Study On Regulators Of Maltocin P28 Biosynthesis

Stenotrophomonas maltophilia is a Gram-negative bacterium and ubiquitous in nature.It is currently listed as an important multi-drug resistant pathogen by the World Health Organization.As an important clinical opportunistic pathogen,it has a high infection rate and mortality.Maltocin P28 is a R-type phage tail-like bacteriocin（PTLB）produced by S.maltophilia P28.It can be developed as a new antibacterial agent for the treatment of S.maltophilia infections.This study mainly focuses on the regulators involved in maltocin P28 biosynthesis,hoping to reveal the biosynthetic regulatory mechanism and physiological function of maltocin P28.orf3、orf5 and orf6 in maltocin P28 gene cluster are related to maltocin P28 synthesis.This study proved that the products of these three genes regulate the transcription of maltocin P28 gene,named mps A,mps R and mps H respectively.Firstly,RT-PCR experiments confirmed that the transcription of maltocin P28 genes were driven by five promoters P_A,P_R,P_H,P₁₀ and P_S,respectively.Then,promoter activity analysis showed that Mps A increased the promoter activity of P_H,Mps H increased the promoter activity of P₁₀ and P_S,and Mps R inhibited the promoter activities of P_A,P_R,P₁₀ and P_S.Electrophoretic mobility shift assay（EMSA）showed that Mps A could bind to P_H,Mps H could bind to P₁₀ and P_S,and Mps R could bind to P_A,P_R,P₁₀ and P_S.The experimental results confirmed that Mps A activated the transcription of mps H,Mps H activated the transcription of structural genes,while Mps R inhibited the transcription of mps A and structural genes,and regulated its own transcription through self-inhibition.This study found that Mps R plays an important role in the response of maltocin P28to MMC induction.Previous studies have found that the deletion of rec A or lex A in P28strain does not affect the synthesis of maltocin P28 in response to mitomycin（MMC）induction.Promoter activity analysis showed that the activity of each promoter increased after induction in P28 strain,but in the strain lacking maltocin gene cluster（P28Δm）,the activity of each promoter was no change after induction,indicating that the factors in response to MMC induction were encoded by maltocin gene clusters.Since Mps R can inhibit the activity of multiple promoters,the expression of fusion protein Mps R-gfp after induction was detected by Western blot.The amount of fusion protein decreased significantly after induction for 30 minutes,and then increased gradually.The results indicated that Mps R plays a key role in the synthesis of maltocin P28 in response to MMC induction,but the specific mechanism still needs to be further analyzed.Since S.maltophilia has strong biofilm formation ability and antibiotic resistance,this study also explored whether maltocin P28 affects the relevant characteristics of the producing bacteria.Compared with wild type strain,the biofilm formation ability of strain P28Δm decreased significantly,and the resistance to kanamycin and gentamicin also decreased significantly.Further experiments showed that the deletion of cleavage gene orf8 and regulatory gene mps H（orf6）would reduce the biofilm formation ability and antibiotic resistance of P28 strain.It suggested that the release of maltocin P28 will promote the producer strain toform biofilm and resist to kanamycin and gentamicin.There are three prophages in the genome of P28 strain,one is the genomic sequence of the reported filament phageΦSHP2,the other two are novel prophages,designed SHP4and SHP5,respectively.It is speculated that mps H may increase the biofilm formation ability and antibiotic resistance of strain P28 by activating the release of these lysogenic phages.Therefore,after the mps H deletion strain（P28Δmps H）was cultured for 12 hours,the genomic amount of lysogenic phages was detected by q RT-PCR.Compared with the wild-type strain,the genomic amount ofΦSHP4 andΦSHP5 in the supernatant of P28Δmps H decreased by about 100 times,andΦSHP2 could not be detected in the culture supernatant or strain P28Δmps H.These results suggested that mps H is related to the synthesis of three lysogenic phages in strain P28.The infection experiment ofΦSHP2 showed thatΦSHP2 could not replicate in strain P28Δmps H.When P28Δmps H was complemented by mps H,ΦSHP2 could replicate in large quantities.The results confirmed that mps H is related to the replication ofΦSHP2.g1,encoding the replication initiation factor ofΦSHP2,could complement the deletion of mps H,suggesting that Mps H may affect the replication ofΦSHP2 by regulating the expression of g1.EMSA showed that Mps H could bind to p1 promoter P_g1.The induced promoter P_amy was used to express Mps H in P28Δmps H.However,the promoter activity of P_g1 could not be detected whether Mps H was expressed or not.Further experiments are needed to determine the effect of Mps H on P_g1 and the specific way in which Mps H affects the replication ofΦSHP2.In conclusion,this dissertation mainly focuses on the regulator of maltocin P28biosynthesis.Mps A,Mps R and Mps H were proved to be new transcriptional regulators through experiments in vivo and in vitro.The specific mode of biosynthesis regulation of maltocin P28 was uncovered.The bacterial lysis by maltocin release contributed to the biofilm formation and resistance to kanamycin and gentamicin of the producer bacteria,and mps H was related to the synthesis of lysogenic phages.Since the maltocin gene cluster is widely present in the genomes of S.maltophilia,this study lays the foundation for the application of maltocin,analyzing its physiological function,and further exploring the mechanism of biofilm formation of S.maltophilia.