Cellular Localization of Predicted Transmembrane and Soluble Chemoreceptors in Sinorhizobium meliloti

Bacterial chemoreceptors primarily locate in clusters at the cell pole, where they form large sensory complexes which recruit cytoplasmic components of the signaling pathway. The genome of the soil bacterium Sinorhizobium meliloti encodes seven transmembrane and two soluble chemoreceptors. We have investigated the localization of all nine chemoreceptors in vivo using genome-encoded fusions to a variant of the enhanced green fluorescent protein and to monomeric red fluorescent protein. Six of the transmembrane (McpT to McpX and McpZ) and both soluble (McpY and IcpA) receptors localize to the cell pole. Only McpS, encoded from the symbiotic plasmid pSymA, is evenly distributed in the cell. While the synthesis of all polar localized receptors is confined to exponential growth correlating with the motility phase of cells, McpS is only weakly expressed throughout cell culture growth. Therefore, motile S. meliloti cells form one major chemotaxis cluster that harbors all chemoreceptors except for McpS. Colocalization and deletion analysis demonstrated that formation of polar foci by the majority of receptors is dependent on other chemoreceptors and that receptor clusters are stabilized by the presence of the chemotaxis proteins CheA and CheW. The transmembrane McpV and the soluble IcpA localize to the pole independently of CheA and CheW. However, in mutant strains McpV formed delocalized polar caps that spread throughout the cell membrane while IcpA exhibited increased bipolarity. Immunoblotting of fractionated cells revealed that IcpA, which lacks any hydrophobic domains, nevertheless is associated to the cell membrane.The chemosensory machinery of Escherichia coli and other bacteria is arranged in large protein clusters (22, 28, 43, 49). One individual signaling unit is formed by a ternary assembly of chemoreceptor dimers, the histidine kinase CheA, and the so-called adaptor protein CheW. E. coli cells contain 20,000 receptor molecules (22). Recent studies suggest that the stoichiometry of such chemosensory complexes is flexible (17, 32). Allosteric interactions among receptors in a chemosensory cluster facilitate amplification and integration of chemotactic stimuli (20, 21, 41, 42).In contrast to E. coli, which has a single set of che genes and only five receptors, some species from the alpha subgroup of the proteobacteria, such as Pseudomonas aeruginosa, Rhodobacter sphaeroides, and Sinorhizobium meliloti, encode multiple chemotaxis-like systems, reflecting their complex lifestyle. The opportunistic pathogen P. aeruginosa possesses four chemotaxis systems that together have 26 known receptor genes (47), while the nonsulfur bacterium R. sphaeroides has three separate che operons with 13 known receptor-like genes (27).The symbiotic soil bacterium S. meliloti possesses eight methyl-accepting chemotaxis proteins (MCPs), McpS to McpZ, and one transducer-like-protein, IcpA, which lacks the conserved Glu or Gln residues that serve as methyl-accepting sites (29). Seven of the MCP proteins are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The S. meliloti mcpS gene is the third gene of the che2 operon located on the symbiotic plasmid pSymA (4). The icpA gene is the first gene of the chromosomal che operon comprising a total of 10 genes (9). This operon is part of the flagellar gene cluster with 56 chemotaxis, motor, and flagellar genes residing on one contiguous 51.4-kb chromosomal region (7, 46). For bacteria with numerous chemoreceptor genes, it is not unusual to find most of them located outside chemotaxis operons. This is the case with six monocistronic S. meliloti mcp genes which are scattered throughout the genome. The remaining mcpW gene is cotranscribed with a putative cheW gene. In this study, we examined the localization of the nine receptor gene products in the S. meliloti cell by fluorescence microscopy in wild-type and various deletion strains. The cellular localization of the two soluble receptors, McpY and IcpA, was also analyzed in vitro using an immunoblot assay on fractionated cell components. Furthermore, timing of chemoreceptor gene expression during exponential and stationary phase was determined.