The molecular genetic analysis of the C₄-dicarboxylate sensor (DcuS) : part of a two component sensor-regulator system (DcuSR) in Escherichia coli

• Main Author: Al-Harbi, Khaled B. J.
• Other Authors: Andrews, Simon C.
• Published: University of Reading 2008
• Subjects: 579.342135
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502486

In E. coli, DcuSR controls gene expression in response to C4-dicarboxylates, including the C4-dicarboxylate carriers DcuB and DctA. The DcuS sensor has a periplasmic PASp domain sensing extracellular C4-dicarboxylates, a cytoplasmic PASc domain of unknown function and a His-kinase domain autophosphorylating during the signaling process. To understand the role of the PASc domain, it was subjected to site-directed mutagenesis and the effects of the mutations on DcuSRdependent regulation determined. 4 site-directed alterations within the PASc domain reduced induction of dcuB and dctA in response to fumarate indicating an essential role in sensing fumarate and/or in transmitting the signal from PASp to the kinase domain. 2 alterations were unable to activate either dcuB or dctA showing an involvement of the PASc domain in the intramolecular signalling pathway of DcuS or preventing protein assembly resulting in inactive DcuS. This was clearly seen when DcuS-E293A had a negative complementation effect on dcuB in a wildtype background. The alterations affected the DcuSR-dependent regulatory response in a manner that was partly reliant on the presence or absence of oxygen suggesting that the PASc domain may sense redox status and adjust the activity of DcuS in response to fumarate, accordingly. The use of the MalE-DcuS constructs showed no complementation effect on dcuB and dctA in the dcuS strain and a clear negative complementation effect in the wildtype strain indicating that the periplasmic domain is required for high kinase activity or significant phosphor-transfer to DcuR. The negative complementati the PASc and Kinase domains indicates that these domains associate in vivo. This is suggestive of inactivation of wildtype DcuS through heterocomplex with the s oteins. The PASc-kinase and PASc domains of DcuS were purified as MBP yielding pure protein, 400 and 5 respectively.