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Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia
A B Flavier1, S J Clough, M A Schell
1Department of Plant Pathology, University of Georgia, Athens 30602, USA.
Abstract:
Expression 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 coordinately reduced by inactivation of phcB, but is restored 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 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 < or =1 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.
Insights
A volatile extracellular factor (VEF) from Ralstonia solanacearum was identified as 3-hydroxypalmitic acid methyl ester (3-OH PAME). This molecule regulates virulence gene expression and may represent a new class of bacterial communication signals.
Area of Science:
- Microbiology
- Plant Pathology
- Biochemistry
Background:
- Bacterial virulence gene expression is tightly regulated by complex networks.
- Ralstonia solanacearum virulence is impaired in phcB mutants.
- A volatile extracellular factor (VEF) restores virulence in phcB mutants.
Purpose of the Study:
- To identify and characterize the VEF involved in regulating Ralstonia solanacearum virulence.
- To elucidate the role of VEF in intercellular communication and virulence gene expression.
Main Methods:
- Purification of VEF from bacterial culture supernatant using distillation, solvent extraction, and chromatography.
- Identification of VEF using gas chromatography and mass spectrometry.
- Assay of VEF and synthetic 3-hydroxypalmitic acid methyl ester (3-OH PAME) activity on virulence gene expression in R. solanacearum mutants.
Main Results:
- 3-hydroxypalmitic acid methyl ester (3-OH PAME) was identified as the major active component of VEF.
- 3-OH PAME restored virulence gene expression, including extracellular polysaccharide (eps) production, in phcB mutants at nanomolar concentrations.
- 3-OH PAME acts as an intercellular signal, detected before virulence gene expression, suggesting autoregulation.
Conclusions:
- 3-OH PAME is a novel, long-distance intercellular communication signal in Ralstonia solanacearum.
- This fatty acid derivative may represent a new class of signaling molecules mediating bacterial communication.
- The PhcB protein is potentially involved in the synthesis of 3-OH PAME.