Construction Of Gene Knockout System For Paenibacillus Polymyxa SC2

Paenibacillus polymyxa SC2is a broad-spectrum antagonistic bacterium that wasscreened from the rhizosphere of pepper. The strain has been widely used in agriculturalindustry because of its ability to promote plant growth and solubilize the insoluble mineralelements phosphorus and potassium. We have completed the whole genome sequencing of P.polymyxa SC2. To explore bio-control related genes function and their molecular mechanism,we constructed the gene knockout system for P. polymyxa SC2.Based on determination of SC2growth curve and research on sporulation law, we knewthat SC2cultured for5-6h is logarithmic phase before mid and not forming spores. Weprepared efficient competent cell using SC2which was cultured for5-6h by the method ofBacillus subtilis. E. coli-B. subtilis shuttle plasmid pHY300PLK was transformed into SC2by electrotransformation and its transformation frequency was high. So it can be used fordetecting the activity of SC2competent cell and selecting suitable resistance gene for SC2.We constructed recombinant plasmids pHY300PLK-Km and pHY300PLK-Cm andtransformed them into P. polymyxa SC2seperately. Kanamycin (Km) cassette could notexpress in SC2, chloroamphenicol (Cm) cassette could express in SC2. We screenedrecombinants using Cm cassette in15μg/mL.pRN5101, a temperature sensitive plasmid, could suicide upon35C. It was used for geneknockout in low-GC-content, gram-positive bacteria such as Bacillus cereus and Bacillusthuringiensis. In theory, pRN5101could be used for P. polymyxa. This study proved thatpRN5101could be transformed into SC2by electrotransformation and suicided under40C.So it was selected as gene knockout plasmid for P. polymyxa SC2. We confirmed theavailability of SC2gene knockout system by knocking out the key polymyxinsynthesis-related gene pmxE. We selected the key polymyxin synthesis-related gene pmxE astarget gene and amplification specific fragment of pmxE. Cm cassette was inserted to pmxEfragment by MluI and BglII restriction enzyme digestion and T4DNA ligase. PmxE fragmentdisrupted by Cm cassette was connected with pRN5101by Hind III and BamH I restrictionenzyme digestion sites and formed gene knockout pRN5101-EC. We transformedpRN5101-EC into SC2and screened pmxE mutant. Three recombinants were screened bycolony PCR. Antibacterial activity assay and high performance liquid chromatography (HPLC) confirmed recombinants lose the ability of synthesizing polymyxin. PmxE gene wasknocked out and this gene knock-out system is available. The gene knock-out system for P.polymyxa SC2was constructed.The research constructs gene knock-out system for P. polymyxa for the first time. It is anessential tool for studying functional genomics of P. polymyxa. We can investigate molecularmechanism of bio-control related genes in P. polymyxa SC2. However，we still need to paymore attention on improving the electro-transformation efficiency and transformants geneticstability in future.