Bacterial Conjugation - Mechanism

Mechanism

The prototypical conjugative plasmid is the F-plasmid, or F-factor. The F-plasmid is an episome (a plasmid that can integrate itself into the bacterial chromosome by homologous recombination) with a length of about 100 kb. It carries its own origin of replication, the oriV, and an origin of transfer, or oriT. There can only be one copy of the F-plasmid in a given bacterium, either free or integrated, and bacteria that possess a copy are called F-positive or F-plus (denoted F+). Cells that lack F plasmids are called F-negative or F-minus (F-) and as such can function as recipient cells.

Among other genetic information the F-plasmid carries a tra and trb locus, which together are about 33 kb long and consist of about 40 genes. The tra locus includes the pilin gene and regulatory genes, which together form pili on the cell surface. The locus also includes the genes for the proteins that attach themselves to the surface of F- bacteria and initiate conjugation. Though there is some debate on the exact mechanism of conjugation it seems that the pili are not the structures through which DNA exchange occurs. This has been shown in experiments where the pilus are allowed to make contact, but then are denatured with SDS and yet DNA transformation still proceeds. Several proteins coded for in the tra or trb locus seem to open a channel between the bacteria and it is thought that the traD enzyme, located at the base of the pilus, initiates membrane fusion.

When conjugation is initiated by a signal the relaxase enzyme creates a nick in one of the strands of the conjugative plasmid at the oriT. Relaxase may work alone or in a complex of over a dozen proteins known collectively as a relaxosome. In the F-plasmid system the relaxase enzyme is called TraI and the relaxosome consists of TraI, TraY, TraM and the integrated host factor IHF. The nicked strand, or T-strand, is then unwound from the unbroken strand and transferred to the recipient cell in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated either independent of conjugative action (vegetative replication beginning at the oriV) or in concert with conjugation (conjugative replication similar to the rolling circle replication of lambda phage). Conjugative replication may require a second nick before successful transfer can occur. A recent report claims to have inhibited conjugation with chemicals that mimic an intermediate step of this second nicking event.

If the F-plasmid that is transferred has previously been integrated into the donor’s genome some of the donor’s chromosomal DNA may also be transferred with the plasmid DNA. The amount of chromosomal DNA that is transferred depends on how long the two conjugating bacteria remain in contact. In common laboratory strains of E. coli the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can then be integrated into the recipient genome via homologous recombination.

A cell culture that contains in its population cells with non-integrated F-plasmids usually also contains a few cells that have accidentally integrated their plasmids. It is these cells that are responsible for the low-frequency chromosomal gene transfers that occur in such cultures. Some strains of bacteria with an integrated F-plasmid can be isolated and grown in pure culture. Because such strains transfer chromosomal genes very efficiently they are called Hfr (high frequency of recombination). The E. coli genome was originally mapped by interrupted mating experiments in which various Hfr cells in the process of conjugation were sheared from recipients after less than 100 minutes (initially using a Waring blender). The genes that were transferred were then investigated.