The biochemical characterization of a novel quorum quenching lactonase from E.Amylovora plant pathogen

Quorum Sensing (QS) is a population density-dependent bacterial regulatory communication mechanism that involves the production, detection, and response to extracellular signaling molecules called autoinducer (AIs). Many bacterial pathogens express their virulence factors through this mechanism. Among gram-negative bacteria, AHLs (N-acyl homoserine lactones) are the most common autoinducers. AHL lactonases, named quorum quenching (QQ) lactonases, catalyze the opening of the AHL ring and thus hydrolyze the lactones. One such AHL lactonase family belongs to the metallo-β-lactamase (MBL) superfamily; containing unique αββα fold with two Zn⁺² ions in their active site, needed for hydrolysis. While several hypotheses prevail regarding their physiological role, it`s still not clear and appears to be species-dependent. We hypothesized that by characterizing their specificity spectrum, we can gain insights regarding their role. Erwinia Amylovora is a gram-negative bacteria and the cause of the “Fire Blight” disease, uses QS to regulate its virulence factor expression. Using sequence and phylogenetic analyses, we have identified a new QQ lactonase gene within the Erwinia Amylovora (dubbed as EaAiiA). We have characterized EaAiiA`s biochemical properties as well as its specificity spectrum, with the aim of determining its sequence and structural determinates towards the understanding of its physiological role. The biochemical characterization indicates that EaAiiA is a metal-dependent lactonase with the highest activity (k_cat values) towards E. Amylovora secreted AHL, while higher affinity (K_M values) was observed towards AHLs secreted by other bacteria. These results suggested that this enzyme is involved in QS self-regulation as well as quenching the signaling of other species, and can be used as quorum quenching lactonase towards other bacterial pathogens.