Summary The aerobic Escherichia coli C4‐dicarboxylate transporter DctA and the anaerobic fumarate/succinate antiporter DcuB function as obligate co‐sensors of the fumarate responsive sensor kinase DcuS under aerobic or anaerobic conditions respectively. Overproduction under anaerobic conditions allowed DctA to replace DcuB in co‐sensing, indicating their functional equivalence in this capacity. In vivo interaction studies between DctA and DcuS using FRET or a bacterial two‐hybrid system (BACTH) demonstrated their interaction. DctA–YFP bound to an affinity column and was able to retain DcuS. DctA shows substantial sequence and secondary structure conservation to GltPh, the Na+/glutamate symporter of Pyrococcus horikoshii with known 3D structure. Topology studies of DctA demonstrated the presence of eight transmembrane helices in an arrangement similar to that of GltPh. DctA contains an additional predicted amphipathic helix 8b on the cytoplasmic side of the membrane that is specific for DctA and not present in GltPh. Mutational analysis demonstrated the importance of helix 8b in co‐sensing and interaction with DcuS, and the isolated helix 8b showed strong interaction with DcuS. In DcuS, deletion and mutation of the cytoplasmic PASC domain affected the interaction between DctA and DcuS. It is concluded that DctA forms a functional unit or sensor complex with DcuS through specific interaction sites.

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...

The membrane-integral sensor kinase DcuS of Escherichia coli consists of a periplasmically located sensory PAS(P) domain, transmembrane helices TM1 and TM2, a cytoplasmic PAS(C) domain and the kinase domain. Stimulus (C(4)-dicarboxylate) binding at PAS(P) is required to stimulate phosphorylation of the kinase domain, resulting in phosphoryl transfer to the response regulator DcuR. PAS(C) functions as a signaling device or a relay in signal transfer from TM2 to the kinase. Phosphorylated DcuR induces the expression of the target genes. Sensing by DcuS requires the presence of the C(4)-dicarboxylate transporter DctA during aerobic growth. DctA forms a sensor unit with DcuS, and a short C-terminal sequence of DctA forming the putative helix 8b is required for interaction with DcuS. Helix 8b contains a LDXXXLXXXL motif that is essential for function and interaction. DcuS requires the PAS(C) domain for signal perception from DctA. Thus, DcuS and DctA form a DctA/DcuS sensory unit, and DcuS perceives stimuli from two different sites (PAS(P) and DctA). The signal transfer pathways are supposed to merge at PAS(C). The fumarate/succinate antiporter DcuB takes over the role as a co-sensor of DcuS under anaerobic growth conditions.

The sensor kinase DcuS of Escherichia coli co-operates under aerobic conditions with the C -dicarboxylate transporter DctA to form the DctA/DcuS sensor complex. Under anaerobic conditions C -dicarboxylate transport in fumarate respiration is catalyzed by C -dicarboxylate/fumarate antiporter DcuB. (i) DcuB interacted with DcuS as demonstrated by a bacterial two-hybrid system (BACTH) and by co-chromatography of the solubilized membrane-proteins (mHPINE assay). (ii) In the DcuB/DcuS complex only DcuS served as the sensor since mutations in the substrate site of DcuS changed substrate specificity of sensing, and substrates maleate or 3-nitropropionate induced DcuS response without affecting the fumarate site of DcuB. (iii) The half-maximal concentration for induction of DcuS by fumarate (1 to 2 mM) and the corresponding K for transport (50 µM) differ by a factor of 20 to 40. Therefore, the fumarate sites are different in transport and sensing. (iv) Increasing levels of DcuB converted DcuS from the permanent ON (DcuB deficient) state to the fumarate responsive form. Overall, the data show that DcuS and DcuB form a DcuB/DcuS complex representing the C -dicarboxylate responsive form, and that the sensory site of the complex is located in DcuS whereas DcuB is required for converting DcuS to the sensory competent state.