Evaluating Transmission Barriers to Escherichia coli x Saccharomyces cerevisiae interkingdom conjugation
Thesis DisciplineBiological Sciences
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Conjugation is a fundamentally important mechanism of horizontal DNA transfer between bacteria, bacteria x archea, and bacteria x eukaryotes. This work has concentrated on conjugation between bacteria x eukaryotes, specifically Escherichia coli x Saccharomyces cerevisiae. Four hypotheses were tested, investigating the barriers to this particular form of DNA transfer. The first investigated if a mutation that altered the cell-surface of the recipient S. cerevisiae could inhibit DNA transfer. The final three utilised a recombination-dependent-conjugation assay to investigate the barrier to DNA transmission through recombination. The hypotheses tested if the frequency of recombination, in this recombination-dependent-conjugation assay, differed when using similar or diverged DNA substrates, if a mismatch repair mutation within the recipient could affect the frequencies of recombination observed, and if the position on the plasmid of the gene of interest affected the frequency of transmission. Transmission of the Ura3 DNA sequence in the recipient S. cerevisiae was used to test all four hypotheses. The cell wall mutants mnn9, knr4, fks1 and kre6 were utilised to investigate if the cell-surface of the recipient could affect the frequency of transmission. The similar and diverged substrates utilised in the investigation of the affect of sequence similarity on recombination were the DNA sequences of ura3 from S. cerevisiae and Saccharomyces carlsbergensis, respectively and the MMR mutants utilised were msh2, pms1 and pol30-52. Cell wall mutants were not found to limit the frequency of transfer once donor-recipient contact was induced through the solid surface mating procedure. Sequence similarity, MMR and the relative position of the ura3 DNA sequence on the conjugative plasmids were shown to have little effect on the frequency of transmission in S. cerevisiae. This suggests that any DNA that enters the nucleus of S. cerevisiae (eukaryotes) can recombine with the chromosome and alter it to the same extent. However, trends within the data also suggest that DNA is transferred into the recipient and then transported to the nucleus to recombine with the chromosome as a single-stranded DNA molecule.