Construction And Preliminary Application Of A Mini-transposon In Streptomyces Coelicolor A3 (2)

The basic researd on Streptomycestes enters the post-genomics era since the published of Streptomyces coelicolor A3 (2) and Streptomyces avermitilis MA-4680 genome. Function of many novel genes or unknown genes is to be identified. Construction and screening of mutant library is an important strategy in functional genomics, and transposon is a useful tool to construct mutant libraries.Transposon is a very useful tool of gene function analysis. However, currently utilized transposons in Streptomyces have many disadvantages. Such as low transposition frequency or complicated to use. Some currently employed transposons (Tn4556 and Tn5 derivatives) had been used to construct mutant libraries, but they are not perfect at all.Aiming at improving currently used transposons, a novel transposon plasmid pHL265 was constructed containing a mini-transposon mini-Tn4560A. The pHL265 has several advantages. First, it a transposase gene tnpA located beyond the mini-transposon mini-Tn4560A. It contains no replication origin region for replication in Streptomyces. In theory a transposition mutagenized strain will not be subject to a second transposition because tnpA will be lost during the first transposition process. Scecond, pHL265 also contains oriT, the origin of transfer from RK2, which can be used to introduce DNA from E. coli to Streptomyces by conjugation. Conjugation will minimize mutation usually occurred in protoplast transformation. Finally, in-vivo transposition using pHL265 was much more concisely or directly, compared to in-vitro Tn5 transposition in which construction of a cosmid genomic library is required and the transposition mutagenized cosmid library has to be reintroduced into Streptomyces and screen for mutants.About one thousand transposition mutants were screened using this minitransposon. Some of them were developmental deficient. Eight strains were selected for Southern Blot analysis and the randomness of the transposition was confirmed. The stableness of the transposons was also confirmed by relaxed cultivation for several generations. The transposon system constructed here has provided a novel approach for the function genomic analysis of Streptomyces.