Plasmids

• Plasmids: naturally occurring extrachromosomal nucleic acids capable of independent replication in the cell (contain origins of replication) (Fig. 7.1)
•  The overwhelming majority: circular, double-stranded DNA molecules

Plasmids found in bacteria.

•  The size of bacterial plasmids ranges from a few kb to more than 500 kb, or from encoding one or two genes to several hundred genes.
•  The genes encoded by a plasmid often provide an additional advantage for the host cell like allowing it to grow in otherwise hostile environments. Examples are genes that confer resistance to antibiotics or to heavy metals, such as mercury and lead.  (Fig. 7.1)
• In the absence of selective pressure plasmids are usually dispensable to the host cell or can even be regarded as a burden because resources must be spent in replicating them.
•  Some bacterial plasmids have the ability to transfer themselves from one cell to another, thus spreading horizontally in a population. (Vertical spreading would be through growth and cell division.) The ability can go even so far that some plasmids transfer themselves from one bacterial species to another.

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.

Replication
Plasmids may also replicate by bi-directional (Fig.7.2) or uni-directional replication normally during the same time period that the chromosomal DNA is undergoing replication. In this way, vertical transmission of plasmids (from parent to progeny) can also occur. Transfer of plasmids by conjugation would be an example of horizontal transmission.

Hfr Plasmids
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 called Hfr cells.
• 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.

Copy number
Low copy # or high copy # of plasmids in a cell is a characteristic of the plasmid.

• F plasmids are always present in only 1 or 2 copies per host cell.
• plasmid replication regulated in coordination with replication of bacterial chromosome
• regulation at the level of initiation of replication
• High copy number plasmids have independent controls
• May have 3-200 copies per cell

• Certain plasmids cannot be maintained together in the same cell. This phenomenon is refered to as plasmid incompatability.
• Incompatible plasmids: use same mechanism to regulate replication initiation. Such two plasmids together will occur in a specific copy number in the cell, but there is no mechanism that ensures their equal number. By chance one plasmid is often lost after a series of cell divisions.

• Plasmids can be used to carry DNA fragments (Fig 7.8).
• Cells transformed with plasmids can be selectively grown on special media (Fig. 7.3).

Activity

Quiz

Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., Darnell, J., 2000, Molecular Cell Biology, 4th Ed., W.H. Freeman and Company, NY, New York.  ISBN 0-7167-3136-3.