Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Palpha system caused detectable mobilization to yeast, giving peak values of 5 x 10(-5) transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of oriT in cis and the provision in trans of the Palpha Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Palpha conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Palpha system in mediating gene transfer from bacteria to eukaryotes.

• PMID: 9851996
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Bates S, Cashmore AM, Wilkins BM

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