Abstract
Retrophosphorylation of the histidine kinase CheA in the chemosensory transduction chain is a widespread mechanism for efficient dephosphorylation of the activated response regulator. First discovered in Sinorhizobium meliloti, the main response regulator CheY2-P shuttles its phosphoryl group back to CheA, while a second response regulator, CheY1, serves as a sink for surplus phosphoryl groups from CheA-P. We have identified a new component in this phospho-relay system, a small 97-amino-acid protein named CheS. CheS has no counterpart in enteric bacteria but revealed distinct similarities to proteins of unknown function in other members of the α subgroup of proteobacteria. Deletion of cheS causes a phenotype similar to that of a cheY1 deletion strain. Fluorescence microscopy revealed that CheS is part of the polar chemosensory cluster and that its cellular localization is dependent on the presence of CheA. In vitro binding, as well as coexpression and copurification studies, gave evidence of CheA/CheS complex formation. Using limited proteolysis coupled with mass spectrometric analyses, we defined CheA(163-256) to be the CheS binding domain, which overlaps with the N-terminal part of the CheY2 binding domain (CheA(174-316)). Phosphotransfer experiments using isolated CheA-P showed that dephosphorylation of CheY1-P but not CheY2-P is increased in the presence of CheS. As determined by surface plasmon resonance spectroscopy, CheY1 binds ∼100-fold more strongly to CheA/CheS than to CheA. We propose that CheS facilitates signal termination by enhancing the interaction of CheY1 and CheA, thereby promoting CheY1-P dephosphorylation, which results in a more efficient drainage of the phosphate sink.