DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system inThe DcuSR (dicarboxylate uptake sensor and regulator) system of Escherichia coli is a typical two-component system consisting of a membranous sensor kinase (DcuS) and a cytoplasmic response regulator (DcuR) (11,26,48). DcuS responds to C 4 -dicarboxylates like fumarate, malate, or succinate (19). In the presence of the C 4 -dicarboxlates, the expression of the genes of anaerobic fumarate respiration (dcuB, fumB, and frdABCD) and of aerobic C 4 -dicarboxylate uptake (dctA) is activated. DcuS is a histidine protein kinase composed of two transmembrane helices with an intermittent sensory PAS domain in the periplasm (PAS P ) that was also termed the PDC domain (for PhoQ/DcuS/DctB/CitA domain or fold) (7,20,32,48). The second transmembrane helix is followed by a cytoplasmic PAS domain (PAS C ) and the C-terminal transmitter domain. PAS C functions in signal transfer from transmembrane helix 2 (TM2) to the kinase domain (9). The C-terminal part of the transmitter domain consists of a catalytic or HATPase (histidine kinase/ATPase) subdomain for autophosphorylation of DcuS (16). The N-terminal part of the transmitter contains two conserved ␣-helical regions, including a conserved His residue which is the site for autophosphorylation. The ␣-helices serve in dimerization and form a four-helix bundle in the kinase dimer (dimerization and histidine phosphotransfer [DHp] domain) (25,35,42,44).The dimeric sensor kinases have been supposed to phosphorylate mutually, by the catalytic domain of one monomer, the His residue of the partner monomer (10). The oligomeric state of the membrane-bound sensor kinases EnvZ and VirA was also deduced from in vivo complementation studies (31,46). In addition, signal transduction across the membrane and along cytoplasmic PAS domains appears to be a mechanical process requiring oligomeric proteins (9, 40). Therefore, His kinases are supposed to be dimeric in the functional state, but a higher oligomeric state has not been tested and is conceivable. Only a limited number of membrane-bound sensor kinases have been studied for their oligomerization in their membrane-bound state. Thus, the oligomeric state of the KdpD and TorS sensor kinases of E. coli have been shown to prevail in the detergent-solubilized state as oligomers, presumably dimers (14, 29). There was indirect information that functional DcuS is a dimer as well. Purified DcuS shows kinase activity only after reconstitution into liposomes, and phosphorylation is stimulated by C 4 -dicarboxylates (16,19). Detergentsolubilized DcuS, on the other hand, shows no kinase activity,

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 membranebound 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 citC-DEFXGT 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. E scherichia coli can grow on a wide variety of substrates under aerobic or anaerobic conditions. Citrate fermentation by E. coli requires the presence of an oxidizable cosubstrate, like glucose or glycerol, which is used as an electron donor (28). Citrate is taken up by the citrate/succinate antiporter CitT (39) and cleaved to acetate and oxaloacetate (OAA) by citrate lyase (CL). Holocitrate lyase and the citrate transporter CitT are encoded by the citCDEFXGT gene cluster. Oxaloacetate then is reduced to malate by malate dehydrogenase (Mdh), and malate subsequently is converted to fumarate by fumarase (FumB). Fumarate finally is reduced to succinate by fumarate reductase (FrdABCD). The twocomponent system CitA/CitB of E. coli is supposed to regulate the expression of the genes for citrate fermentation in response to external citrate under anaerobic conditions (20, 52), similarly to the citrate-responsive two-component system CitA/CitB of Klebsiella pneumoniae (6). CitA/CitB represents a typical extracytoplasmic-sensing two-component system consisting of a membrane-bound sensory histidine kinase, CitA, and the cognate response regulator CitB (30, 50). The perception of the stimulus leads to the autophosphorylation of a conserved histidine residue (His 347 ) in the kinase domain of the sensor CitA. The phosphoryl...

Signal transduction in prokaryotes is frequently accomplished by two-component regulatory systems in which a histidine protein kinase is the sensory component. Many of these sensory kinases control metabolic processes that do not show an obvious requirement for inhomogeneous distribution within bacterial cells. Here, the sensory kinases DcuS and CitA, two histidine kinases of Escherichia coli, were investigated. Both are membrane-integral and involved in the regulation of carboxylate metabolism. The two-component sensors were fused with yellow fluorescent protein (YFP) and live images of immobilized cells were obtained by confocal laser fluorescence microscopy. The fluorescence of the fusion proteins was concentrated at the poles of the cells, indicating polar accumulation of the sensory kinases. For quantitative evaluation, line profiles of the imaged fluorescence intensities were generated; these revealed that the fluorescence intensity of the polar bright spots was 2.3-8.5 times higher than that of the cytoplasm. With respect to the cylindrical part of the membrane, the values were lower by about 40 %. The polar accumulation was comparable to that of methyl-accepting chemotaxis proteins (MCPs) and MCP-related proteins. The degree of DcuS-YFP localization was independent of the presence of MCP and the expression level of dcuS-yfp (or DcuS concentration). The presence of effector (fumarate or citrate, respectively) increased the polar accumulation by more than 20 %. Cell fractionation demonstrated that polar accumulation was not related to inclusion body formation. Therefore, sensory kinases DcuS and CitA, which regulate metabolic processes without obvious polar function, exhibit polar accumulation.