The prototype for conjugative plasmids is the F-plasmid, also called the F-factor. The F-plasmid is an episome (a plasmid that can integrate itself into the bacterial chromosome by homologous recombination) of about 100 kb length. It carries its own origin of replication, the oriV, as well as an origin of transfer, or oriT. There can only be one copy of the F-plasmid in a given bacterium, either free or integrated (two immediately before cell division). The host bacterium is called F-positive or F-plus (denoted F+). Strains that lack F plasmids are called F-negative or F-minus (F-).
Among other genetic information, the F-plasmid carries a tra and a 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, polymeric proteins that can attach themselves to the surface of F- bacteria and initiate the conjugation. Though there is some debate on the exact mechanism, the pili themselves do not seem to be the structures through which the actual exchange of DNA takes place. This has been shown through tests in which the pilus are allowed to make contact, then denatured by SDS detergent. DNA transformation still proceeds. Several proteins coded for in the tra or trb loci seem to open a channel between the bacteria. It is thought that the traD enzyme located at the base of the pilus is involved in DNA exchange, by iniating membrane fusion. .
When conjugation is initiated, via a mating signal, a relaxase enzyme creates a nick in one plasmid DNA strand at the origin of transfer, or oriT. The 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 transferred, or T-strand, is unwound from the duplex plasmid and transferred into the recipient bacterium 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 necessitate 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 becomes integrated into the host genome, donor chromosomal DNA may be transferred along with plasmid DNA.The certain amount of chromosomal DNA that is transferred depends on how long the bacteria remain in contact; for common laboratory strains of E. coi the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can be integrated into the recipient genome via homologous recombination.
A culture of cells containing non-integrated F plasmids usually contains a few that have accidentally become integrated, and these are responsible for those low-frequency chromosomal gene transfers which do 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) and investigating which genes were transferred
Among other genetic information, the F-plasmid carries a tra and a 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, polymeric proteins that can attach themselves to the surface of F- bacteria and initiate the conjugation. Though there is some debate on the exact mechanism, the pili themselves do not seem to be the structures through which the actual exchange of DNA takes place. This has been shown through tests in which the pilus are allowed to make contact, then denatured by SDS detergent. DNA transformation still proceeds. Several proteins coded for in the tra or trb loci seem to open a channel between the bacteria. It is thought that the traD enzyme located at the base of the pilus is involved in DNA exchange, by iniating membrane fusion. .
When conjugation is initiated, via a mating signal, a relaxase enzyme creates a nick in one plasmid DNA strand at the origin of transfer, or oriT. The 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 transferred, or T-strand, is unwound from the duplex plasmid and transferred into the recipient bacterium 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 necessitate 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 becomes integrated into the host genome, donor chromosomal DNA may be transferred along with plasmid DNA.The certain amount of chromosomal DNA that is transferred depends on how long the bacteria remain in contact; for common laboratory strains of E. coi the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can be integrated into the recipient genome via homologous recombination.
A culture of cells containing non-integrated F plasmids usually contains a few that have accidentally become integrated, and these are responsible for those low-frequency chromosomal gene transfers which do 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) and investigating which genes were transferred
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