Structural and functional stability of IncP plasmids during stepwise transmission by trans-kingdom mating: promiscuous conjugation of Escherichia coli and Saccharomyces cerevisiae.
In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes. A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E. coli harboring pAY101, and E. coli carrying a helper plasmid with mob and tra. In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10(-5) to 10(-3) per recipient cell. This E. coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes. The mob and tra genes were trans-acting elements as in an E. coli conjugation system. The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation. DNA analysis indicated that the plasmid pAY101 was transferred from E. coli to S. cerevisiae, and retained its original structure and function in yeast host cells.
Nishikawa
M
M
Botanical Institute, Faculty of Science, Hiroshima University.
Suzuki
K
K
Yoshida
K
K
D015139:Blotting, Southern; D003227:Conjugation, Genetic; D003433:Crosses, Genetic; D004926:Escherichia coli / Q000235:genetics*; D005800:Genes, Fungal; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection