Engineering chimeric two-component system into <Emphasis Type="Italic">Escherichia coli</Emphasis> from <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> to sense methanol

Escherichia coli does not have the methanol sensing apparatus, was engineered to sense methanol by employing chimeric two-component system (TCS) strategy. A chimeric FlhS/EnvZ (FlhSZ) chimeric histidine kinase (HK) was constructed by fusing the sensing domain of Paracoccus denitrificans FlhS with the catalytic domain of E. coli EnvZ. The constructed chimeric TCS FlhSZ/OmpR could sense methanol by the expression of ompC and gfp gene regulated by ompC promoter. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed the dynamic response of the chimeric TCS to methanol. The expression of ompC and the gfp fluorescence was maximum at 0.01 and 0.5% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric HK FlhSZ. This strategy can be employed for the construction of several chimeric TCS based bacterial biosensors for the development of biochemical producing recombinant microorganisms.     

Construction of malate-sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.     

C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain

C₄-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C₄-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C₄-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C₄ succinate, and a complex with C₃ malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C₄-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.     

CitA/CitB two-component system regulating citrate fermentation in Escherichia coli and its relation to the DcuS/DcuR system in vivo

Citrate fermentation by Escherichia coli requires the function of the citrate/succinate antiporter CitT (citT gene) and of citrate lyase (citCDEFXG genes). Earlier experiments suggested that the two-component system CitA/CitB, consisting of the membrane-bound sensor kinase CitA and the response regulator CitB, stimulates the expression of the genes in the presence of citrate, similarly to CitA/CitB of Klebsiella pneumoniae. In this study, the expression of a chromosomal citC-lacZ gene fusion was shown to depend on CitA/CitB and citrate. CitA/CitB is related to the DcuS/DcuR two-component system which induces the expression of genes for fumarate respiration in response to C(4)-dicarboxylates and citrate. Unlike DcuS, CitA required none of the cognate transporters (CitT, DcuB, or DcuC) for function, and the deletion of the corresponding genes showed no effect on the expression of citC-lacZ. The citAB operon is preceded by a DcuR binding site. Phosphorylated DcuR bound specifically to the promoter region, and the deletion of dcuS or dcuR reduced the expression of citC. The data indicate the presence of a regulatory cascade consisting of DcuS/DcuR modulating citAB expression (and CitA/CitB levels) and CitA/CitB controlling the expression of the citCDEFXGT gene cluster in response to citrate. In vivo fluorescence resonance energy transfer (FRET) and the bacterial two-hybrid system (BACTH) showed interaction between the DcuS and CitA proteins. However, BACTH and expression studies demonstrated the lack of interaction and cross-regulation between CitA and DcuR or DcuS and CitB. Therefore, there is only linear phosphoryl transfer (DcuS→DcuR and CitA→CitB) without cross-regulation between DcuS/DcuR and CitA/CitB.     

Identification and Characterization of a Two-Component Sensor-Kinase and Response-Regulator System (DcuS-DcuR) Controlling Gene Expression in Response to C4-Dicarboxylates in Escherichia coli

The dcuB gene of Escherichia coli encodes an anaerobic C₄-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition, dcuB expression is strongly induced by C₄-dicarboxylates, suggesting the presence of a novel C₄-dicarboxylate-responsive regulator in E. coli. This paper describes the isolation of a Tn10 mutant in which the 160-fold induction of dcuB expression by C₄-dicarboxylates is absent. The corresponding Tn10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to the dcuB gene at ~93.5 min in the E. coli chromosome. The yjdHG genes (redesignated dcuSR) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the dcuSR operon restored the C₄-dicarboxylate inducibility of dcuB expression in the dcuS mutant to levels exceeding those of the dcuS⁺ strain by approximately 1.8-fold. The dcuS mutation affected the expression of other genes with roles in C₄-dicarboxylate transport or metabolism. Expression of the fumarate reductase (frdABCD) operon and the aerobic C₄-dicarboxylate transporter (dctA) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C₄-dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal. However, under aerobic conditions with C₄-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant. Studies employing a dcuA dcuB dcuC triple mutant unable to transport C₄-dicarboxylates anaerobically revealed that C₄-dicarboxylate transport is not required for C₄-dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ~140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C₄-dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae. DcuS is not closely related to the C₄-dicarboxylate-sensing DctS or DctB protein of Rhodobacter capsulatus or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.     

Polar Localization of a Tripartite Complex of the Two-Component System DcuS/DcuR and the Transporter DctA in Escherichia coli Depends on the Sensor Kinase DcuS

The C₄-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of E. coli is membrane-bound and reveals a polar localization. DcuS uses the C₄-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when expressed without DcuS, but co-localize with DcuS when co-expressed at appropriate levels. Thus, DcuS is required for location of DctA and DcuR at the poles and formation of tripartite DctA/DcuS/DcuR sensor/regulator complexes. Vice versa, DctA, DcuR and the alternative succinate transporter DauA were not essential for polar localization of DcuS, suggesting that the polar trapping occurs by DcuS. Cardiolipin, the high curvature at the cell poles, and the cytoskeletal protein MreB were not required for polar localization. In contrast, polar localization of DcuS required the presence of the cytoplasmic PAS_C and the kinase domains of DcuS.     

Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli

In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [³H]-NaBH₄ indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.     

Function of DcuS from Escherichia coli as a fumarate-stimulated histidine protein kinase in vitro

The two-component regulatory system DcuSR of Escherichia coli controls the expression of genes of C(4)-dicarboxylate metabolism in response to extracellular C(4)- dicarboxylates such as fumarate or succinate. DcuS is a membrane-integral sensor kinase, and the sensory and kinase domains are located on opposite sides of the cytoplasmic membrane. The intact DcuS protein (His(6)-DcuS) was overproduced and isolated in detergent containing buffer. His(6)-DcuS was reconstituted into liposomes made from E. coli phospholipids. Reconstituted His(6)-DcuS catalyzed, in contrast to the detergent-solubilized sensor, autophosphorylation by [gamma-(33)P]ATP with an approximate K(D) of 0.16 mm for ATP. Up to 7% of the reconstituted DcuS was phosphorylated. Phosphorylation was stimulated up to 5.9-fold by C(4)-dicarboxylates, but not by other carboxylates. The phosphoryl group of DcuS was rapidly transferred to the response regulator DcuR. Upon phosphorylation, DcuR bound specifically to dcuB promoter DNA. The reconstituted DcuSR system therefore represents a defined in vitro system, which is capable of the complete transmembrane signal transduction by the DcuSR two-component system from the stimulus (fumarate) to the DNA, including signal transfer across the phospholipid membrane.     

DNA interaction and phosphotransfer of the C4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C(4)-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C(4)-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-(32)P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C(4)-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 micro M for untreated DcuR and < or =1 to 2 microM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.     

Hysteretic and graded responses in bacterial two-component signal transduction

Bacterial two-component systems (TCS) are key signal transduction networks regulating global responses to environmental change. Environmental signals may modulate the phosphorylation state of sensor kinases (SK). The phosphorylated SK transfers the phosphate to its cognate response regulator (RR), which causes physiological response to the signal. Frequently, the SK is bifunctional and, when unphosphorylated, it is also capable of dephosphorylating the RR. The phosphatase activity may also be modulated by environmental signals. Using the EnvZ/OmpR system as an example, we constructed mathematical models to examine the steady-state and kinetic properties of the network. Mathematical modelling reveals that the TCS can show bistable behaviour for a given range of parameter values if unphosphorylated SK and RR form a dead-end complex that prevents SK autophosphorylation. Additionally, for bistability to exist the major dephosphorylation flux of the RR must not depend on the unphosphorylated SK. Structural modelling and published affinity studies suggest that the unphosphorylated SK EnvZ and the RR OmpR form a dead-end complex. However, bistability is not possible because the dephosphorylation of OmpR approximately P is mainly done by unphosphorylated EnvZ. The implications of this potential bistability in the design of the EnvZ/OmpR network and other TCS are discussed.     

Molecular cloning, sequence analysis and structure modeling of OmpR, the response regulator of Aeromonas hydrophila

The ability of bacteria to survive and proliferate in changing environmental conditions, and during host cell invasion is the key to their pathogenicity. In order to achieve this, the bacteria use a signal transduction system, the two component regulatory system, which consists of a sensor kinase and a response regulator. The EnvZ/OmpR system regulates the porin genes ompF/ompC in response to changes in osmolarity. In the present study, the ompR gene of Aeromonas hydrophila (isolate Ah17) was cloned, sequenced and characterized. Further an attempt was made to analyze the structural characteristics of the OmpR protein from Aeromonas hydrophila. The three dimensional structure of the protein was predicted by homology modeling and the modeled structure was compared to other members of two component response regulators. This study would be helpful for structure based drug design approaches to generate drugs against this harmful pathogen to control its proliferation in both human and fish hosts.     

Co-regulation of polysaccharide production,motility, and expression of type III secretion genes by EnvZ/OmpR and GrrS/GrrA systems in Erwinia amylovora

The EnvZ/OmpR and GrrS/GrrA systems, two widely distributed two-component systems in gamma-Proteobacteria, negatively control amylovoran biosynthesis in Erwinia amylovora, and the two systems regulate motility in an opposing manner. In this study, we examined the interplay of EnvZ/OmpR and GrrS/GrrA systems in controlling various virulence traits in E. amylovora. Results showed that amylovoran production was significantly higher when both systems were inactivated, indicating that the two systems act as negative regulators and their combined effect on amylovoran production appears to be enhanced. In contrast, reduced motility was observed when both systems were deleted as compared to that of grrA/grrS mutants and WT strain, indicating that the two systems antagonistically regulate motility in E. amylovora. In addition, glycogen accumulation was much higher in envZ/ompR and two triple mutants than that of grrS/grrA mutants and WT strain, suggesting that EnvZ/OmpR plays a dominant role in regulating glycogen accumulation, whereas levan production was significantly lower in the grrS/grrA and two triple mutants as compared with that of WT and envZ/ompR mutants, indicating that GrrS/GrrA system dominantly controls levan production. Furthermore, both systems negatively regulated expression of three type III secretion (T3SS) genes and their combined negative effect on hrp-T3SS gene expression increased when both systems were deleted. These results demonstrated that EnvZ/OmpR and GrrS/GrrA systems co-regulate various virulence factors in E. amylovora by still unknown mechanisms or through different target genes, sRNAs, or proteins, indicating that a complex regulatory network may be involved, which needs to be further explored.     

Regulatory Protein OmpR Influences the Serum Resistance of Yersinia enterocolitica O:9 by Modifying the Structure of the Outer Membrane

The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.     

Differentiation of DctA and DcuS function in the DctA/DcuS sensor complex of Escherichia coli: function of DctA as an activity switch and of DcuS as the C4‐dicarboxylate sensor

The C₄‐dicarboxylate responsiveness of the sensor kinase DcuS is only provided in concert with C₄‐dicarboxylate transporters DctA or DcuB. The individual roles of DctA and DcuS for the function of the DctA/DcuS sensor complex were analysed. (i) Variant DctA(S380D) in the C₄‐dicarboxylate site of DctA conferred C₄‐dicarboxylate sensitivity to DcuS in the DctA/DcuS complex, but was deficient for transport and for growth on C₄‐dicarboxylates. Consequently transport activity of DctA is not required for its function in the sensor complex. (ii) Effectors like fumarate induced expression of DctA/DcuS‐dependent reporter genes (dcuB–lacZ) and served as substrates of DctA, whereas citrate served only as an inducer of dcuB–lacZ without affecting DctA function. (iii) Induction of dcuB–lacZ by fumarate required 33‐fold higher concentrations than for transport by DctA (K_m = 30 μM), demonstrating the existence of different fumarate sites for both processes. (iv) In titration experiments with increasing dctA expression levels, the effect of DctA on the C₄‐dicarboxylate sensitivity of DcuS was concentration dependent. The data uniformly show that C₄‐dicarboxylate sensing by DctA/DcuS resides in DcuS, and that DctA serves as an activity switch. Shifting of DcuS from the constitutive ON to the C₄‐dicarboxylate responsive state, required presence of DctA but not transport by DctA.