Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 03 Jan 2019 08:16:15 Identification and characterization of chemosensors for D- malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum Mattana Tunchai, Akiko Hida, Shota Oku, Yutaka Nakashimada, Takahisa Tajima and Junichi Kato* Abstract Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable D-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for D-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to D-malate, suggesting the existence of another D-malate chemosensor. McpM previously had been identified as a chemosensor for L- malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to D-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for D-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to L-tartrate and D-malate and moderately to D-tartrate, but not to L-malate or succinate. K18 harbouring mcpM responded strongly to L-malate and D-tartrate and moderately to succinate, fumarate and D-malate. Ps29 utilizes L-malate and L-tartrate, but not D-malate. We therefore concluded that L-tartrate and L-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward D-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild- type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection. INTRODUCTION Chemotaxis is one of the most important behaviours in bac- teria, allowing these organisms to sense environmental changes and appropriately respond to such changes [1]. Because most chemotactic attractants are growth substrates [2–5], chemotaxis is believed to assist bacteria in efficiently moving toward environments suitable for growth. Bacterial chemotaxis can be also viewed as an important prelude to ecological interactions such as symbiosis, infection, root col- onization and metabolism [6]. The molecular mechanisms that underlie bacterial chemotaxis have been studied inten- sively in Escherichia coli and Salmonella enterica serovar Typhimurium [7, 8]. Chemotactic ligands are detected by cell surface chemoreceptors called methyl-accepting chemo- taxis proteins (MCPs). Upon binding a chemotactic ligand, an MCP generates chemotaxis signals that are communi- cated to the flagellar motor via a series of chemotaxis (Che) proteins. E. coli possesses five MCPs and six Che proteins (CheA, CheB, CheR, CheW, CheY and CheZ). Ralstonia solanacearum is a Gram-negative and motile plant pathogenic bacterium that causes bacterial wilt in economi- cally important crops, including tomato, potato, eggplant, tobacco and banana [9, 10]. This soil-borne bacterium usu- ally enters plant roots through wounds, root tips and sec- ondary root emergence points, from which the organism invades the xylem vessels and spreads to the aerial parts [11]. R. solanacearum is a heterogeneous species and termed as ‘the R. solanacearum species complex’ [12, 13]. The R. solanacearum species complex can be subdivided into four phylotypes [14]. Safni et al. [15] have proposed to emend the description of R. solanacearum and reclassify current R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis and current R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum. By this Received 30 August 2016; Accepted 1 December 2016 Author affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi- Hiroshima, Hiroshima 739-8530, Japan. *Correspondence: Junichi Kato, jun@hiroshima-u.ac.jp Keywords: chemotaxis; D-malate; Ralstonia pseudosolanacearum; methyl-accepting chemotaxis protein; plant infection; bacterial wilt disease. Abbreviation: MCP, methyl-accepting chemotaxis protein. The GenBank/EMBL/DDBJ accession number for the mcpT gene sequence of Ralstonia pseudosolanacearum MAFF106611 is KX537646. Two supplementary figures are available with the online Supplementary Material. RESEARCH ARTICLE Tunchai et al., Microbiology 2017;163:233–242 DOI 10.1099/mic.0.000408 000408 ã 2017 The Authors 233