Construction of random transposition mutagenesis system in Rhodococcuserythropolis using IS1415

Recent studies on the metabolic activities of genus Rhodococcus have shown rhodococci to be of important use in industrial, pharmaceutical and environmental biotechnology. The increasing economic significance of Rhodococcus encourages renewed efforts to characterize their genetic systems, as Rhodococcus genetics are still poorly understood. The goal of this study is to adapt a transposon system for use in creating random mutagenesis in Rhodococcus erythropolis. A plasmid carrying IS1415, a member of IS21 family identified from Rerythropolis, has been constructed and designated as pTNR. pTNR is a non-replicating transposon tool introduced into target cells by electroporation. During its transposition, the transposable-marker gene is separated from the open reading frames (istAB) of IS1415, which should avoid secondary transposition. Transposition of pTNR into wild-type R. erythropolis created mutagenesis with a high efficiency of 1.23x10(6)mutants per microgram plasmid DNA. However, it could also be transposed into other Rhodococcus spp. at lower frequencies in comparison with that of R. erythropolis. It has been indicated by Southern hybridization that the generated kanamycin-resistant mutants were resulted from single transposition event of pTNR. The results also revealed that the transposable-marker gene of pTNR was randomly inserted into the chromosomal DNA of R. erythropolis. The affected DNA regions carrying the transposed DNA element could be conveniently recovered for further characterization using a plasmid rescue procedure. Sequence data of the insertion sites of 40 random mutants analyzed indicated that transposition of pTNR generated 6-bp direct target duplications in 36 cases, while in the remaining four mutants; it generated 5- or 7-bp target duplications (two cases each). This study concluded that pTNR could be served as an efficient genetic tool for construction of random mutagenesis system in Rhodococcus species.