Exploration On The Lysis Genes Of Maltocin P28

Maltocin P28 is a bacteriocin produced by Stenotrophomonas maltophilia P28,its structure is similar to that of Myoviridae phage tail.The gene cluster of maltocin P28 is predicted to contain 23 genes,of which the putative genes from orf1 through orf9 are non-structural genes,orf10-orf23 are structural genes.The genes orf17 and orf18 are the main structural genes,encoding tail sheath and tail tube protein,respectively.This study focuses on exploring the genes related to the release of maltocin P28.Bioinformatics analysis suggested that the putative holin and endolysin,encoded by orf7 and orf8,should consist of a lysis system to release maltocin P28.The transcripts of orf6-orf9 were firstly detected by RT-PCR.The results showed that orf6,orf7,orf8 and orf9 were all transcribed,and orf6-orf9 were co-transcribed.Previous studies found that orf6 and orf5 were not co-transcribed,orf9 and orf10 were not co-transcribed,either.Therefore,orf6-orf9 were in the same operon.Then,in order to explore whether orf7-orf9 were related to the release of maltocin P28,the double gene deletion strains P28?7-8 and P28?8-9,and three gene deletion strain P28?7-9 were constructed.These three mutant strains and three single gene deletion strains P28?7,P28?8 and P28?9 were cultured respectively.The growth of these strains had no significant difference.Compared with strain P28,the bactericidal activities of the orf7 or orf8 deletion strain showed a delay of about 2 h,double gene deletion of orf7 and orf8 resulted in a delay of about 4 h.Just deleting orf9 had no obvious effect.The results indicated that orf7 and orf8 were associated with the release of maltocin P28,and orf9 was unrelated to the release of maltocin P28.The sequence of orf4-orf6 have been deleted in strain P28?4-6N.The bactericidal activity and the tail sheath protein could not been detected in the supernatant.However,but a large amount of tail sheath protein were detected inside P28?4-6N by Western blot.It suggested that the release of maltocin P28 might be blocked.The structural genes orf10-orf23 has two predicted promoters,the promoter P₁₀of orf10 and the promoter P₁₇of orf17.The promoter P₁₀was replaced with the strong promoter P₃in the strain?4-6N(P₁₀-P₃).The supernatant of strain?4-6N(P₁₀-P₃)had obvious bactericidal activity,showing that maltocin P28 of?4-6N(P₁₀-P₃)was successfully synthesized and released into the extracellular environment.This suggested that the lack of orf4-orf6 would not affect the release of maltocin P28.To verify the lysis genes in the cluster,the 9 non-structural genes orf1-orf9 were knocked out to construct the strain P28?1-9.Just like the strain P28?4-6N,the bactericidal activity and the tail sheath protein could not be detected in the supernatant of P28?1-9,but the tail sheath protein was detected within the strain.The supernatant had bactericidal activity when the promoter P₁₀was replaced with promoter P₃in strain P28?1-9.Apparently,deletion of orf1-orf9 could not block the release of maltocin P28,implying the existence of other release-related genes.Finally,the release-related genes of maltocin P28 were screened by the transposon random mutation method.But the relevant mutants have not been obtained yet.In conclusion,orf7 and orf8 were identified as the genes related to the release of maltocin P28.And they encoded holin and endolysin,respectively.Meanwhile,in addition to orf7 and orf8,there are other genes involved in the release of maltocin P28in S.maltophilia P28.The function of these release-related genes need to be further studied.