The Fumarate/Succinate Antiporter DcuB of Escherichia coli Is a Bifunctional Protein with Sites for Regulation of DcuS-dependent Gene Expression

Abstract

DcuB of Escherichia coli catalyzes C 4 -dicarboxylate/succinate antiport during growth by fumarate respiration. The expression of genes of fumarate respiration, including the genes for DcuB (dcuB) and fumarate reductase (frdABCD) is transcriptionally activated by C 4 -dicarboxylates via the DcuS-DcuR two-component system, comprising the sensor kinase DcuS, which contains a periplasmic sensing domain for C 4 -dicarboxylates. Deletion or inactivation of dcuB caused constitutive expression of DcuSregulated genes in the absence of C 4 -dicarboxylates. The effect was specific for DcuB and not observed after inactivation of the homologous DcuA or the more distantly related DcuC transporter. Random and site-directed mutation identified three point mutations (T394I, D398N, and K353A) in DcuB that caused a similar derepression as dcuB deletion, whereas the transport activity of the DcuB mutants was retained. Constitutive expression in the dcuB mutants depended on the presence of a functional DcuS-DcuR two-component system. Mutation of residues E79A, R83A, and R127A of DcuB, on the other hand, inactivated growth by fumarate respiration and transport of [ 14 C]succinate, whereas the expression of dcuBlacZ was not affected. Therefore, the antiporter DcuB is a bifunctional protein and has a regulatory function that is independent from transport, and sites for transport and regulation can be differentiated.The fumarate/succinate antiporter DcuB (dicarboxylate uptake) of Escherichia coli catalyzes the uptake of external C 4 -dicarboxylates like fumarate, L-malate, or aspartate (1-3). Fumarate is used as an electron acceptor in fumarate respiration by fumarate reductase, which carries the active site at the cytoplasmic side of the membrane (for reviews, see Refs. 4 -6). L-Malate and aspartate are converted to fumarate by fumarase and aspartase, respectively, and are then metabolized in the same way as fumarate. Fumarate respiration results in the generation of a proton potential and drives ATP synthesis and growth of the bacteria. The product of fumarate respiration, succinate, is not further catabolized in anaerobic growth and is excreted by DcuB. DcuB, therefore, is responsible for the substrate/product antiport of fumarate, L-malate, or aspartate against succinate. DcuB and fumarase B are encoded by the dcuB fumB gene cluster (1, 7).

CitA/CitB Two-Component System Regulating Citrate Fermentation in Escherichia coli and Its Relation to the DcuS/DcuR System In Vivo

“…In the strains deficient in CitT, DcuC, or DcuB (Table 3), the induction of citC-LacZ depended on citrate comparably to the citrate-dependent induction of citC-lacZ in the wild-type strain. This is in contrast to the expression of dcuB-lacZ, which requires fumarate for induction in the wild-type strain, whereas the induction is fumarate independent in the dcuB mutant strain (IMW370), in agreement with earlier observations (22). Thus, lacking the requirement for a transporter as a cosensor for CitA is a clear difference to the otherwise structurally and functionally similar DcuS.…”

“…In E. coli the fumarate/succinate antiporter DcuB exerts a regulatory effect on genes regulated by DcuS/DcuR (22). For normal regulatory function, the DcuS/DcuR system requires the presence of DcuB.…”

“…In addition, DcuS requires the fumarate/succinate antiporter (DcuB) as a cosensor and becomes constitutively active when DcuB is missing (22). For cosensing, DcuB contains specific sites that are not essential for transport.…”

Regulation of tartrate metabolism by TtdR and relation to the DcuS–DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli

“…First, DcuS responds to the presence of C 4 -dicarboxylates (Golby et al, 1999;Kleefeld et al, 2009;Kneuper et al, 2005;Zientz et al, 1998), which regulate phosphorylation of DcuS and DcuR. In addition, NarXL/nitrate and CRP-cAMP/ glucose control the expression and level of DcuS and DcuR (Goh et al, 2005;Oyamada et al, 2007).…”

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

“…4 and branches with the other known and predicted C 4 -dicarboxylate-specific TRAP transporters (Shaw et al, 1991;Forward et al, 1997). It is also striking that most of these TRAP transporters cluster with the dctS and dctR genes (annotated as dctDB in P. aeruginosa), which form a well-studied two-component regulatory system for C 4 -dicarboxylates (Hamblin et al, 1993;Golby et al, 1999;Kleefeld et al, 2009). …”