Quorum sensing inhibition through site-directed mutation by deletion PCR

Quorum sensing induces biofilms and virulence factors that are adverse industrially and medically. Nowadays, quorum sensing inhibitions focus on signal analogs or signal degradation, but these methods have several downsides, which are temporal and affected by several environmental factors. In this research, we used deletion PCR to perform site-directed mutagenesis on the quorum sensing pathway gene and then analyzed its effects on quorum sensing. Serratia fonticola DSM 4576 strain was utilized as the research strain, and the gram-negative bacteria's universal quorum sensing pathway, which is conducted by acyl-homoserine lactone (AHL), was analyzed. The structure and active site of the AHL synthase enzyme encoded by S. fonticola DSM 4576's luxI-type gene were predicted. The gene's partial section solely encodes the enzyme's active site. By using sequence and ligation-independent cloning, the obtained mutagenic gene was cloned into the suicide vector pEX18Ap. The recombinant vector was used to transform wild-type S. fonticola DSM 4576 strains, and the mutants were determined through two-step selections and PCR genotyping. The gene expression level and biofilm formation were quantitatively analyzed through RT-PCR and biofilm assay, and no significant difference was noted in the gene expression between wild types and mutants. However, when mutants were compared to wildtypes, there was a significant decrease in biofilm formation as a result of quorum sensing induced bioreaction. Thus, we propose a quorum sensing inhibitory technique based on enzyme mutation on the quorum sensing pathway, and we proved the feasibility of enzyme active site's site-directed mutation through deletion PCR.