Identification of a third secondary carrier (DcuC) for anaerobic C4-dicarboxylate transport in Escherichia coli: roles of the three Dcu carriers in uptake and exchange

Abstract

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 VC1777–VC1779 proteins are members of a sialic acid-specific subfamily of TRAP transporters (SiaPQM) and constitute the sole route of sialic acid uptake in the human pathogen Vibrio cholerae

“…Three more genes, vc2699, vcA0205 and vcA0665, in V. cholerae N16961 encode putative DcuABC family proteins that clustered with the anaerobic C 4 -dicarboxylate transporter of E. coli (N. Chowdhury & E. F. Boyd, unpublished data; Six et al, 1994;Zientz et al, 1996). In Pseudomonas aeruginosa, DctA (PA1183) and DctPQM (PA6167-PA5169) systems co-ordinately work for C 4 -dicarboxylate uptake (Valentini et al, 2011).…”

The l -Tartrate/Succinate Antiporter TtdT (YgjE) of l -Tartrate Fermentation in Escherichia coli

“…After various times, the reaction was stopped by the addition of 0.9 ml of ice-cold 0.1 M LiCl followed by rapid vacuum filtration through membrane filters (mixed cellulose ester, type ME 24; diameter, 25 mm; 0.2-m pore size) (Schleicher & Schuell MicroScience). The filters were washed three times with ice-cold 0.1 M LiCl, transferred to scintillation vials with 4 (32).…”

“…Due to repression of the citric acid cycle under these conditions, succinate cannot be oxidized further and is excreted (for reviews, see references 2, 12, 19, and 27). The transport of the C 4 -dicarboxylates is effected by carriers which are specifically produced under anaerobic conditions (5,12,26,32,33,34). DcuB functions as a C 4 -dicarboxylate/succinate antiporter which catalyzes electroneutral antiporting of the external C 4 -dicarboxylates (generally fumarate, malate, and aspartate) against succinate as the end product of fumarate respiration (6).…”

“…DcuA is homologous to DcuB but is expressed constitutively (8). DcuC normally functions as a succinate efflux carrier during glucose fermentation, but it can take over fumarate/succinate antiporting when required (32,34).…”

A Na+-coupled C4-dicarboxylate transporter (Asuc_0304) and aerobic growth of Actinobacillus succinogenes on C4-dicarboxylates

“…The genome sequence revealed that A. succinogenes possesses a total of 2199 genes (McKinlay et al, 2010), including various transporters that potentially function in aerobic or anaerobic C 4 -dicarboxylate transport (Table S1, available in the online Supplementary Material). The genome of A. succinogenes encodes three carrier proteins of the Dcu (DcuAB; C 4 -dicarboxylate uptake) or DcuC (C 4 -dicarboxylate uptake C) family which are good succinate exporter candidates under conditions of succinate production from sugar fermentation, as shown for Escherichia coli (Six et al, 1994;Zientz et al, 1996;Golby et al, 1998;Zientz et al, 1999;Chen et al, 2014). One other carrier protein of the CitT family might also catalyse transport of C 4 -dicarboxylate, as shown for CitT and TdtT in E. coli (Pos et al, 1998;Kim & Unden, 2007).…”