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Plasmids and conjugation
©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
VI. GENE TRANSFER SYSTEMS
The following sections on plasmids and conjugation, transformation, and transduction will provide some background information on how and why the systems work, but the main emphasis will be on their use in gene transfer and amplification.
VI A. PLASMIDS AND CONJUGATION
(This section treats movement of DNA without cell-cell contact. Please realize that Plasmids and conjugation mixes rather different things: conjugation can certainly occur without plasmids, and many plasmids cannot be moved by conjugation.)
A plasmid is defined as a replicon, or replicating piece of DNA, that is stably inherited in an extrachromosomal state. In older literature, the term episome was used for plasmids capable of integration into the chromosome, but this term has largely gone into disuse. It typically exists as a covalently closed circular piece of double stranded DNA that has the capability of replicating autonomously and it is this property that leads to its isolation and physical recognition. The closed covalent nature of their structure allows them to be separated from chromosomal DNA by either gel electrophoresis or cesium chloride buoyant density gradients.
There are two features held in common by all plasmids:
They all have replication functions. In the simplest case, this consists of one or more origins of replication with the trans-acting proteins necessary for replication either being encoded by the plasmid itself or "borrowed" from the normal host replication machinery. The broad host range of some plasmids is at least in part explained by their multiple replication systems that allow them to function in a variety of dissimilar hosts. (Question for the reader: when does a plasmid become a "chromosome"?)
All plasmids fall into only one of the many existing incompatibility groups. Two plasmids are incompatible if either is less stable in the presence of the other than it was by itself. There are more than 30 incompatibility groups thus far described with no upper limit in sight. Incompatibility, whose genotypic designation is inc, is often a necessary consequence of a plasmid's desire to maintain a certain copy number in the cell. If plasmids of a given incompatibility group have a certain copy number that they attempt to maintain, then a competition will result when two plasmids of the same incompatibility group are found in the same cell. Whichever plasmid is able to replicate faster, or has some other advantage, will be represented to a disproportionate degree among the copies allowed by the incompatibility system. Surprisingly, plasmids can also be incompatible when they both possess the same functions for partitioning themselves into daughter cells (see paragraph 5 below).
There are a variety of features that are not found in all plasmids but which are common enough that they deserve mention:
Many plasmids contain genes uninvolved in either replication or incompatibility. Such genes can encode properties like antibiotic resistance (and therefore give rise to the terms "resistance" or "R" factors), degradation of complex macromolecules, production of bacteriocins, resistance to various heavy metals, synthesis of antibiotics, or virulence factors necessary for infection of animal or plant hosts. This is by no means an exhaustive list of the various functions that can be encoded on a plasmid. Clearly, virtually any gene can be found on a plasmid.
A second common property is the ability to promote the transfer of the plasmid itself from one cell to another, termed conjugative ability. Conjugation is defined as the unidirectional transfer of genetic information between cells by cell-to-cell contact. As such, it is not restricted to plasmids, but can occur with any DNA so long as the critical elements below are present in the cell (there are even conjugative transposons). This latter requirement for contact distinguishes conjugation from transduction and transformation which will be discussed below. The term "unidirectional" refers to the fact that a copy of the plasmid is transferred from one cell, termed the "donor", to another cell, termed the "recipient". There are two dissimilar functions involved in conjugative ability: the first is a site of initiation of transfer that is called either oriT or mob. The former term is a mnemonic for "origin of transfer" and the second is short for "mobility". In each case they refer to a site on the DNA and not to a diffusible product. The second group of functions are those proteins that act at these sites and cause the range of functions necessary for mobilization to occur. These are encoded by the tragenes and have a variety of functions including the formation of the pilus that makes contact with the recipient cell and seems to be involved in drawing the donor and recipient cells together. This brings about a region of membrane contact and it appears a conjugation bridge of some sort is formed. The products of the tra genes are involved in both the regulation and the physical construction of these events. Some event in this sequence triggers the nicking of a site, termed oriT, by a specific single-strand nuclease and a subsequent binding of one or more "pilot" proteins to the free 5' end of the DNA. These proteins seem to function in the subsequent replication of the transferred DNA with one serving as a "primase". A single-strand is then transferred from this end to the recipient while a "rolling circle" form of replication occurs in the donor. If the DNA being transferred is a plasmid, it is made double-stranded and circularized in the recipient, whereupon it can presumably replicate. If the transfer DNA is chromosomal, circularization cannot occur, but in some way a complementary strand is generated and homologous recombination with the chromosome can occur (in any case, the incoming DNA must become associated with a replicon if it is to be inherited). It is possible for a plasmid to be non-conjugative and yet mobilizable (if the traproducts are supplied by another plasmid) so long as an oriT site is encoded on the plasmid (whoever has the oriT site is transferred). Finally, a plasmid lacking both the trafunctions and oriT functions would be non-conjugative and non-mobilizable. Many applications in inverse genetics employ small plasmids containing oriT regions, where the tra functions are supplied by another plasmid in the cell.
Plasmids have mechanisms that increase the likelihood that, following cell division, both daughter cells will contain a copy of the plasmid. The partition functions (often termed par) responsible for this work by a variety of mechanisms including monomerization of plasmid multimers (better to have many monomers than a few multimers) and association of the plasmid with membranes (which apparently helps physically separate the plasmids). While we refer to a plasmid being "lost" by a cell, the actual mechanism is almost certainly that the cell never received the plasmid at the previous cell division due to inappropriate partitioning. Such loss is termed segregation. For both low and high copy-number plasmids, this "loss" occurs at (very roughly) l% frequency, though some exceptionally stable plasmids have been found, presumably because of a set of different par functions. Some plasmids have evolved a system, with effects like the par systems above, that "prevents" segregation by killing any daughter cell that has not received a plasmid. They do this by producing a relatively long-lived killing function (kil) and a short-lived kill override (kor) function. A daughter without the plasmid will have the kil product, but will not be able to maintain the necessary amount of kor product to survive. To the experimenter, these systems will look like partitioning systems, since, in mutants lacking these, plasmid-free segregants will be more frequently detected. They can also appear to be inc functions.
Occasionally, it is necessary to isolate plasmid-free derivative of a strain currently containing a plasmid, a procedure termed curing. These can be sought (i) spontaneously (perhaps replica printing isolated colonies if the plasmid confers a scorable phenotype); (ii) following an enrichment (again, if the plasmid confers a growth phenotype); (iii) by selecting a different, but incompatible, plasmid into the cell; (iv) or by treatment with elevated temperature or chemicals such as acridines, ethidium bromide, sodium dodecyl sulfate and novobiocin (since the first two chemicals are known as mutagens, they should be used with restraint).
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