Direct plasmid transfer from replica-plated E. coli colonies to competent B. subtilis cells

Abstract

We have cloned the hisH tyrA wild-type genes of Bacillus subtilis with the aid of the chimeric plasmid pBJ194, which replicates both in B. subtilis and Escherichia coli. Primary cloning was done in E. coli. The original E. coli clone, carrying the recombinant plasmid (pGR1) which complements hisH tyrA mutants of B. subtilis, was selected directly from a mixture of plated E. coli clones by replicaplating these clones onto minimal agar plates without tyrosine, spread just before with competent B. subtilis cells. After overnight incubation clusters of small colonies had developed exclusively in the E. coli [pGR1] colony prints. The Tyr+ minicolonies were shown to be B. subtilis carrying pGR1 because (i) their appearance depended linearly on the number of B. subtilis cells plated, (ii) they produced extracellular protease and amylase and (iii) plasmids could be reisolated from the minicolonies and used to transform B. subtilis recE4 tyrA1 both to Cmr and Tyr+. Plasmid pGR1 transfer through replica plating was compared with plasmid transfer in liquid. Both systems depended on transformable B. subtilis strains and were sensitive to DNAseI. However, whereas integration of the tyrA+ gene into the chromosome and concomitant loss of plasmids occurred frequently during regular plasmid transformation of Rec+ B. subtilis, this was a rare event during plasmid transfer through replica plating.

Horizontal Gene Exchange in Environmental Microbiota

“…Later, however, it became clear that the mechanism of DNA acquisition in the biofilm is more complex and was, therefore, described as a “cell-to-cell transformation,” which also involves a peptide pheromone as a regulator (Etchuuya et al, 2011). Other circumstantial evidences of DNA transfer due to close cell-to-cell contact (Van Randen and Venema, 1984; Wang et al, 2007) may be interpreted within the frames of this hypothesis, although the transfer in these cases was between the genera, and the role of pheromones was not shown. The language of intercellular communication used in regulation of various processes within the complex structure of biofilms is indeed the language of pheromones such as N -acyl- L -homoserine lactones, furanosyl borate diester, and peptide autoinducers (Dickschat, 2010).…”

Cloning and sequence analysis of tryptophan synthetase genes of an extreme thermophile, Thermus thermophilus HB27: plasmid transfer from replica-plated Escherichia coli recombinant colonies to competent T. thermophilus cells

“…Randen and Venema reported the cloning of the Bacillus subtilis tyrA gene by a new cloning method of direct plasmid transfer from E. coli to competent B. subtilis (17). This methodology, with some modification, was found to be applicable to cloning of T. thermophilus HB27 genes.…”

Protoplast Transformation of Bacillus licheniformis MC14

“…Other transformation systems developed for various Bacillus species, incuding cell mating in liquid culture (Landman & Pepin, 1982), filter mating with E. coli (Van Randen & Venema, 1984), and direct plasmid transfer (Gonzalez & Carlton, 1982), were also unsuccessful.…”