Low temperature protocol for efficient transformation of Mycobacterium smegmatis spheroplasts.
Spheroplasts of Mycobacterium 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 x 10(8) to 1.0 x 10(9) spheroplasts to saturating DNA (1 microgram) for 15 min at 5 degrees C resulted in a transfection efficiency of approximately 0.009% . The transfer of the beta-lactamase marker with DNA purified from strain LM15 to spheroplasts of a beta-lactamase-negative mutant, strain LM144, was achieved. The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillin-resistant cells than the nontreated control culture. Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for beta-lactamase Cell-free extracts of penicillin-resistant transformants contained beta-lactamase activity that ranged from 0.046 to 0.134 micromol of benzylpenicillin hydrolyzed/min per mg protein. This low temperature procedure is recommended for high efficiency transformation of M. smegmatis.
Naser
S A
SA
Department of Biology, New Mexico State University, Las Cruces.
McCarthy
C M
CM
Smith
G B
GB
Tupponce
A K
AK
D009160:Mycobacteriophages / Q000235:genetics; D009161:Mycobacterium / Q000166:cytology / Q000235:genetics*; D010403:Penicillin Resistance; D013104:Spheroplasts / Q000235:genetics*; D013696:Temperature; D014162:Transfection; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000235:genetics
D001618:beta-Lactamases