Abstract
Sinorhizobium meliloti is a soil bacterium that can establish beneficial symbiosis with legume plants. The fadD gene encodes a long-chain fatty acyl-coenzyme A (CoA) synthetase. Inactivation of FadD in S. meliloti leads to a pleiotropic phenotype, including the overproduction of several volatile methylketones (MKs). One of them, 2-tridecanone (2-TDC), was found to act as an infochemical that affects important bacterial traits and hampers plant-bacteria interactions. Knowledge about bacterial genes involved in MK production is limited. In wild tomato species, MK synthesis requires intermediates of fatty acid biosynthesis and the activity of the methylketone synthase 2 (MKS2), a thioesterase belonging to the hotdog-fold family. In this study, we have identified SMc03960, a conserved hypothetical protein with homology to bacterial YbgC-like thioesterases, as an ortholog of MKS2 in S. meliloti. Heterologous expression of smc03960 in Escherichia coli results in the formation of several MKs, including 2-TDC, and causes the accumulation of free fatty acids. Purified His-SMc03960 showed thioesterase activity for different acyl groups linked either to acyl carrier protein (ACP) or to CoA with preference for C14-long substrates. Moreover, formation of 2-TDC in vitro was achieved by using His-SMc03960 and 3-oxo-myristoyl-ACP. Although deletion of smc03960 in the wild type or in the fadD mutant does not significantly alter the amount of MKs released by S. meliloti, overexpression of the gene results in increased production of 2-TDC in these two strains. Overall, our data demonstrate that SMc03960 is an acyl-ACP/acyl-CoA thioesterase with broad substrate specificity that contributes to 2-TDC formation.