Identification of 3‐hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum

Abstract

SummaryExpression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is co-ordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wildtype strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at Յ1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum. Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.

A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection

“…3b). Transcription from the eps promoter -which remained high at all stages -slowly and steadily increased over time, likely due to accumulation of the inducing molecule 3-OH-PAME (Flavier et al, 1997). In contrast, PhrpB transcription showed a biphasic pattern: it increased dramatically during exponential growth and rapidly decreased when bacteria reached the stationary phase of growth.…”

“…We prove here that this is a reliable system to measure bacterial promoter activity during plant colonization. Up to now, expression studies in R. solanacearum were semi-quantitative or used long-lived reporters such as lacZ (Flavier et al, 1997;Kang et al, 1999). In our system, luminescence was shown to be efficient, sensitive and extremely easy to use, providing quantitative and qualitative information in real time.…”

Global regulation of pathogenicity mechanism of Ralstonia solanacearum

“…At the center of this network is PhcA, a LysR family transcriptional regulator (Brumbley et al 1993), which, directly or through intermediary regulatory genes, coordinates the expression of several virulence factors such as EPS and various CWDEs (Huang et al 1995). Active PhcA is regulated in response to cell density by a quorum-sensing mechanism that involves the specific autoinducer molecule 3-hydroxy palmitic acid methyl ester (3-OH PAME) (Flavier et al 1997). At a low cell density, presumably corresponding to saprophytic life and early plant colonization, PhcA is not expressed in culture, leading to expression of early disease virulence factors, including several polygalacturonases and both twitching and swimming motility (Liu et al 2001;Kang et al 2002;Tans-Kersten et al 2004).…”

The Ralstonia solanacearum pathogenicity regulator HrpB induces 3-hydroxy-oxindole synthesis

“…The increased production of HDF in the presence tryptophan might thereby be of relevance during plant infection, where the amino acid might become available to the pathogen from the host (29). The production of a probably relatively high local concentration of HDF that we expect in situ is in line with a role of interfering with quorum-sensing circuits of coexisting bacteria rather than acting on the endogenous ones (22,23). Because V. fischeri, the LuxR receptor of which is so far the only protein that we found inducible by HDF is not expected to coexist in the same habitat as R. solanacearum, it will be interesting to test other plant-associated bacteria that might profit from the pathogen's plant attack during infection for luxR-mediated HDF effects and phenotypes during plant infections.…”

“…Besides these classical acyl-HSL molecules, R. solanacearum was shown to produce 3-hydroxypalmitic acid methyl ester (3-OH PAME) as a molecule involved in cell-cell signaling (23). To investigate whether HDF acts on its endogenous quorum-sensing systems, we quantified acyl-HSL and 3-OH PAME, respectively, under various conditions.…”