Team:UCSC/Conjugation

Conjugation

What is Conjugation?

Bacterial conjugation, a form of horizontal gene transfer (HGT), propagates genetic material through a population via plasmid-mediated transfer [1], as seen in the figure below. This is essential in bacterial evolution as it often leads to genetic diversity and increased fitness. Antibiotic resistance, for example, spreads through a mixed population of bacteria through conjugation [2]. To deliver our gene-editing machinery, we will use conjugation in conjunction with bacteriophages to propagate our genetic payloads through a bacterial population. Conjugation only occurs when there is direct cell-to-cell contact between a donor and recipient. Donor cells are those that contain an F, or “fertility” plasmid (F+), and recipients are those that lack an F plasmid (F-). The F plasmid contains genes to generate pili, cellular projections that attach to possible recipients and draw them in to make contact with the donor. Once contact is made, replicated plasmid or chromosomal DNA can pass through a pore between the two cells [3].

Conjugation relies on the expression of transfer (tra) genes that code for essential components such as a relaxosome and the type 4 secretion system (T4SS) proteins, also known as the mating pair formation (mpf) apparatus [4][5]. The relaxosome is composed of a relaxase (formed by a group of proteins known as tra1) and three accessory proteins (TraY, TraM, IHF). It nicks the dsDNA at a nic site located in the origin of replication for transfer (oriT). A type 4 coupling protein (T4CP) connects to the relaxosome, which is still attached to the 5’ end of the nicked DNA, in order to begin transfer of the nicked strand (the transfer strand) through the T4SS on into a recipient cell [5][5]—all of which are essential for bacterial conjugation. After transportation, the remaining ssDNA in the donor is replicated to restore the complementary strand while the recipient cell replicates the transferred ssDNA, resulting in a dsDNA in both the donor and recipient after conjugation [6].

Why Do We Need to Utilize Conjugation?

The stx2 gene is in the chromosome, so any mutation in it will be inherited by the next generation of bacteria - a process known as vertical gene transfer. Thus far the Progenie system can spread in a population vertically but not horizontally. Horizontal gene transfer occurs when genes are passed directly between adjacent cells. Many foodborne pathogens require only a few functional cells in order to make people sick - for example, in an interview with Dr. Alison Weiss we learned only 30 individual STEC cells are needed to induce food poisoning symptoms in most people. Therefore we need to be able to eliminate the toxin in as many cells as possible, as quickly and thoroughly as we can. To do so, we need to make our system capable of transferring and propagating horizontally within a population of STEC.

Bacterial conjugation allows a donor cell (left) to share a plasmid it carries with a recipient cell (right). A donor cell’s conjugative pilus makes contact with a recipient cell, pulling the cells together. When the cells’ membranes fuse, a single replicated strand of a mobilizable plasmid is transferred to the recipient cell. The cells then dissociate, both of which now carry the plasmid.

Self-transmissible vs. mobilizable plasmids

There are two main types of conjugative plasmids: self-transmissible plasmids, and mobilizable plasmids. Self transmissible plasmids are those that have the complete T4SS genes to form pili and transfer over their genetic material on their own [8]. In contrast, mobilizable plasmids only encode parts of the relaxosome and the oriT [9]; mobilizable plasmids can only be conjugated if the cell already has a different self-transmissible plasmid.

Plasmid incompatibility

When constructing our mobilizable plasmid, φMINTO, for these experiments, plasmid incompatibility is the primary concern. Plasmid incompatibility is the tendency for certain plasmids to be incapable of coexisting in a cell line together. It is determined by the similarity of the plasmids’ vegetative replicon, which encodes and recruits replicative machinery for plasmid replication during cell growth and division [12]. If two plasmids within the same incompatibility group (Inc) are together in a cell, they compete for the same replicative machinery, which destabilizes the host cell, results in lower copy numbers of both incompatible plasmids, and/or results in the expulsion of one of the plasmids. Therefore, it is critical that Progenie is designed such that our phagemid is not incompatible with any other plasmids native to the target cells. See our contribution page to learn more specifics on plasmid incompatibility.

Application

We made our gene-eliminating plasmid mobilizable by hijacking existing conjugative machinery in host cells by adding an oriT to the plasmid. This new plasmid is called φMINTO. For preliminary experiments on testing the conjugation ability of Progenie, we will use a known F’ cell line, E. coli DH5α F’. F’ strains contain the genes for conjugation on a plasmid that was once integrated into the chromosome of the cell. To hijack the encoded F’ relaxosome replication machinery and mpf, we will add the F’ oriT to our phagemid. The presence of the oriT on the phagemid will allow it to be recognized and replicated by the DH5α F’ relaxosome, and transferred to recipient cells via the F’ mpf transfer system. In doing so, we have transformed our phagemid into a “mobilizable” unit that can act as a passenger in conjugation.