Coloning And Related Analysis Of Quorum-sensing Gene Cluster In Bacillus Amyloliquefaciens PEBA20

In this study, we cloned the genes about Quorum Sensing of Bacillus amyloliquefaciens PEBA20, and make the bioinformatics analysis, in order to understand the kind of intraspecific evolution in Bacillus. We got the mutant strain △comA using the method of homologous recombination. We found the deletion of the gene comA not only affected the functions of quorum sensing, but also lost the ability of antibacterial activity, biofilm formation.The results were as follows:1. We have successfully cloned the gene cluster comQXPA of quorum sensing in Bacillus amyloliquefaciens PEBA20. So, we made the sequence alignment analysis of comXQPA throughout the genus Bacillus. We found that the conservation of the genes followed the same order: comA > comP > comQ > comX, and comX showed the highest within-clade variability.2. We created mutant strain △comA. We connected the fragments of comAF and comAB cloned from B. amyloliquefaciens PEBA20 using DNA ligase. We have obtained recombinant suicide plasmid of comAF-comAB-pk18 mobsacB and transformed it into PEBA20. After homologous recombination, to verify that plasmid integration occurred at the locus, this region was amplified by PCR using genomic DNA isolated from Kan as the template and flanking primers comAF1 and comAB2.3. We also detected the ability of biofilm formation of mutant strain △comA. We observed that △comA mutant formed as the small white colony firstly, it had smooth surface without fold. Then the edge of colony became radical, while a white circle with evident boundaries. With the time went on, colony margins expanded significantly faster than the smaller circle inside; then the colony stopped expanding, the edge of colony showed obvious differentiation. In 12 hours, the interface of air- liquid appear turbid, when it was 24 h, it began to appear a thin pellicle, but interestingly, the pellicle became to be a state of degradation. From the persistent observation, it showed that △comA mutant can’t form a complete biofilm.4. Finally, we detected the ability of antibacterial activity of mutant strain △ comA. Compared the wild type of B. amyloliquefaciens PEBA20, we observed that antibacterial activity of mutant strain △comA was decreased, even had little inhibitory effect on some fungus. And therefore, deletion of the gene comA affected ability of antibacterial activity of B. amyloliquefaciens PEBA20.