Identification and Characterization of a Two-Component Sensor-Kinase and Response-Regulator System (DcuS-DcuR) Controlling Gene Expression in Response to C 4 -Dicarboxylates in Escherichia coli

Abstract

ABSTRACT The dcuB gene of Escherichia coli encodes an anaerobic C 4 -dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition, dcuB expression is strongly induced by C 4 -dicarboxylates, suggesting the presence of a novel C 4 -dicarboxylate-responsive regulator in E. coli . This paper describes the isolation of a Tn 10 mutant in which the 160-fold induction of dcuB expression by C 4 -dicarboxylates is absent. The corresponding Tn 10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to the dcuB gene at ∼93.5 min in the E. coli chromosome. The yjdHG genes (redesignated dcuSR ) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the dcuSR operon restored the C 4 -dicarboxylate inducibility of dcuB expression in the dcuS mutant to levels exceeding those of the dcuS + strain by approximately 1.8-fold. The dcuS mutation affected the expression of other genes with roles in C 4 -dicarboxylate transport or metabolism. Expression of the fumarate reductase ( frdABCD ) operon and the aerobic C 4 -dicarboxylate transporter ( dctA ) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C 4 -dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal. However, under aerobic conditions with C 4 -dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant. Studies employing a dcuA dcuB dcuC triple mutant unable to transport C 4 -dicarboxylates anaerobically revealed that C 4 -dicarboxylate transport is not required for C 4 -dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ∼140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C 4 -dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae . DcuS is not closely related to the C 4 -dicarboxylate-sensing DctS or DctB protein of Rhodobacter capsulatus or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.