Soil metagenome-derived 3-hydroxypalmitic acid methyl ester hydrolases suppress extracellular polysaccharide production in Ralstonia 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 bio-control strategy against bacterial wilt.