Sinorhizobium meliloti CheA Complexed with CheS Exhibits Enhanced Binding to CheY1, Resulting in Accelerated CheY1 Dephosphorylation

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 ␣ 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 ϳ100-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.

The domain of unknown function DUF1521 exhibits metal ion-inducible autocleavage activity - a novel example from a putative effector protein ofVibrio coralliilyticusATCC BAA-450

“…The full-length proteins studied here and the C-terminal cleavage product migrated significantly slower in an SDS polyacrylamide gel than expected from their predicted molecular weights. Highly charged proteins have been reported to exhibit migration anomalies in SDS gels (Graceffa et al, 1992;Hu & Ghabrial, 1995;Dogra et al, 2012). For example, b-lactamase of Actinomadura R39 appears as a c. 55 kDa protein after SDS-PAGE but has a calculated molecular weight of 29 kDa (Matagne et al, 1991).…”

Polarity of bacterial magnetotaxis is controlled by aerotaxis through a common sensory pathway

“…7). cheOp1 encodes a hybrid CheA kinase and no CheZ, suggesting signal termination is through a phosphate sink, as in S. meliloti 35 . Owing to the absence of an MCP gene in cheOp1 and the great number of putative MCP-encoding genes (Supplementary Table 3), an energy/oxygen sensor in M. gryphiswaldense has not been identified.…”

A cheZ -Like Gene in Azorhizobium caulinodans Is a Key Gene in the Control of Chemotaxis and Colonization of the Host Plant

“…For example, CheC and FliY, belonging to another class of phosphatases, are found in Bacillus subtilis, and CheX is found in spirochetes (7). The situation in Sinorhizobium meliloti, a member of Alphaproteobacteria, shows another level of complexity, because this bacterium possesses two genes encoding the CheY proteins, cheY1 and cheY2 (8,9). CheY2 is the actual response regulator, which once phosphorylated by CheAϳP, interacts with the flagellar motor.…”

“…Modulation of CheY2 phosphorylation is also achieved by CheA, which can retransfer the phosphoryl group to CheY1. Thus, CheY1 functions only as a sink for phosphoryl groups, and CheA together with another protein, CheS, can achieve dephosphorylation of CheY1 (8,9). In addition, other proteins such as CheV and hybrid CheA kinases, which have additional REC domains, may also play a role in CheYϳP dephosphorylation (10).…”