Study On Gene Expression Regulation Of Maltocin P28

Maltocin P28 is a kind of phage tail-like bacteriocin(PTLB)produced by Stenotrophomonas maltophilia P28 strain,which can kill multiple strains of S.maltophilia.The relationship between maltocin P28 gene expression and environmental stress is unclear,and the regulation of ORF3,ORF5,and ORF6 proteins encoded by maltocin P28 gene cluster remains to be resolved.Based on these two points,this paper studied the impact of various environmental stresses on the regulation of maltocin P28genes expression.Subsequently,it was clarified whether Rec A and Lex A proteins in SOS response were involved in the induction and regulation of maltocin P28 synthesis;At the same time,the regulation ways of OFR3,ORF5 and ORF6 proteins were studied,revealing the mechanism of maltocin P28 genes expression regulation.Under different environmental stresses,the amount of expression of intracellular tail sheath protein ORF17 was detected by Western blot and the bactericidal activity assay was used to detect the bactericidal activity of culture supernatant.The results showed that H₂O₂,ultraviolet and mitomycin C were strong inducers,which could induce expression of intracellular tail sheath protein to increase by 6-10 times,and the bactericidal activity of the strain increased significantly;While ethanol was a mild inducer,which could induce expression of intracellular tail sheath protein to increase2-3 times;In addition,changing osmotic stress did not have significant effect on expression of maltocin P28 genes;While high temperature,acid and alkali pH would inhibit synthesis of maltocin P28;H₂O₂,UV and MMC can activate bacterial SOS response.Rec A and Lex A are the main regulatory proteins of SOS response and usually participate in the induction and regulation of bacteriocin synthesis.Our laboratory already had lexA-deleted P28 strain PDlexA and recA-deleted P28 strain PDrecA,under normal culture conditions,the bactericidal activity of strain PDlexA did not change significantly compared to wild strain P28,while the bactericidal activity of strain PDrecA increased 8 times.Under induction conditions,the bactericidal activity of strain PDlexA and P28 increased by 4-32 times,and the bactericidal activity of strain PDrecA increased only by 2-16 times.The results show that Lex A is not involved in induction of maltocin P28 genes regulation;Rec A may be a repressor of maltocin P28 genes expression.In order to further determine the function of Rec A,real-time quantitative PCR was used to detect expression of each gene on the maltocin P28 gene cluster in PDrecA and P28 strain.The results showed that deletion of recA and inducers can cause the increase of the expression level of orf3,orf5,orf6 and orf17,all of which the increase of the expression level of orf6 is the highest,followed by orf17,orf3 and orf5.The above results indicated that recA deletion and inducers may regulate the expression of maltocin P28 genes through the same mechanism.ORF5-GFP fusion protein was expressed in strain P28and PDrecA,and changes of the ORF5-GFP fusion protein under induction and non-induction conditions were detected.It was found that under recA-deletion and induction conditions,ORF5 would not be hydrolyzed,indicating induction of maltocin P28 genes expression may be not achieved by degradation of ORF5,the regulation of maltocin P28 genes expression is different from those of known bacteriocins under induction.Real-time quantitative PCR and promoter activity methods were used to analyze the regulation of orf3,orf5 and orf6.Real-time quantitative PCR detected expression levels of the orf3,orf5,orf6,orf10 and orf17 genes in deletion strains.The results showed that expression levels of orf6,orf10 and orf17 in PD3 strain decreased,while in PD6(x)strain,only the expression levels of orf10 and orf17 decreased,indicating that ORF3 can activate the expression of orf6,and ORF6 can activate the expression of orf10 and orf17.In PD5-6 strain deleting orf5-orf6,the expression levels of orf3 and orf10 increased about 10 times and 4 times compared with wild strain P28,respectively.It is speculated that ORF5 can repress expression of orf3 and orf10.To further prove the above speculation,the interaction between regulatory protein and promoter was studied by using lacZ as reporter gene to detect the activity of promoters.In Stenotrophomonas maltophilia,ORF3 can increase the promoting activity of P_orf6from 7.0 U/m L to 8098.2 U/m L,ORF5 reduced the promoting activity of P_orf3to about 1/44 of the initial activity,and ORF5 can also repress the activity of its own promoter P_orf5,reducing it from 1915.8 U/m L to 400.5 U/m L.In E.coli DH5?,ORF6 can increase the activity of P_orf17by about 25 times.These results indicate that ORF5represses the transcription of orf3 and orf5,ORF3 can activate the transcription of orf6,and ORF6 can activate the transcription of orf17.In summary,this dissertation demonstrates that H₂O₂,UV and MMC treatment can induce maltocin P28 synthesis,and Lex A and Rec A proteins are not involved in this induction and regulation process,but Rec A may be an inhibitor of maltocin P28genes expression;ORF3 and ORF6 proteins encoded by maltocin P28 gene cluster are activation factors,and ORF5 protein is a repressor.Among them,ORF6 directly activates expression of structural genes,ORF3 can activate expression of orf6,and ORF5 represses expression of orf3 and itself,thereby regulating the synthesis of maltocin P28 in S.maltophilia P28.