Location And Analysis Of Replication And Conjugation Region Of The Plasmid PJTU112in Micoromonospora Sp.40027

Micromonospora belong to rare actinomycetes, could produce wide varieties ofantibiotics. In recent years, with the deepening development of marine resources, thediscovery of new antibiotics generated by marine Micromonospora is increasing yearby year. These new antibiotics with unique structure and biological activity attractedgreat attention around the world. However, research on genetic operation system of theMicromonospora, especially on gene cloning system is rarely reported. PlasmidpJTU112isolated from Micromonospora sp.40027has the function of autonomousreplication, integration and conjugation. In this study, the complete sequence ofpJTU112was obtained. The main purpose is to conduct studies on the replicationregion and conjugation region.The complete sequence of plasmid pJTU112was determined and analysed.Sequence analysis showed that the circular plasmid pJTU112is13,648bp in total sizewith74.6%G+C content.13open reading frames (ORFs) with putativeribosome-binding sites were identified, pJTU112.3, pJTU112.4and pJTU112.5wererelated to plasmid replication; pJTU112.1, pJTU112.2, pJTU112.12and pJTU112.13were related to conjugation; pJTU112.6were related to site-specific recombinant; andpJTU112.9were related to antibiotic resistance.A series of recombinant plasmids were constructed by the ligation of differentfragments of pJTU112and vector pHZ199or pOJ260. These plasmids were introducedby conjugation into Micromonospora sp.LXH20. Plasmids containing1.4kb DNABamHI fragment (such as pJTU111, pJTU116and pSCU207) transferred,transconjugants with resistance of thiostreptone or apramycin were obtained. Plasmidswithout1.4kb DNA BamHI fragment (such as: pJTU113) transferred, transconjugantswere not obtained. It indicated that1.4kb BamHI DNA fragment of pJTU112isessential for plasmid replication. Plasmid pSCU207containing4.7kb SacI-KpnI DNAfragment of pJTU112could replicate in Micromonospora LXH20, indicating that thereplication region of pJTU112was located in the4.7kb SacI-KpnI DNA fragment.The analysis of amino acid sequence of pJTU112.5protein showed thatpJTU112.5has57%identity with Rep protein of pSG5of S.ghanaensis. Meanwhile,there were iterons at upstream of pJTU112.5in pJTU112. A similar nick at1647bpexisted in the pJTU112, compared with pMEA300, pMEA100, pSG5and pSE21,Plasmid pSCU213, pSCU214and pSCU215were constructed by the ligation ofdifferent fragments of pJTU112and vector pHZ199, and recombinants were introduced by conjugation (from E.coli to Micromonospora) into Micromonospora sp.LXH20togenerate donor strains together with the recipient strain Micromonospora sp.LXH22for conjugation experiment (from Micromonospora to Micromonospora), testing theconjugal transfer of pJTU112.The analysis of amino acid sequence of pJTU112.1, pJTU112.2and pJTU112.12protein showed that pJTU112.1(302aa) has32%identity with cell division FtsK;pJTU112.2(478aa) has69%identity with cell division FtsK/SpoIIIE, meanwhile,pJTU112.2contains a conserved FtsK/SpolIIE domain, a conserved amino acid motifRAAGI and a conserved nucleic acid binding site GAGKS; pJTU112.12(785aa) has32%identity with major plasmid transfer protein of S.avermitilis and containsFtsk_gamma domain.