Genetic recombination by spheroplast fusion in Escherichia coli K12

Genetic transfer mediated by spheroplast formation and fusion in Escherichia coli was studied. Recombination did not occur from spheroplasts prepared by the combined glycine and lysozyme-EDTA treatment described by Coetzee et al. (1979). In contrast, when bacteria were exposed to a sub-inhibitory concentration of polymyxin B (0.5 microgram/ml) during the spheroplast generation phase, recombinants arose at frequencies of 1 x 10(-8) to 2.6 x 10(-8). The incidence of genetic transfer was further increased by adding 0.01 M CaCl2 to the polyethylene glycol fusion mixture (from 9 x 10(-7) to 9 x 10(-8). Finally, when the effects of both polymyxin B and calcium chloride were combined recombinants arose at frequencies of 3.4 x 10(-6) to 7.3 x 10(-7). These findings suggest that the detergent-like action of polymyxin B in removing a large part of the outer membrane enhances plasma membrane availability where fusion can take place.