic Escherichia coli Virulence.

ABSTRACTEnteric pathogens with low infectious doses rely on the ability to orchestrate the expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of ler, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential.

Diversity of regulatory strategies for two-component system response regulators in bacteria

Two-component systems (TCS), composing of a histidine kinase (HK) and a cognate response regulator (RR), are the most important signal transduction devices employed by bacteria to sense and response to a variety of environmental cues. Upon receiving a physical or chemical signal, the sensor HK is activated and then phosphorylates its cognate RR. As the terminal of TCS signal transduction system, RR typically consists of a highly-conserved N-terminal receiver domain (REC) and avariable C-terminal effector domain (ED). In the typical TCS, phosporylated RRs with the enhanced DNA-binding ability, could directly regulate transcriptional levels of the target genes in the form of homodimers. However, the regulatory strategies of RRs are actually more complex and diverse. First, phosphorylation of RRs sometimes induces their detachment from the target DNA, and different RRs also could form heterodimers. Then, except for the DNA-binding domain, the effector module also could be the RNA-binding, protein-binding or catalytic domain. Finally, some RRs only consist of single receiver domains. In this review, we present the recent advances in the different regulatory strategies of RRs, which expands our knowledge on the complexity and flexibility of response action for TCS signal transduction system.

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

GlnR, an OmpR-like orphan two-component system response regulator, is a master regulator of nitrogen metabolism in the genus Streptomyces In this work, evidence that GlnR is also directly involved in the regulation of antibiotic biosynthesis is provided. In the model strain Streptomyces coelicolor M145, an in-frame deletion of glnR resulted in markedly increased actinorhodin (ACT) production but reduced undecylprodigiosin (RED) biosynthesis when exposed to R2YE culture medium. Transcriptional analysis coupled with DNA binding studies revealed that GlnR represses ACT but activates RED production directly via the pathway-specific activator genes actII-ORF4 and redZ, respectively. The precise GlnR-binding sites upstream of these two target genes were defined. In addition, the direct involvement of GlnR in antibiotic biosynthesis was further identified in Streptomyces avermitilis, which produces the important anthelmintic agent avermectin. We found that S. avermitilis GlnR (GlnRsav) could stimulate avermectin but repress oligomycin production directly through the respective pathway-specific activator genes, aveR and olmRI/RII To the best of our knowledge, this report describes the first experimental evidence demonstrating that GlnR regulates antibiotic biosynthesis directly through pathway-specific regulators in Streptomyces Our results suggest that GlnR-mediated regulation of antibiotic biosynthesis is likely to be universal in streptomycetes. These findings also indicate that GlnR is not only a master nitrogen regulator but also an important controller of secondary metabolism, which may help to balance nitrogen metabolism and antibiotic biosynthesis in streptomycetes.

Construction of a Streptococcus agalactiae phoB mutant and evaluation of its potential as an attenuated modified live vaccine in golden pompano, Trachinotus ovatus

Streptococcus agalactiae is a Gram-positive pathogen that can survive inside professional phagocytes and nonphagocytic cells to cause septicemia and meningoencephalitis in freshwater and marine fish. However, vaccines based on extracellular products (ECP) and formalin-killed whole S. agalactiae cells, as well as subunit vaccine are unable to protect fish from infection by variant serotypes S. agalactiae. The search for live attenuated vaccine with highly conserved and virulent-related genes is essential for producing a vaccine to help understand and control streptococcosis In this study, the phoB gene was cloned from pathogenic S. agalactiae TOS01 strain and the mutant strain SAΔphoB was constructed via allelic exchange mutagenesis. The results showed that the deduced amino acid of S. agalactiae TOS01 shares high similarities with other Streptococcus spp. and has high conserved response regulator receiver domain (REC) and DNA-binding effector domain of two-component system response regulators (Trans_reg_C). Cell adherence and invasion assays, challenge experiments and histopathological changes post-vaccination were performed and observed, the results showed that the mutant strain SAΔphoB has a lower adherence and invasion rate and less virulent than the wild type strain in golden pompano, and it doesn't induce clinical symptoms and obvious pathological changes in golden pompano, thereby indicating that the deletion of phoB affects the virulence and infectious capacity of S. agalactiae. Golden pompano vaccinated via intraperitoneal injection SAΔphoB had the relative percent survival value of 93.1% after challenge with TOS01, demonstrating its high potential as an effective attenuated live vaccine candidate. Real-time PCR assays showed that the SAΔphoB was able to enhance the expression of immune-related genes, including MHC-I, MyD88, IL-22 and IL-10 after vaccination, indicating that the SAΔphoB is able to induce humoral and cell-mediated immune response in golden pompano over a long period of time.

Proteome-wide alterations in an industrial clavulanic acid producing strain of Streptomyces clavuligerus

The usefulness of genetic/metabolic engineering for further improvement of industrial strains is subject of discussion because of the general lack of knowledge on genetic alterations introduced by iterative cycles of random mutagenesis in such strains. An industrial clavulanic acid (CA)-overproducer Streptomyces clavuligerus DEPA was assessed to understand proteome-wide changes that have occurred in a local industrial CA overproducer developed through succesive mutagenesis programs. The proteins that could be identified corresponded to 33 distinct ORFs for underrepresented ones and 60 ORFs for overrepresented ones. Three CA biosynthetic enzymes were overrepresented in S. clavuligerus DEPA; carboxyethylarginine synthase (Ceas2), clavaldehyde dehydrogenase (Car) and carboxyethyl-arginine beta-lactam-synthase (Bls2) whereas the enzymes of two other secondary metabolites were underrepresented along with two important global regulators [two-component system (TCS) response regulator (SCLAV_2102) and TetR-family transcriptional regulator (SCLAV_3146)] that might be related with CA production and/or differentiation. γ-butyrolactone biosynthetic protein AvaA2 was 2.6 fold underrepresented in S. clavuligerus DEPA. The levels of two glycolytic enzymes, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and phosophoglycerate kinase were found decreased while those of dihydrolipoyl dehydrogenase (E3) and isocitrate dehydrogenase, with two isoforms were found as significantly increased. A decrease of amino acid metabolism, methionine biosynthesis in particular, as well as S-adenosylmethionine synthetase appeared as one of the prominent mechanisms of success of S. clavuligerus DEPA strain as a prolific producer of CA. The levels of two enzymes of shikimate pathway that leads to the production of aromatic amino acids and aromatic secondary metabolites were also underrepresented. Some of the overrepresented stress proteins in S. clavuligerus DEPA included polynucleotide phosphorylase/polyadenylase (PNPase), ATP-dependent DNA helicase, two isoforms of an anti-sigma factor and thioredoxin reductase. Downregulation of important proteins of cell wall synthesis and division was recorded and a protein with β-lactamase domain (SCLAV_p1007) appeared in 12 isoforms, 5 of which were drastically overrepresented in DEPA strain. These results described herein provide useful information for rational engineering to improve CA production in Streptomyces clavuligerus.

An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus.

Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium.

Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness at low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. We discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp. The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB.

VirR-Mediated Resistance of Listeria monocytogenes against Food Antimicrobials and Cross-Protection Induced by Exposure to Organic Acid Salts.

Formulations of ready-to-eat (RTE) foods with antimicrobial compounds constitute an important safety measure against foodborne pathogens such as Listeria monocytogenes. While the efficacy of many commercially available antimicrobial compounds has been demonstrated in a variety of foods, the current understanding of the resistance mechanisms employed by L. monocytogenes to counteract these stresses is limited. In this study, we screened in-frame deletion mutants of two-component system response regulators associated with the cell envelope stress response for increased sensitivity to commercially available antimicrobial compounds (nisin, lauric arginate, ε-polylysine, and chitosan). A virR deletion mutant showed increased sensitivity to all antimicrobials and significantly greater loss of membrane integrity when exposed to nisin, lauric arginate, or ε-polylysine (P < 0.05). The VirR-regulated operon, dltABCD, was shown to be the key contributor to resistance against these antimicrobial compounds, whereas another VirR-regulated gene, mprF, displayed an antimicrobial-specific contribution to resistance. An experiment with a β-glucuronidase (GUS) reporter fusion with the dlt promoter indicated that nisin does not specifically induce VirR-dependent upregulation of dltABCD. Lastly, prior exposure of L. monocytogenes parent strain H7858 and the ΔvirR mutant to 2% potassium lactate enhanced subsequent resistance against nisin and ε-polylysine (P < 0.05). These data demonstrate that VirRS-mediated regulation of dltABCD is the major resistance mechanism used by L. monocytogenes against cell envelope-damaging food antimicrobials. Further, the potential for cross-protection induced by other food-related stresses (e.g., organic acids) needs to be considered when applying these novel food antimicrobials as a hurdle strategy for RTE foods.

Genome-wide DNA binding pattern of two-component system response regulator RhpR in Pseudomonas syringae.

Although Pseudomonas syringae uses the two-component system RhpRS to modulate the expression of type III secretion system (T3SS) genes and pathogenicity, the molecular mechanisms and the regulon of RhpRS have yet to be fully demonstrated. We have performed a genome-wide analysis of RhpR binding to DNA prepared from P. syringae pv. phaseolicola in order to identify candidate direct targets of RhpR-mediated transcriptional regulation, as described in our recent article [1]. The data are available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE58533. Here we describe the detailed methods and data analyses of our RhpR ChIP-seq dataset.

Construction and verification of the transcriptional regulatory response network of Streptococcus mutans upon treatment with the biofilm inhibitor carolacton.

BackgroundCarolacton is a newly identified secondary metabolite causing altered cell morphology and death of Streptococcus mutans biofilm cells. To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed. A systems biological approach integrating time-resolved transcriptomic data, reverse engineering, transcription factor binding sites, and experimental validation was carried out.ResultsThe co-expression response network constructed from transcriptomic data using the reverse engineering algorithm called the Trend Correlation method consisted of 8284 gene pairs. The regulatory response network inferred by superimposing transcription factor binding site information into the co-expression network comprised 329 putative transcriptional regulatory interactions and could be classified into 27 sub-networks each co-regulated by a transcription factor. These sub-networks were significantly enriched with genes sharing common functions. The regulatory response network displayed global hierarchy and network motifs as observed in model organisms. The sub-networks modulated by the pyrimidine biosynthesis regulator PyrR, the glutamine synthetase repressor GlnR, the cysteine metabolism regulator CysR, global regulators CcpA and CodY and the two component system response regulators VicR and MbrC among others could putatively be related to the physiological effect of carolacton. The predicted interactions from the regulatory network between MbrC, known to be involved in cell envelope stress response, and the murMN-SMU_718c genes encoding peptidoglycan biosynthetic enzymes were experimentally confirmed using Electro Mobility Shift Assays. Furthermore, gene deletion mutants of five predicted key regulators from the response networks were constructed and their sensitivities towards carolacton were investigated. Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.ConclusionThe network approach used in this study revealed important regulators and interactions as part of the response mechanisms of S. mutans biofilm cells to carolacton. It also opens a door for further studies into novel drug targets against streptococci.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-362) contains supplementary material, which is available to authorized users.

Escherichia coli Isolate for Studying Colonization of the Mouse Intestine and Its Application to Two-Component Signaling Knockouts

The biology of Escherichia coli in its primary niche, the animal intestinal tract, is remarkably unexplored. Studies with the streptomycin-treated mouse model have produced important insights into the metabolic requirements for Escherichia coli to colonize mice. However, we still know relatively little about the physiology of this bacterium growing in the complex environment of an intestine that is permissive for the growth of competing flora. We have developed a system for studying colonization using an E. coli strain, MP1, isolated from a mouse. MP1 is genetically tractable and does not require continuous antibiotic treatment for stable colonization. As an application of this system, we separately knocked out each two-component system response regulator in MP1 and performed competitions against the wild-type strain. We found that only three response regulators, ArcA, CpxR, and RcsB, produce strong colonization defects, suggesting that in addition to anaerobiosis, adaptation to cell envelope stress is a critical requirement for E. coli colonization of the mouse intestine. We also show that the response regulator OmpR, which had previously been hypothesized to be important for adaptation between in vivo and ex vivo environments, is not required for MP1 colonization due to the presence of a third major porin.

Understanding the sequential activation of Type III and Type VI Secretion Systems in Salmonella typhimurium using Boolean modeling

BackgroundThree pathogenicity islands, viz. SPI-1 (Salmonella pathogenicity island 1), SPI-2 (Salmonella pathogenicity island 2) and T6SS (Type VI Secretion System), present in the genome of Salmonella typhimurium have been implicated in the virulence of the pathogen. While the regulation of SPI-1 and SPI-2 (both encoding components of the Type III Secretion System - T3SS) are well understood, T6SS regulation is comparatively less studied. Interestingly, inter-connections among the regulatory elements of these three virulence determinants have also been suggested to be essential for successful infection. However, till date, an integrated view of gene regulation involving the regulators of these three secretion systems and their cross-talk is not available.ResultsIn the current study, relevant regulatory information available from literature have been integrated into a single Boolean network, which portrays the dynamics of T3SS (SPI-1 and SPI-2) and T6SS mediated virulence. Some additional regulatory interactions involving a two-component system response regulator YfhA have also been predicted and included in the Boolean network. These predictions are aimed at deciphering the effects of osmolarity on T6SS regulation, an aspect that has been suggested in earlier studies, but the mechanism of which was hitherto unknown. Simulation of the regulatory network was able to recreate in silico the experimentally observed sequential activation of SPI-1, SPI-2 and T6SS.ConclusionsThe present study integrates relevant gene regulatory data (from literature and our prediction) into a single network, representing the cross-communication between T3SS (SPI-1 and SPI-2) and T6SS. This holistic view of regulatory interactions is expected to improve the current understanding of pathogenesis of S. typhimurium.

Identification of a putative cognate sensor kinase for the two‐component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris

The bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response (HR). The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC-type transcriptional regulator whose expression is controlled by a two-component system (TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open-reading frame XC_3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacted with HrpG. Phos-tag™ SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpG in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC_3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.

Two-Component System Response Regulators Involved in Virulence of Streptococcus pneumoniae TIGR4 in Infective Endocarditis

Streptococci resident in the oral cavity have been linked to infective endocarditis (IE). While other viridans streptococci are commonly studied in relation to IE, less research has been focused on Streptococcus pneumoniae. We established for the first time an animal model of S. pneumoniae IE, and examined the virulence of the TIGR4 strain in this model. We hypothesized that two-component systems (TCS) may mediate S. pneumoniae TIGR4 strain virulence in IE and examined TCS response regulator (RR) mutants of TIGR4 in vivo with the IE model. Thirteen of the 14 RR protein genes were mutagenized, excluding only the essential gene SP_1227. The requirement of the 13 RRs for S. pneumoniae competitiveness in the IE model was assessed in vivo through use of quantitative real-time PCR (qPCR) and competitive index assays. Using real-time PCR, several RR mutants were detected at significantly lower levels in infected heart valves compared with a control strain suggesting the respective RRs are candidate virulence factors for IE. The virulence reduction of the ΔciaR mutant was further confirmed by competitive index assay. Our data suggest that CiaR is a virulence factor of S. pneumoniae strain TIGR4 for IE.

A proteomic analysis of the regulon of the NarP two-component regulatory system response regulator in the bovine pathogen Mannheimia haemolytica A1

BackgroundThe response of the NarQP two-component signal transduction system regulon in response to the presence of nitrate for the bovine pathogen Mannheimia haemolytica A1 was investigated by proteomic analysis. Total proteins from a narP mutant and the parent SH1217 grown with or without NaNO3 supplement were examined by ISO-DALT 2D electrophoresis and liquid chromatography-mass spectrometry.ResultsSeventeen proteins were differentially expressed in the parent strain SH1217 in response to the addition of NaNO3 to the growth media. These responses were absent in the narP mutant, indicating that the altered production of these proteins is mediated by NarPMh. Interestingly, NarPMh mediated the increased production of some proteins which are not generally associated with nitrate respiration, such as the iron transporters FbpA and YfeA. The increased production of proteins such as superoxide dismutase, SodA, and GAPDH were also observed. The increased production of these iron-regulated proteins by NarPMh is thought to enhance the swift establishment of the nitrate respiration mechanism of M. haemolytica during pathogenesis.ConclusionThe data suggested NarPMh acts as an important regulator which regulates the expression of a small set of proteins in response to nitrate availability. This may contribute to the prevalence of M. haemolytica A1 in its host during pathogenesis of BPP, through enhancing the effectiveness of nitrate respiration either directly or indirectly.

Gene Regulation in Borrelia burgdorferi

Borrelia burgdorferi, the spirochete that causes Lyme disease, is maintained in nature via an enzootic cycle that comprises a tick vector and a vertebrate host. Transmission from the tick to the mammal, acquisition from the mammal back to the tick, and adaptation to the two disparate environments require sensing signals and responding by regulating programs of gene expression. The molecular mechanisms utilized to effect these lifestyle changes have begun to be elucidated and feature an alternative sigma factor cascade in which RpoN (σ(54)) and RpoS (σ(S)) globally control the genes required for the different phases of the enzootic cycle. The RpoN-RpoS pathway is surprisingly complex, entailing Rrp2, an unusual enhancer-binding protein and two-component regulatory system response regulator activated by acetyl phosphate; BosR, an unorthodox DNA-binding protein; DsrA(Bb), a small noncoding RNA; and Hfq and CsrA, two RNA-binding proteins. B. burgdorferi also has a c-di-GMP signaling system that regulates the tick side of the enzootic cycle and whose function is only beginning to be appreciated.

The expression of two-component system response regulator in multidrug-resistant Mycobacterium tuberculosis

Objective To screen out the two-component system associated with drug resistance of Mycobacterium tuberculosis by detecting the differential expression of two-component system regulator genes between multidrug resistant Mycobacterium tuberculosis strains and drug sensitive strains. Methods Total RNA of MTB was extracted from cultured MTB during the logarithmic phase in the 7H9 brook medium, and then its purity was identified. Reverse transcription was further completed. The expressing levels of TCS response regulators were quantified using SYBR Green I qRT-PCR, which aimed at finding the differential expressions between multidrug resistant strains and sensitive strains. Finally, all of differentially expressed TCS were screened out under the stress of INH, SM and LFA. Results Compared with sensitive strains,multidrug resistant strains of Rv0491, Rv3133c, Rv3143 and Rv3246c were up-regulated 1. 03, 7.11,3.48and 1.37 folds, respectively (t/t' =5. 623, -4. 196, -3. 559 and -3. 016, respectively, P ＜0. 01 ). The expressing level of other regulators had no statistical significance between muhidrug resistant strains and drug sensitive strains. Under the antibiotic pressure, the expression of Rv1027c, Rv3246c and Rv3143 showed significant changes compared with no antibiotic group. Conclusion Rv3246c and Rv3143 may be associated with MTB drug resistance and the differentially expressed genes in multi-drug resistant strains may be used as potential drug targets against drug resistant tuberculosis. Key words: Mycobacterium tuberculosis; Drug resistance, bacterial; Polymerase chain reaction; Signal transduction

The Atypical Two-component Sensor Kinase Lpl0330 from Legionella pneumophila Controls the Bifunctional Diguanylate Cyclase-Phosphodiesterase Lpl0329 to Modulate Bis-(3′-5′)-cyclic Dimeric GMP Synthesis

A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.

The two-component sensor response regulator RoxS/RoxR plays a role in Pseudomonas aeruginosa interactions with airway epithelial cells

Pseudomonas aeruginosa is an opportunistic pathogen that infects the lungs of patients with cystic fibrosis causing aberrant and destructive neutrophil (PMN)-dominated inflammation of airways. Interaction of P. aeruginosa with the lung epithelial barrier resulting in trans-epithelial PMN migration likely represents a key event during PMN recruitment. To investigate bacterial factors involved in interactions with lung epithelial cells, a mutant library of two-component system response regulators was evaluated to identify mutants exhibiting defects in the ability to induce PMN trans-epithelial migration. Of forty-eight mutants, five reproducibly demonstrated a reduced PMN trans-epithelial migration response. All five mutants also exhibited a decreased ability to interact with lung epithelial cells. One mutant identified lacks the response regulator gene roxR, which has not previously been reported to be involved regulating factors that facilitate interactions with lung epithelial cells. This finding suggests that RoxR likely regulates genes with relevance to P. aeruginosa mediated lung disease.