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DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C(4) -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C(4)-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 A apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mm for the C(4)-dicarboxylates and 0.5 mm for 3-nitropropionate or d-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg(107)-X(2)-His(110)-X(9)-Phe(120)-X(26)-Arg(147)-X-Phe(149)) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or d-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C(4)-dicarboylate binding domain. The amino acid residues responding to fumarate or d-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C(4)-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.

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.

Bacteria contain secondary carriers for the uptake, exchange or efflux of C4-dicarboxylates. In aerobic bacteria, dicarboxylate transport (Dct)A carriers catalyze uptake of C4-dicarboxylates in a H+- or Na+-C4-dicarboxylate symport. Carriers of the dicarboxylate uptake (Dcu)AB family are used for electroneutral fumarate:succinate antiport which is required in anaerobic fumarate respiration. The DcuC carriers apparently function in succinate efflux during fermentation. The tripartite ATP-independent periplasmic (TRAP) transporter carriers are secondary uptake carriers requiring a periplasmic solute binding protein. For heterologous exchange of C4-dicarboxylates with other carboxylic acids (such as citrate:succinate by CitT) further types of carriers are used. The different families of C4-dicarboxylate carriers, the biochemistry of the transport reactions, and their metabolic functions are described. Many bacteria contain membraneous C4-dicarboxylate sensors which control the synthesis of enzymes for C4-dicarboxylate metabolism. The C4-dicarboxylate sensors DcuS, DctB, and DctS are histidine protein kinases and belong to different families of two-component systems. They contain periplasmic domains presumably involved in C4-dicarboxylate sensing. In DcuS the periplasmic domain seems to be essential for direct interaction with the C4-dicarboxylates. In signal perception by DctB, interaction of the C4-dicarboxylates with DctB and the DctA carrier plays an important role.