Low temperature protocol for efficient transformation ofMycobacterium smegmatis spheroplasts

Abstract

Spheroplasts ofMycobacterium smegmatis LM15, strain 607, were prepared by a combined treatment with glycine and lysozyme. The spheroplasts were tested for ability to take up and express purified mycobacteriophage DNA. Exposure of 1.0×10⁸ to 1.0×10⁹ spheroplasts to saturing DNA (1 μg) for 15 min at 5°C resulted in a transfection efficiency of approximately 0.009%. The transfer of the β-lactamase marker with DNA purified from strain LM15 to spheroplasts of a β-lactamase-negative mutant, strain LM144, was achieved. The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillinresistant cells than the nontreated control culture. Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for β-lactamase. Cell-free extracts of penicillin-resistant transformants contained β-lactamase activity that ranged from 0.046 to 0.134 μmol of benzylpencillin hydrolyzed/min per mg protein. This low temperature procedure is recommended for high efficiency transformation ofM. smegmatis.