Lecture 31 - Molecular and Cell Biology

Natural process of plasmid transfer is called conjugation.

Best understood for the E. coli F (fertility factor) plasmid (about 95 kb in size, 1 to 2 copies per cell).

E. coli cells carrying the F plasmid are called "male" and those that do not carry the F plasmid are called "female". The F plasmid from male cells can be transfered to female cells changing them into male cells.

F plasmid contains a tra (transfer) operon with 20 structural genes necessary for conjugation:

• Donor (male) cells mixed with recipient (female) cells ----- mating pairs form joined by a special structure called conjugation bridge (EM Figure). Conjugation bridge comes about because certain F genes encode proteins that form a specialized tubular extension on the cell surface of a donor cell called pilus that then establishes cell-cell contact with recipient cell.
  
• Transfer occurs because the plasmid is able to replicate by a process known as rolling circle replication. In this process, one strand of the F plasmid DNA is nicked at a specific sequence [the ori sequence] by a particular endonuclease encoded in the tra operon.
  
• Other proteins then bind to the 5' end of the nicked strand and guide that single strand (strand displacement) into the recipient cell (Figure). DNA polymerase can replicate a new daughter strand to replace the displaced strand.
  
• Once the displaced DNA strand crosses the conjugation tube and enters the recipient cell, a copy strand is replicated to yield double-stranded DNA which is finally converted into the usual circular and supercoiled form.
  
• In the end the original recipient cell is transformed into a donor cell and can now take part in spreading the plasmid.

Another property of F+ plasmids is that they may occasionally integrate into the bacterial chromosome and behave as though they were part of the chromosome.

Bacterial cells which contain an integrated plasmid are termed:
Hfr males
Hfr stands for high frequency of recombination.
These males can undergo conjugation when mixed with F- female bacteria. As conjugation begins, the entire bacterial chromosome (with integrated plasmid) begins to undergo rolling circle replication. It takes 90-100 minutes for an entire bacterial chromosome to undergo rolling circle replication and normally mating cells break apart (the conjugation tube breaks) before the entire displaced strand can cross. Thus the F- cell receives a large fragment of the bacterial chromosome and a small functionless fragment of the F plasmid. The F- bacterium remains F- {and would only become F+ if the entire chromosome + plasmid crossed the conjugation tube}. At a high frequency, homologous recombination occurs between the bacterial chromosome and the introduced DNA.

The integrated plasmid DNA (termed an episome) can be excised from the bacterial chromosome and be re-established in a plasmid form. Adjacent bacterial genes are often excised too. A plasmid the contains some DNA from the bacterial chromosome is termed an F' plasmid. The F' plasmid can be transfered to an F- female bacterium and complete transfer normally occurs. The recipients are converted into F+ males and there is usually stable retention of the F plasmid and the chromosomal genes.

Comments or questions should be directed to Cheryl.Davis@wku.edu
Last Modified: May 8, 1998
All contents copyright (C) 1997.
Western Kentucky University.