title: Differentiation of DctA and DcuS function in the DctA/DcuS sensor complex of Escherichia coli: function of DctA as an activity switch and of DcuS as the C4-dicarboxylate sensor.
abstract: The C4-dicarboxylate responsiveness of the sensor kinase DcuS is only provided in concert with C4-dicarboxylate transporters DctA or DcuB. The individual roles of DctA and DcuS for the function of the DctA/DcuS sensor complex were analysed. (i) Variant DctA(S380D) in the C4-dicarboxylate site of DctA conferred C4-dicarboxylate sensitivity to DcuS in the DctA/DcuS complex, but was deficient for transport and for growth on C4-dicarboxylates. Consequently transport activity of DctA is not required for its function in the sensor complex. (ii) Effectors like fumarate induced expression of DctA/DcuS-dependent reporter genes (dcuB-lacZ) and served as substrates of DctA, whereas citrate served only as an inducer of dcuB-lacZ without affecting DctA function. (iii) Induction of dcuB-lacZ by fumarate required 33-fold higher concentrations than for transport by DctA (Km  = 30 μM), demonstrating the existence of different fumarate sites for both processes. (iv) In titration experiments with increasing dctA expression levels, the effect of DctA on the C4-dicarboxylate sensitivity of DcuS was concentration dependent. The data uniformly show that C4-dicarboxylate sensing by DctA/DcuS resides in DcuS, and that DctA serves as an activity switch. Shifting of DcuS from the constitutive ON to the C4-dicarboxylate responsive state, required presence of DctA but not transport by DctA.
authors.affiliation: Institute for Microbiology and Wine Research, Johannes Gutenberg-University, 55099, Mainz, Germany.
authors.forename: Philipp Aloysius
authors.initials: PA
authors.lastname: Steinmetz
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wörner
authors.forename: Gottfried
authors.initials: G
authors.lastname: Unden
mesh_terms: D029382:Dicarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D003998:Dicarboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005650:Fumarates / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C000589721:DctA protein, E coli; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D029968:Escherichia coli Proteins; D005650:Fumarates; D011494:Protein Kinases; C469359:DcuS protein, E coli