Expression And Characterization Of AiiA Genes From Bacillus Subtilis BS-2

Quorum sensing is a mechanism in which bacteria coordinate the expression of certain genes in response to their population density by producing, releasing and detecting signal molecules called autoinducers. Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. AiiA is a protein which can block the bacterial quorum sensing by hydrolyzing AHL-lactone, and could greatly attenuate the disease caused by many bacterial pathogens in which quorum sensing regulate the expression of virulence genes. In this study, a pair of primers were designed from the conserved sequences of aiiA gene in the genomes of several Bacillus strains, and the gene was amplified from Bacillus subtilis BS-2 by PCR. AiiA gene was cloned to a cloning vector pMD18-T and sequenced. The open reading frame of the aiiA gene was 753 bp, and the similarity of aiiA gene from B. subtilis BS-2 to other sequences in the GenBank was as high as 95%. This gene was subcloned into an expression vector pET29a, and the recombinant AiiA protein was overexpressed in Escherichia coli BL21 (DE3). The molecular weight of the expressed AiiA protein was estimated to be 28 kDa by SDS - PAGE. The lysate of the recombinant E. coli could not only repress the pigment synthesize of the quorum sensing system reporter strain JAZ1, but also attenuate the effect of soft rot symptoms. This study was important for further research on the disruption of infections caused by Gram-negative bacteria in agriculture.