IDENTIFICATION OF 3-HYDROXYPALMITIC ACID METHYL-ESTER AS A NOVEL AUTOREGULATOR CONTROLLING VIRULENCE IN RALSTONIA-SOLANACEARUM

Expression of virulence genes in Ralstonia solanacearum, a phytopathog enic bacterium, is controlled by a complex regulatory network that int egrates multiple signal inputs. Production of several virulence determ inants is co-ordinately reduced by inactivation of phcB, but is restor ed by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liq uid chromatography. Gas chromatography and mass spectroscopy identifie d 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major compone nt in the single peak of VEF activity. Authentic 3-OH PAME and the pur ified VEF were active at less than or equal to 1 nM, and had nearly eq uivalent specific activities for stimulating the expression of eps (th e biosynthetic locus for extracellular polysaccharide) in a phcB mutan t. Authentic 3-OH PAME also increased the production of three virulenc e factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased express ion of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylm ethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically activ e concentrations of extracellular 3-OH PAME were detected before the o nset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacea rum. Other than acyl-homoserine lactones, 3-OH PAME is the only endoge nous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-dist ance intercellular communication.