Transformation of the thermophilic bacterium, Geobacillus debilis, by conjugation with the mesophilic bacterium, Escherichia coli.

Abstract

A method for transformation of Geobacillus debilis by conjugation was developed using a recombinant plasmid, pNW33N-pxyl-bs2-mob, derived from pNW33N. The plasmid includes the mob region of RP4 for mobilization, is mobilized from E. coli S17-1 to G. debilis, and can stably propagate in G. debilis trans-conjugants grown at 50 oC and 55 oC, in the presence of thiamphenicol. Successful conjugation was depended on the cell density and viability of G. debilis when harvested for conjugation, as well as the metabolic activity of E. coli S17-1 used for conjugation. Substantial reduction in size of the plasmid DNA was observed when G. debilis transconjugants were cultured at 60 oC in the presence of thiamphenicol, and uniform rearrangement of the plasmid DNA was observed after culturing G. debilis transconjugants in the presence of spectinomycin, even at 50 oC.