Delected Mutant Construction Of YplQ And YtjAa Gene In Bacillus Subtilis 224 And Influence On Hemolysis

Bacillus subtilis 224 was a non-pathogenic Bacillus that separated from human body. Accord- ing to the Microecology principle and the antagonism among the microorganism, White Swan aerosol made of live BS224 was a effective medicine for burn external using, it could treat infected burn wounds and promote wound healing. But because of hemolysis, it has been stopped using in medicine now.Up to now, no investigation was performed on the Bacillus subtilis 224 genome sequencing, but Bacillus subtilis 168 genome sequencing has been sure, eight genes were possibe related to hemolysis. yplQ(642bp),ytjAα(228bp)were two of them, and expression product of ytjAαwas similar to alpha-hemolysin.Here, yplQ and ytjAαDNA fragments were amplified by PCR with chromosomal DNA of BS224 as the template and inserted into cloning vector pMD18-T to construct the recombinant plasmids pMD18-T-yplQ,pMD18-T-ytjAα. The primers of PCR amplification reaction were designed according to the genome sequence of Bacillus subtilis 168. The recombinant plasmids were transformed into competent cells of E.coli JM109. Positive clones were screened from the LB solid medium plates including Ampicillin. The positive recombinants were identified by restriction enzyme digest and finally sequenced. The results suggested that the homology between the nucleotide sequence of the cloned yplQ,ytjAαand the reported ones were separately 99.8%,99.4%. So it could be sure that the cloning of yplQ and ytjAαgenes is right in the genome of BS224.An integration plasmid pMD18-T-neo-yplQ for the yplQ gene disruption in Bacillus subtilis 224 was constructed. The plasmid contained upstream region of the yplQ gene 1.0kb and the yplQ gene downstream 0.5kb homologous fragments with insertion of the expression unit of the neom- ycin resistance gene between them. Upstream and downstream DNA fragments were synthesized by PCR from Bacillus subtilis 224 chromosomal DNA. The constructed gene targeting vector was linearized with HindⅢ, and electroporated into competent cells of BS224. Positive transformers were screened from the 50μg·ml-1 neomycin resistance plates. 36 transformers were analyzed by PCR to identify yplQ gene deletion.An integration plasmid pMD18-T-neo-ytjAαfor the ytjAαgene disruption in Bacillus subtilis 224 was constructed. The plasmid contained the ytjAαgene downstream 0.5kb and the ytjAαgene upstream 0.4kb homologous fragments with insertion of the expression unit of the neomycin resistance gene between them. Upstream and downstream DNA fragments were synthesized by PCR from Bacillus subtilis 224 chromosomal DNA. The constructed gene targeting vector was linearized with PstⅠ,EcoRⅠ, and electroporated into competent cells of BS224. Positive transformers were screened from the 50μg·ml-1 neomycin resistance plates. 12 transformers were analyzed by PCR to identify ytjAαgene deletion.Hemolysis of yplQ and ytjAαgene deleted mutants were separately identified on 5% sheep blood agar plates. The results suggested that the two genes deleted mutants could all induce hemolysis. It indicated that knockout of single gene had no influence or no main influence on hemolysis of BS224.Based on knockouting of single gene yqhB, yrkA, yugS, yhdT, yhdP had no influence on hemolysis of BS224, the test was futher study on Bacillus subtilis 224 other hemolytic genes. yplQ and ytjAαgenes deleted mutants were constructed by homologous recombination and their hemolysis were identified to make sure hemolytic genes or main genes inducing hemolysis, and one key step was forward taken for finally determines on Bacillus subtilis 224 hemolytic genes and studies on hemolysis.