IncP plasmids are unusually effective in mediating conjugation of Escherichia coli and Saccharomyces cerevisiae: involvement of the tra2 mating system

Abstract

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.

FIG. 1

Construction of mobilizable shuttle vectors. Shuttle vectors were based on YEp13. Replication and selection in E. coli are conferred by the oriV, Tc^r, and Ap^r elements of pBR322. The ORI/STB region of the 2μm yeast plasmid allows stable replication in yeast. Selection in yeast is achieved by leucine prototrophy of a leucine auxotroph. Mobilizable vectors were constructed by inserting the oriT region of ColIb-P9, RP4, and F into the Tc^r gene of YEp13. Restriction sites are as follows: B, BamHI; Bg, BglII; H, HindIII; P, PstI; S, SalI; Sp, SphI; X, XmaIII; Xb, XbaI.

FIG. 2

Capacity of yeast cells to receive plasmids at different stages in the growth cycle. YEPD complete medium was inoculated with a sample of stationary-phase yeast cells of strain S150-2B to give an initial concentration of 4 × 10⁶ cells ml⁻¹. The culture was sampled at 1-h intervals to determine cell titer (○). Samples, concentrated to give 2 × 10⁸ cells ml⁻¹, were mixed with BW103(pUB307 pAC88) donors for 1 h to determine transconjugant production (•).

FIG. 3

E. coli-yeast shuttle plasmids containing the Tra1 region (pSB12) and Tra1 core (pSB13) of RP4. The constructs are based on the yeast centromeric plasmid YCp50, allowing detection of transfer to yeast by uracil prototrophy. Horizontal boxes shown in black indicate Tra1 core genes essential for transfer between E. coli K-12 strains when the donor strain also expresses Tra2 functions in trans. Other Tra1 genes are indicated in gray. Addition of traM to the Tra1 core genes increases the transfer efficiency (35). Restriction sites shown are as follows: B, BamHI; H, HindIII.