Variable Lead Green Streptomyces 66 Within The Source Plasmid Slp2 And Slp3, Research

SLP2 was a Ca. 50kb endogenous conjugative linear plasmid ofStreptomyces lividans66, pQC542 with two right-orientation telomeres was abi-functional plasmid derived from SLP2 with 23kb fragment from SLP2.pQC542 could stable replicate as linear structure in Streptomyces and transferefficiently. Plasmid with one or no telomere derived from pQC542 still couldreplicate as circular structure and transfer efficiently. Studies on gene functionsuggested there were at least three kinds of genes: circular and linear plasmidreplication related genes, stable replication as high copy number related genes orfactors and high frequency conjugation related genes.Shut-gun DNA sequences analysis were carried out for the 23kb fragment.G+C content is 68.3%. Frame analysis indicated there were at least 25 probableprotein-coding open reading frames (ORFs) in this DNA fragment. 3 of themwere unknown protein. There were 5 ORFs (A, 10, 11, 21, 22) shown conservedsimilarity with some hypothytical proteins in the protein data bank. It wasremarkable that ORF19 shown similarity with Rep1 of pSLA2 involved in thereplication. ORF6 showed similarity with Tra protein of pIJ101 involved in theplasmid transfer.In order to identify the minimal replication region of SLP2, subclones wereconstructed with the ori-proble vector pQC156. It was shown ORF18 andORF19 consisted of the minimal replication region (the corresponding plasmid ispXQ20). Sequence analysis showed that there were lots of direct repeats, indirectrepeats and palindrome also an AT-rich region within ORF18. ORF19 showedhigh similarity (56%) with DNA helicase. The transform frequency of pXQ20which was isolated from E. coli DH5α was 3×105/μgDNA, but it was instable inStreptomyces lividans ZX7. The inherited stability of pXQ20 is Ca. 1%. Thestructure of replication region of SLP2 was somehow similar to that of pSLA2from S. roches, it was reported the basic replication region of pSLA2 consistedof 2 overlapped genes in one operon: rep1 and rep2. In our case, the result ofRT-PCR transcription analysis of ORF18 and ORF19 showed that ORF18 did nottranscript. This was differing from the basic replication region of pSLA2. ORF18and 19 were not located in the center of the linear structure of SLP2 instead ofclosing to one end.With 23kb fragment of SLP2, pQC542 could conjugate efficiently inStreptomyces. The transfer frequency of pQC542 was Ca. 60% when detectedwith TK23 as a recipient. The subclones of the 23kb fragment from SLP2 wereconstructed by the vector pQC578 which can stable replication in Streptomyceswithout transfer ability. A recombination plasmid, pXQ102, cloned a Ca. 8.4kbfragment from pQC542 was shown to transfer efficiently (the transfer frequency(TF)≥10%), sequence analysis of the 8.4kb fragment shown that there were 6ORFs which transcript in the same direction. Blast results showed that ORF6encode a Tra-like protein with a conserved FtsK/SpoIIIE domain in it. It wassuggested the transfer mechanism of circular plasmid maybe somehow similar tolinear plasmid. An essential gene ORF1 which encoded a protein with a VirB4conserved domain in it was first identified in our study. VirB4 related with thetransfer energy is a component of type IV secretion system. Deletions of the8.4kb fragment were carried out. It was shown that the deletion of ORF6 lead theconjugation transfer frequency reduced greatly and closed to zero. The deletionof the other ORFs would result the reduce of transfer frequency in some degree.Transcription analysis result by RT-PCR showed the 6 ORFs transcript as aoperon. These characteristic differed from that of pIJ101 whose essentialelements of conjugation transfer were Tra and clt only. It was first shown thegenes involved in the conjugation of Streptomyces linear plasmid were identified.Also it was first shown the T4SS related VirB4-like protein: ORF1 was theessential gene for conjugative transfer of linear plasmid SLP2.According to the sequence and functional analysis result some ORFs wasselected to overexpress in E.coli.