Trans-kingdom plasmid transfer from bacteria to yeast

• Main Author: Bates, Steven
• Published: University of Leicester 1997
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696293

Bacterial conjugation is a plasmid-encoded process resulting in the horizontal transfer of DNA from a donor to a recipient cell. However, the process of conjugation is not limited to transferring DNA between related prokaryotes. Indeed conjugation can result in the transfer of DNA to a wide range of organisms, even eukaryotes. Two such trans-kingdom transfer events have been reported, the transfer of the Ti plasmid of Agrobacterium tumefaciens to plant cells, and transfer from Escherichia coli to the budding yeast Saccharomyces cerevisiae the subject of this thesis. The aim of this study was to identify the genetic factors, both in the donor bacterium and the recipient yeast cell, required for trans-kingdom transfer to occur. The evolutionary implications of trans-kingdom transfer will also be discussed. This work describes the successful development and optimisation of an efficient protocol for E. coli-yeast plasmid transfer. Using this optimised protocol the ability of a range of conjugative plasmids to mediate transfer to yeast was compared. Only the promiscuous plasmid RP4 appeared capable of mediating transfer to yeast using this protocol. A genetic analysis of RP4 was carried out to determine the factors required for transfer to yeast and to identify the basis of RP4 promiscuity. This analysis revealed that the same set of genes essential for transfer between E. coli cells are also required for transfer to yeast. Therefore, no additional factors are required for the promiscuous transfer of RP4 to yeast. Hence, the promiscuous nature of RP4 is inherent in its transfer machinery. To determine the yeast factors required for successful transfer, mutants were isolated that displayed an altered ability to receive and establish a plasmid by trans-kingdom conjugation. Five UV induced yeast mutants (conI-5) were identified that displayed a decrease in transfer. Genetic complementation of conI with a genomic library was successfully carried out. CONI is allelic to the previously uncharacterised ORF YHR185c, located on the right arm of chromosome 8. CONI encodes a predicated protein of 237 amino acids, and is unique in the yeast genome. The function of CONIp is unclear, it may be required for the cellular interactions between cells or for the establishment of the transferred DNA strand. Analysis of these mutants will provide a tool to determine the molecular basis of trans-kingdom DNA transfer.