Cloning and characterization of Aiia, an acylhomoserine lactonase from Bacillus cereus RC1 to control soft rot causing pathogen Lelliottia amnigena RCE.

Abstract

Bacterial pathogenesis-associated characteristics such as biofilm formation, synthesis of hydrolyzing enzymes, and toxins are regulated by Acyl Homoserine Lactones (AHLs), small peptides and diffusing signal factors (DSF). Lelliottia amnigena is gram negative bacteria and its pathogenicity is regulated by the luxR andluxIclass of quorum sensing. The signaling molecules and their concentrations are essential for the virulence of the pathogenic bacterium. To suppresses the pathogenicity; the concentration of signalling molecules must be controlled or degraded. The lactonase have the ability to hydrolyze lactonesof different chain length. The present study deals with a newer approach to control the pathogenesis of Lelliottia amnigena through isolation and characterization of Aiia lactonase from Bacillus cereus RC1. Aiia lactonase specific primers were used to amplify the gene, and the sequence thus obtained was submitted to the Genbank database under accession # OK643884.1. The gene was cloned in pBE-S shuttle vector and transformed in the recombinant host. The expressed and purified protein had a molecular weight of 28.00 KDa and exhibited its optimum activity at 37℃ by inhibiting the violacein pigment of the monitor strain Chromobacterium violaceum MTCC 2656. The proteinaceous nature of the purified molecule was confirmed by incubating it in the presence of proteinase K for 1h. The activity of the pathogenesis-related protein, polygalacturonase was drastically reduced in the presence of the purified Aiia protein. The purified protein also showed a zone of inhibition when plated together with Lelliottia amnigena RCE (MZ712952.1). Searches of the Conserved Domain Database suggested that this protein belonged to the Metallo-beta-lactamase superfamily and is closely related to Aiia from B. thuringiensis serovar kurstaki. Modeling of the protein structure was done using I-TASSER; a C-score of 0.55 suggested that the model was of good quality. To be used commercially, this recombinant protein needs to be purified at an industrial scale; it can then be used to repress the growth of soft rot causing bacteria in horticultural crops during their storage period.