Composition and function of the DctA/DcuS sensor complex of E. coli

点击翻译按钮获取中文摘要

Two-component system consisting of a membrane-bound sensor kinase and a cytoplasmic response regulator represent the major devices of bacteria for sensing environmental stimuli. Many membrane-bound sensor kinases require accessory proteins or independent proteins like transporters for function. The molecular function of the transporters in controlling the sensors is not known. The sensor kinase DcuS (C4-dicarboxylate uptake sensor) of E. coli requires the transporters DctA or alternatively DcuB for function. In the absence of the transporters is DcuS permanent in the active state, that is even without the effector fumarate. The transporters thus transfer DcuS to the fumarate-responsive state. Preparatory work showed that (a) DctA forms a sensor complex with DcuS (DctA/DcuS), (b) DctA interacts by a cytoplasmic helix (H8b) with DcuS, (c) stimulus perception is effected only via DcuS but not by DctA in the complex, and (d ) DctA is no flux sensor since transport deficient variants still form functional DctA/DcuS sensor complexes. According to the present model of function, transforms DctA the sensor DcuS by structural changes to the competent form. Preparatory work showed that transmembrane signal transfer by DcuS relies on a piston-type translocation of transmembrane helix 2 (TM2). TM2 is in the periplasmic position when it is in the activated state and in the cytoplasmic position when it is in the inactive state. Methods for differentiation both positions have been developed.After establishing a system for studying carrier-controlled sensor kinases, (i) the composition of the DctA/DcuS complex and the site of DctA-DcuS interaction, and (ii) the effect of DctA (or DcuB) on structure and function of DcuS shall be characterized. The DctA/DcuS complex was not isolated so far. For (i), the composition of the complex shall be studied by new procedures of mass spectrometry, defined cross-linking of DctA with DcuS and identifying of the interacting peptides. For studies of the mechanism of interaction, the interacting areas and domains of DcuS and DctA will be mapped. For (ii), the position of TM2 of DcuS will be determined in variants of E. coli lacking either DctA or DcuB, or contain only regulatory deficient variants of DcuB. This will show if DctA (or DcuB) control the function of DcuS by affecting the position of TM2 and shift it to the ground state as predicted by the actual working model. The experiments will be performed in part by DcuB in addition to DctA since some experiments can be done in a more clear-cut manner with DcuB, and since well-defined regulation deficient variants of DcuB (but not of DctA) are available.