Differentiation of DctA and DcuS function in the DctA / DcuS sensor complex of E scherichia coli : function of DctA as an activity switch and of DcuS as the C 4 ‐dicarboxylate sensor

Summary The C 4 ‐dicarboxylate responsiveness of the sensor kinase DcuS is only provided in concert with C 4 ‐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 C 4 ‐dicarboxylate site of DctA conferred C 4 ‐dicarboxylate sensitivity to DcuS in the DctA / DcuS complex, but was deficient for transport and for growth on C 4 ‐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 ( K m  = 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 C 4 ‐dicarboxylate sensitivity of DcuS was concentration dependent. The data uniformly show that C 4 ‐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 C 4 ‐dicarboxylate responsive state, required presence of DctA but not transport by DctA .