The Identification Of Regulatory Genes On Maltocin P28 Locus

Maltocin P28,which produced by Gram-negative bacterium Stenotrophomonas maltophilia P28,is a kind of phage tail-like bacteriocin composed of multiple proteins and can inhibit many bacteria belonging to S.maltophilia.Maltocin P28 contains two major structure proteins,tail sheath and tail tube.Maltocin P28 includes 23 open reading frames(ORFs),orf10-orf23 are associate with encoding structure proteins of maltocin P28,while the orfl-orf9 respond for encoding the non-structure proteins.This study focus on determining the regulatory genes,located in orfl-orf9,of maltocin P28.First,we tested if the non-structure genes predicted transcription applying reverse-transcription polymerase chain reaction(RT-PCR).The result indicated that besides orf2,other genes are transcribed and orf6-9 is co-transcription.Then we constructed a serials of mutants,P28?orf1l,P28?orf6(N),P28?orf5-6 and P28?orf4-6(N).After getting mutants,their bactericidal action of culture supernatant and expression level of sheath protein were tested.Compared with the strain P28,P28?orf1 and P28?orf5-6 strains' bactericidal activity did not changed but P28?orf6(N)and P28?orf4-6 strains had no bactericidal activity.Previously,our laboratory had already constructed five mutants that deleted orf3,orf4,orf8,orf9 respectively,in which orf3 deleted strain loss the bactericidal activity while others mutants still have bactericidal activity with no decrease.Western blot results indicated that P28?orf3 and P28?orf6(N)strains didn't expressed sheath proteins,the expression level of sheath protein increased in P28?orf5-6,the expression level of sheath protein has no changed in other mutants,compared with P28 strain.Based on the results above,three key genes associate with the production of sheath of maltocin P28 have been confirmed,orf3 as well as orf6 promote the expression of sheath while orf5 may suppress the expression of it.To excluding the polar effect of gene knock out affecting the analysis for experimental results,several complement plasmids were constructed,including pRK415-ORF3,pRK415-ORF5,pRK415-ORF6.Through the method of conjugation,the complement plasmids were transferred into certain mutants.We found that pRK415-ORF6 cannot restore the bactericidal activity of P28?orf6(N)strain,but the expression of sheath protein was detected through western blot in the complementation strain contains pRK415-ORF6 but in the P28?orf6(N)strain.This result indicated that deleted ORF6 might affect downstream genes and lead to maltocin P28 cannot resemble completely or release from P28?orf6(N)strain.On the other hand,we surprising discovered that pRK415-ORF6 can restore the bactericidal activity of P28?orf3 strain.This result suggested that orf3 located at the upstream of orf6 and can promote the latter's expression.Then we investigated the relative gene expression level of orf3?orf5?orf6?orfl 7 in different mutants and P28 using real-time quantitative PCR.The results showed that the transcription level of orf5 has no change in different strains,the transcription level of orf3 has no change in P28?orf6(N)strain while its' transcription level increased by 61.25 folds in P28?orf5-6 strain,which indicated that orf5 suppresses the transcription of orf3.The transcription level of orf6 decreased by 99 folds in P28?orf3,indicated that orf3 promotes the transcription of orf6.We had detected no mRNA of orfl 7 in P28?orf6(N)strain and the transcription level of orfl 7 decreased by 99 folds in P28?orf3 strain while its' transcription level increased by 5.32 folds in P28?orf5-6 strain,indicated that orf3 and orf6 are promote the expression of orfl 7 while orf5 restrains its' expression.In order to determine whether the orf3 directly regulate the expression of orf6,orf6 and orf5 directly control the expression of sheath,we validated by EMSA.First,we built the expression vector,pET26-ORF3,pET28-ORF5,pET26-ORF6,heterologous expressed those recombinant plasmids in E.coli BL21(DE3),obtained recombinant proteins with his-tag.Then,by PCR method,different promoters' DNA fragments were acquired.Purified proteins and target DNA fragments were added into particular buffer,reacting freely in certain condition.By native PAGE,we observed ORF6 can combine with the promoter of sheath.This result strongly supports that orf6 regulates the expression of sheath.It is likely that experimental condition not good optimization,we hadn't detected ORF5 or ORF3 combined with target DNA fragments.Enventually,we confirmed 3 genes associated with the expression and regulation of maltocin P28,orf3 promotes the expression of orf6,orf6 promotes the expression of sheath directly while orf5 suppresses the expression of orf3 and sheath.