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.

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di-and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The fumarate sensor DcuS is a prototype for a two component sensory histidine kinase with signal perception in the periplasm, transmembrane signal transfer (1, 2), and autophosphorylation of a His residue in the kinase domain in the cytoplasm (3). DcuS belongs to the CitA family of sensors that are specific for sensing di-and tricarboxylates (1, 2, 4, 5). The periplasmic domain of the histidine autokinase CitA works as a highly specific citrate receptor, whereas DcuS uses any type of C 4 -dicarboxylate, like fumarate, succinate, and malate, as a stimulus (1, 4 -6). DcuS is predicted to consist of two transmenbrane helices and of a periplasmic sensory domain enclosed by the transmembrane helices. The second transmembrane helix is followed by a cytoplasmic PAS 1 domain of unknown function and the kinase with the consensus histidine residue for autophosphorylation. The periplasmic citrate binding domain of CitA is conserved in DcuS and presumably responsible for binding of fumarate and other C 4 -dicarboxylates. Preliminary results suggest that fumarate sensing occurs by this domain in the periplasm (2, 4, 5). After phosphorylation by DcuS the response regulator DcuR of the DcuSR system activates the expression of the target genes like dcuB and frd-ABCD encoding an anaerobic fumarate carrier DcuB and fumarate reductase (4, 5). Despite their prevalence no structural information is available for transmembranous sensory kinases, in particular not for signal perception and transmission across the membrane. Only the structures of cytoplasmic sensory kinases, or of domains not involved in transmembrane signaling, have been determined.Purified DcuS is active after reconstitution in proteoliposomes and capable of transmembranous stimulation of the kinase by fumarate (2). For a more detailed understanding of signal perception representing the first step of signal transduction in transmembranous histidine kinases of two-component systems, the structure of the periplasmic C 4 -dicarboxylate binding domain of DcuS (DcuS-pd) was determined after stable over-production of the domain. ("DcuS-pd")-The sequence of dcuS coding for the periplasmic domain of DcuS (DcuS or DcuS-pd) enclosed by the two transmembrane helices was cloned into the NdeI and HindIII sites of plasmid pET28a (Novagen) resulting in plasmid pMW145. The DNA fragment was amplified with oligonucleotides pdcus-NdeII (ATT TAC TTC TCG CAT ATG AGT GAT ATG) and pdcuSHind (GAC CAG ATA AAG CTT CAG CGA CTG) by PCR of genomic Escheric...

In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C 4 dicarboxylates in anaerobic growth were known. Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C 4 dicarboxylates which is functional in anaerobic growth. The dcuC gene is located at min 14.1 of the E. coli map in the counterclockwise orientation. The dcuC gene combines two open reading frames found in other strains of E. coli K-12. The gene product (DcuC) is responsible for the transport of C 4 dicarboxylates in DcuA-DcuB-deficient cells. The triple mutant (dcuA dcuB dcuC) is completely devoid of C 4 -dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration. DcuC, however, is not required for C 4 -dicarboxylate uptake in aerobic growth. The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers. DcuC shows sequence similarity to the two major anaerobic C 4 -dicarboxylate carriers DcuA and DcuB. Mutants producing only DcuA, DcuB, or DcuC were prepared. In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode.In Escherichia coli, various transport activities for C 4 dicarboxylates are known. Under aerobic growth conditions, unidirectional uptake of C 4 dicarboxylates (fumarate, succinate, and malate) and aspartate, but no export, is catalyzed (5, 12). This transport is effected by a binding protein-dependent carrier or by a secondary carrier which is driven by the electrochemical H ϩ gradient over the membrane (10, 17). The dctA and dctB genes have been shown to be related to the aerobic carriers (3, 17, 23). The dctA gene has been sequenced (23), but none of the carriers has been clearly defined so far by genetic or biochemical means. Bacteria grown under anaerobic conditions, on the other hand, catalyze exchange, uptake, and efflux of C 4 dicarboxylates (5, 6). Fumarate/succinate exchange is required during fumarate respiration where the acceptor fumarate has to be taken up and the product succinate has to be excreted. Net C 4 -dicarboxylate uptake is required for anaerobic growth with C 4 dicarboxylates as the C source. Citrate fermentation, on the other hand, which produces 1 succinate per citrate, depends on a C 4 -dicarboxylate efflux system. The exchange reaction of the C 4 dicarboxylates is an electroneutral process, whereas uptake and efflux are electrogenic symport reactions, presumably of the dicarboxylate 2Ϫ with 3 H ϩ (6). The anaerobic transport activities were found only in bacteria grown under anaerobic conditions, and the synthesis requires intact FNR (named FNR for fumarate nitrate reductase regulator), the transcriptional regulator of anaerobic metabolism (5, 6, 25).Recently two homologous genes (dcuA and dcuB) were identified in E. coli which encode two C 4 -dicarboxylate carriers, DcuA and DcuB (22). The carriers (DcuA and DcuB) were complementary to each other, and each was sufficient for fumarate/s...

The histidine protein kinase DcuS of Escherichia coli senses C 4 -dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C 4 -dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C 4 -dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C 4 -dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C 4 -dicarboxylate-specific sensor (DcuS DC ) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (DcuS Cit ). DcuS DC and DcuS Cit had complementary effector specificities and responded either to C 4 -dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the C 4 -dicarboxylates) via the C 4 -dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of C 4 -dicarboxylates; sites C1 and H are required for other essential purposes.Escherichia coli is able to use C 4 -dicarboxylates as substrates for anaerobic growth by fumarate respiration, which requires the synthesis of fumarate reductase (frdABCD genes) and the fumarate/succinate antiporter DcuB (dcuB gene) (for reviews, see references 6, 15, and 28). Expression of the frdABCD and dcuB genes is stimulated by the DcuSR two-component system (12,14,15,29). DcuS responds to C 4 -dicarboxylates and related compounds through a periplasmic sensing domain (19). The two carboxylic groups of the C 4 -dicarboxylates are crucial for stimulus perception, whereas other parts of the C 4 -dicarboxylates such as ligands at position C2 or C3 are of minor significance. The apparent K D values of C 4 -dicarboxylates for stimulating the expression of DcuS-regulated genes are in the range of 0.45 to 3 mM.DcuS is a member of the CitA/DcuS family of sensory histidine kinases and shares significant sequence similarities with CitA (4,5,15,17,18). CitA is the highly specific and highaffinity citrate sensor kinase of the CitAB two-component system that controls expression of the citrate fermentation genes in Klebsiella pneumoniae and E. coli. The CitA and DcuS sensors have similar membrane topologies, with a periplasmic sensory domain and a cytoplasmic kinase domain (15,18,29). CitA and DcuS are typical members of the periplasmic sensing histidine kinases (20). The structure of the periplasmic domain of DcuS has been solved by nuclear magnetic resonance (NMR) spectroscopy, and that of CitA from K. pneumoniae has been solved by crystallography and X-ray analysis (23, 25).The overall structures for the two domains are similar and resemble the PAS (Per-Arnt-Sim)...

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