Interaction of the Escherichia coli transporter DctA with the sensor kinase DcuS: presence of functional DctA/DcuS sensor units.
The aerobic Escherichia coli C(4) -dicarboxylate transporter DctA and the anaerobic fumarate/succinate antiporter DcuB function as obligate co-sensors of the fumarate responsive sensor kinase DcuS under aerobic or anaerobic conditions respectively. Overproduction under anaerobic conditions allowed DctA to replace DcuB in co-sensing, indicating their functional equivalence in this capacity. In vivo interaction studies between DctA and DcuS using FRET or a bacterial two-hybrid system (BACTH) demonstrated their interaction. DctA-YFP bound to an affinity column and was able to retain DcuS. DctA shows substantial sequence and secondary structure conservation to Glt(Ph), the Na(+)/glutamate symporter of Pyrococcus horikoshii with known 3D structure. Topology studies of DctA demonstrated the presence of eight transmembrane helices in an arrangement similar to that of Glt(Ph) . DctA contains an additional predicted amphipathic helix 8b on the cytoplasmic side of the membrane that is specific for DctA and not present in Glt(Ph). Mutational analysis demonstrated the importance of helix 8b in co-sensing and interaction with DcuS, and the isolated helix 8b showed strong interaction with DcuS. In DcuS, deletion and mutation of the cytoplasmic PAS(C) domain affected the interaction between DctA and DcuS. It is concluded that DctA forms a functional unit or sensor complex with DcuS through specific interaction sites.
Institute for Microbiology and Wine Research, University of Mainz, Germany.
Julian
J
Witan
Julia
J
Bauer
Ilka
I
Wittig
Philipp Aloysius
PA
Steinmetz
Wolfgang
W
Erker
Gottfried
G
Unden
D029382:Dicarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D043902:Pyrococcus horikoshii / Q000235:genetics / Q000378:metabolism
D029382:Dicarboxylic Acid Transporters; D029968:Escherichia coli Proteins; D011494:Protein Kinases; C469359:DcuS protein, E coli