Knockout Of YhdP,yugS,yhdT And YqhB Gene In Bacillus Subtilis 224 And Influence On Hemolysis

Bacillus subtilis 224 was separated from human body and it was non-pathogenic. BS224 wasnot only widely used as engineer strain, but also could diminish inflammation, resist germs and cureburn and scald wounds. On the grounds of the Microecology principle, Yi jun sheng and WhiteSwan aerosol made of live BS224 could improve Microecology environment of wounds, resistgerms, diminish inflammation and improve wound healing by antagonistic action among microbe.But they impaired wounds in a way because of hemolysis, when they were used for curing burn andscald wounds. Therefore their uses was much limited.Since Bacillus subtilis 168 genome sequencing was finished, eight genes possibly related tohemolysis have been found. They were yqhB (1329bp) ,yrkA (1305bp) ,yugS (1305bp) ,yhdT(1386bp) ,yhdP (1335bp), yqxC (810bp), yplQ (642bp) and ytjAα(228bp), the homologyof the fore five genes was 69.83 %, and expression product of ytjAαwas similar to alpha-hemolysin.The finding of unconfirmed hemolytic genes made it possible that hemolytic genes and molecularmechanism of hemolysis were ascertained.Here, yhdP, yugS, yhdT and yqhB DNA fragments were amplified by PCR with chromosomalDNA of BS224 as the template and inserted into cloning vector pMD18-T to construct therecombinant plasmid pMD 18-T-yhdP, pMD 18-T- yugS, pMD 18-T-yhdT and pMD 18-T-yqhB. Theprimers of PCR amplification reaction were designed according to the genome sequence of Bacillussubtilis 168. The recombinant plasmids were transformed into competent cells of E.coli JM109.Positive clones were screened from the LB solid medium plate including Ampicillin, IPTG andX-gal. The positive recombinants were identified by restriction enzyme digest and finallysequenced .The results suggested that the homology between the nucleotide sequence of the clonedyhdP, yugS, yhdT and yqhB and the reported one were separately 99.9%, 99.6%, 99.9% and 99.9%.So it could be made sure that yhdP, yugS, yhdT and yqhB existed in the genome of BS224.To construct skeleton plasmid of targeting vector, the neomycin resistance gene includingself-promoter were amplified by PCR with pEGFP-N1 plasmid DNA as the template . DNApolymerase needed in the reaction was high fidelity Ex Taq and primers were designed according tothe sequence of pEGFP-N1. The amplified DNA fragments were inserted into cloning vectorpMD18-T. The positive recombinants were identified by restriction enzyme digest and finallysequenced. The results suggested that the nucleotide sequence of the cloned neomycin resistancegene was identical with the reported one in Genbank. Thus recombinant plasmid pMD18-T-neo was successfully constructed.Then long and short arms of targeting vector were cloned. Restriction enzyme cutting siteSalⅠand PstⅠwere introduced in the 5'flank of the primers of homologous short arms of yhdP,yugS, yhdT and yqhB genes. KpnⅠand BamHⅠwere separately introduced in the 5'flank of theprimers of homologous long arms of yhdP, yugS and yqhB genes, and KpnⅠand XbaⅠwereintroduced in that of yhdT gene. These homologous arms were amplified by PCR and inserted intovector pMD18-T simple, and objective fragments carried restriction enzyme cutting site wereobtained from the above constructed recombinant plasmids. Then these DNA fragments wereligated into targeting vector in certain order.Two ligation methods were chosen. The first method: at first,skeleton plasmid pMD18-T-neowith single enzyme digestion of SalⅠand PstⅠseparately, were treated with Alkaline Phosphatase(CLAP) ,then it was ligated with homologous short arm digested from the above constructedrecombinant plasmid with SalⅠand PstⅠ. Ligation products were transformed into competentcells of E.coli JM109 to increase, In a similar way , homologous long arms were ligated ,thustargeting vector was successfully constructed. The targeting vector of yhdP gene was constructed inthis method; The second method: big linear pMD18-T fragment were recovered after skeletonplasmid pMD18-T-neo was digested with KpnⅠand PstⅠ, then small linear neomycin resistancegene fragment was recovered after skeleton plasmid pMD18-T-neo was digested with SalⅠandBamHⅠ(XbaⅠ). The tow recovered fragments were ligated with homologous long and short armsdigested from the formerly constructed recombinant plasmid. thus targeting vector wassuccessfully constructed. The targeting vectors of yugS, yhdT and yqhB gene were constructed inthe second method.The constructed gene targeting vector was linearized with KpnⅠand PstⅠ, and electroporatedinto competent cells of BS224. Positive transformers were screened from the neomycin resistanceplate, yhdP, yugS, yhdT and yqhB gene deletion were identified by PCR. Primers of identificationreaction of yhdP gene deletion were designed according to the upstream sequence of the fore armand the downstream sequence of the back arm. Whereas primers of identification reaction of yugS,yhdT and yqhB gene deletion were designed according to the downstream sequence of neomycinresistance gene and the downstream sequence of back arm.96,163,105 and 87 transformers wereanalyzed by PCR to identify yhdP, yugS, yhdT and yqhB gene deletion, and finally single yhdP,yugS, yhdT and yqhB gene deleted mutant were separately found.Hemolysis of yqhB, yugS,yhdT and yhdP gene deleted mutants was separately identified on5% sheep blood agar plate .The results suggested that the four gene deleted mutants could allinduce hemolysis, It indicated that knockout of single gene had no influence on hemolysis ofBS224.Possible hemolytic genes of BS224 were first cloned in this experiment, since Bacillus subtilis168 genome sequencing was finished. Moreover yhdP, yugS, yhdT and yqhB gene deleted mutantswere constructed by homologous recombination and their hemolysis were identified to make sure hemolytic gene or main gene inducing hemolysis and to discuss molecular mechanism of hemolysisof Bacillus subtilis, finally modified BS224 was widely used in medicine. At the same time, aneffective method in which unknown functional genes in Bacillus subtilis genome were researchedwere provided, and one important step was forward taken for studies on proteomics of BacillusSubtilis in the future.