The chemotaxis signalling chain of Sinorhizobium meliloti is distinguished from the well-studied enterobacterial system in at least three features - reception, response regulation und motor function. The main focus of this proposal is the characterization of the initial step in the chemotactic process - perception and processing of chemoeffectors. S. meliloti possesses six transmembrane and two cytosolic receptors. We have shown that a strain deleted in all eight receptor genes was negatively affected to compete for nodulation with the wild type. The receptor(s) involved in the establishment of symbiosis will be identified by competitive nodulation assays of single knockout mutants accompanied by chemotaxis assays with root exsudates. Swimming speed and pattern of individual knockout strains will be compared to wild type as a measure for their chemotactic capability. The function of a putative second chemotaxis operon on the pSymA replicon will be analyzed in a similar way. We will examine the cellular localization of all receptors in S. meliloti-cells using GFP-fusions as reporter. Two receptors, McpY and IcpA, are of particular interest, since they are lacking a periplasmic sensing and transmembrane spanning domains. McpY contains two PAS-domains known to bind FAD, as well as a putative ATPase motif. We propose that IcpA and McpY might sense the metabolic status of the cell. We will analyze both cytosolic receptors in greater detail using isolated proteins for enzymatic studies.
