Transformation and transposition of the genome of Mycobacterium marinum

Objective-To develop and evaluate protocols for genetic manipulations (tran sformation and transposition) of the fish pathogen, Mycobacterium marinum. Sample Population-Isolates of M marinum obtained from fish and humans. Procedure-Electrocompetent cells were prepared from isolates of M marinum g rown to various growth phases at several temperatures and with or without t he addition of ethionamide or cycloheximide. Mycobacterial cells were trans formed by electroporation with a replicative Escherichia coli-mycobacteria shuttle vector (pYUB18) as well as suicide vectors (pYUB285 and pUS252) tha t carried transposable elements (IS1096 and IS6110, respectively). Mutants from both isolates of M marinum were recovered on 7H10 agar plates suppleme nted with kanamycin. Transformation and transposition efficiencies for vari ous protocols were compared. Southern hybridization analysis was performed on mycobacterial mutants to confirm transposition events. Results-Competent cells prepared at room temperature (23-25 C) from organis ms in late-exponential growth phase yielded higher transposition efficiency , compared with cells prepared at 4 C or from organisms in early- or mid-ex ponential growth phase. Naturally developing kanamycin-resistant colonies o f M marinum were not detected. Only the IS1099-derived transposition was ab le to efficiently mutate M marinum. Southern hybridization of M marinum mut ants revealed random integration of IS1096 into the M marinum genome. Conclusions-Transposition and transformation efficiencies were comparable, suggesting that the limiting factor in transposition is the transformation step. Most of the experiments resulted in transposition of IS1096; however, better approaches are needed to improve transposition efficiency.