3-hydroxy Palmitic acid methyl ester (3-hydroxy PAME) is an esterized long-chain fatty acid involved in quorum sensing in R. solanacearum, a bacteria that causes lethal wilting in plants. 3-hydroxy-PAME (175 nM) increases levels of PhcA-regulated virulence factors, greater than 20-, 30-, and 25-fold for EPS I, EGL, and PME, respectively, in the AW1-83 strain of R. solanacearum
Autoinducers are indispensable for bacterial cell-cell communication. However, due to the reliance on culture-based techniques, few autoinducer-hydrolyzing enzymes are known. In this study, we characterized soil metagenome-derived unique enzymes capable of hydrolyzing 3-hydroxypalmitic acid methyl ester (3-OH PAME), an autoinducer of the plant pathogenic bacterium Ralstonia solanacearum. Among 146 candidate lipolytic clones from a soil metagenome library, 4 unique enzymes capable of hydrolyzing the autoinducer 3-OH PAME, termed ELP86, ELP96, ELP104, and EstDL33, were selected and characterized. Phylogenetic analysis revealed that metagenomic enzymes were novel esterase/lipase candidates as they clustered as novel subfamilies of family I, V, X, and family XI. The purified enzymes displayed various levels of hydrolytic activities towards 3-OH PAME with optimum activity at 40-50 degrees C and pH 7-10. Interestingly, ELP104 also displayed N-(3-oxohexanoyl)-L-homoserine lactone hydrolysis activity. Heterologous expression of the gene encoding 3-OH PAME hydrolase in R. solanacearum significantly decreased exopolysaccharide production without affecting bacterial growth. mRNA transcription analysis revealed that genes regulated by quorum-sensing, such as phcA and xpsR, were significantly down-regulated in the stationary growth phase of R. solanacearum. Therefore, metagenomic enzymes are capable of quorum-quenching by hydrolyzing the autoinducer 3-OH PAME, which could be used as a biocontrol strategy against bacterial wilt.
        
Title: Characterization of bacteria degrading 3-hydroxy palmitic acid methyl ester (3OH-PAME), a quorum sensing molecule of Ralstonia solanacearum Achari GA, Ramesh R Ref: Lett Appl Microbiol, 60:447, 2015 : PubMed
UNLABELLED: Bacterial wilt pathogen Ralstonia solanacearum causes severe crop loss of eggplant, which is of economic importance in India. 3-hydroxy palmitic acid methyl ester (3OH-PAME) is the main quorum sensing molecule governing the expression of virulence factors in R. solanacearum. Ability of 164 bacterial isolates from the xylem of eggplant (Solanum melongena L.), chilli pepper (Capsicum annuum L.) and wild eggplant (Solanum torvum Sw.) to degrade 3OH-PAME was tested by disc diffusion assay. Enzymatic degradation of 3OH-PAME by five bacteria was confirmed by High-Performance Liquid Chromatography-Mass Spectrometry analysis. 3OH-PAME degrading bacteria were identified as Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Rhodococcus corynebacterioides. 3OH-PAME degrading bacteria reduced the expression of virulence factors (exopolysaccharides and endoglucanase) of R. solanacearum in vitro and reduced wilt incidence in eggplant seedlings under greenhouse conditions. Isolates with quorum quenching activity successfully re-colonized eggplant seedlings. Quorum quenching bacteria produced antagonistic compounds, which may act synergistically with quorum quenching in reducing bacterial wilt in eggplant. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on endophytic bacteria of class Gammaproteobacteria and phylum Actinobacteria having 3OH-PAME degrading activity. This study demonstrates the potential use of endophytic bacteria as quorum quenching biocontrol agents for management of bacterial wilt in eggplant.
        
Title: Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum Flavier AB, Clough SJ, Schell MA, Denny TP Ref: Molecular Microbiology, 26:251, 1997 : PubMed
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.