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.

Philipp Aloysius Steinmetz, Sebastian Wörner, Gottfried Unden

Molecular microbiology 2014 Oct

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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. © 2014 John Wiley & Sons Ltd.

Citation

Philipp Aloysius Steinmetz, Sebastian Wörner, Gottfried Unden. 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. Molecular microbiology. 2014 Oct;94(1):218-29