| Stenotrophomonas maltophilia is an important global opportunistic pathogen.With high infectivity and lethality,S.maltophilia also shows strong resistant towards many antibiotics,such as ?-lactam,macrolides,cephalosporins,etc.Its multi-drug resistance(MDR)makes the normal antibiotic therapy for S.maltophilia infection to be less efficient.So it is urgent demand to find novel antibiotic alternatives,which might be bacteriocin,virulent phage,microbial preparation,plant extract and enzymic preparations.A novel bacteriocin named maltocin P28 has been isolated and identified in S.maltophilia P28,which was isolated from the soil sample.Maltocin P28 is the first bacteriocin produced by S.maltophilia and shows bactericidal activity to some S.maltophilia strains.Maltocin P28 has a contractile tail like myoviridae phages.Its size is 120×20 nm.Its gene cluster is comprised of 23 genes.Genes from orf1 through orf9 are nonstructural genes,encoding regulator and lysis genes.Genes from orf10 to orf23 are structural genes,encoding structural proteins and chaperones for tail assembly.Functions of all genes can be predicted based on sequence analyses except for orf10 and orf19.Due to complex regulatory mechanism,biosynthesis of maltocin P28 is at low level.In order to synthesize maltocin P28 at high level in Escherichia coli,the two unknown genes orf10 and orf19 were studied at first.Deletion strains P28?10 ? P28?19 were constructed by knocking out orf10 and orf19,respectively.Deletion of orf10 let the bactericidal activity of maltocin P28 decrease by 87.5%.However,deletion of orf19 made no bactericidal activity.Western blot analysis of tail sheath protein showed that orf10 or orf19 deletion didn't affect the strain to synthesize and release maltocin P28.Electron microscope observed that the length of maltocin P28 produced by strain P28?10 was longer.The results showed that orf10 could affect the length of maltocin.The complementation of orf10 in strain P28?10 could rescue the antibacterial activity of maltocin P28.The complementation of orf19 in P28?19 could not recover the antibacterial activity,but the complementation of orf19.5 could.It indicated that a frameshift translation happened in the translation of orf19.Under electron microscope maltocin P28 produced by P28?19 showed longer hollow tails,while maltocin P28 produced by P28?19/orf19 showed longer solid tails.It suggested that orf19 was relative to tail tube assembly.Then lac Z was used as a reporter gene to measure the ratio of frameshift in S.maltophilia P28.The frameshift ratio in orf19.5/orf19 of maltocin P28 was 4.87%.The results showed that orf19 and orf19.5 were required for correct assembly of maltocin P28.Next,RT-PCR was used to analyze the transcript of structural genes.Result showed that all structural genes could be grouped into two operons.Genes from orf10 to orf16 was the first operon,and genes from orf17 to orf23 consisted of the second operon.The two operons were cloned and ligated to expression vector p ET-26(+),respectively.With the induction of IPTG,bactericidal activity can be detected within the cell.When induced by both of IPTG and MMC,the bactericidal activity of the recombinant strain culture supernatant was 3200 AU/m L,indicating heterologous expression of maltocin structural genes was successful and maltocin P28 could not be released by itself.As the bactericidal activity of maltocin P28 in E.coli was not as high as that of S.maltophilia.The express conditions in E.coli still need to be optimized.Altogether,this study confirmed that orf10,orf19 and orf19.5 were indispensable genes for maltocin biosynthesis.At the meantime,maltocin P28 had been successfully biosynthesized in E.coli.This may pave the way for its application in anti-infective therapy of S.maltophilia infection. |
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