Involvement of many chemotaxis sensors in negative chemotaxis to ethanol in Ralstonia pseudosolanacearum Ps29.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.