14. Multiple systems ensure plasmid survival in bacterial populations

12.14 Multiple systems ensure plasmid survival in bacterial populations

When a genome consists of a single replicon, it must also contain a system for segregating the progeny to daughter cells. Here we consider these systems and their connection with replication itself.

We have mentioned that each type of plasmid is maintained in its bacterial host at a characteristic copy number:

• Single-copy control systems resemble that of the bacterial chromosome and result in one replication per cell division. A single-copy plasmid effectively maintains parity with the bacterial chromosome.
  
• Multicopy control systems allow multiple initiation events per cell cycle, with the result that there are several copies of the plasmid per bacterium. Multicopy plasmids exist in a characteristic number (typically 10 V20) per bacterial chromosome.

Copy number is primarily a consequence of the type of replication control mechanism. The system responsible for initiating replication determines how many origins can be present in the bacterium. Since each plasmid consists of a single replicon, the number of origins is the same as the number of plasmid molecules.

Single-copy plasmids have a system for replication control whose consequences are similar to that governing the bacterial chromosome. A single origin can be replicated once; then the daughter origins are segregated to the different daughter cells.

Multicopy plasmids have a replication system that allows a pool of origins to exist. If the number is great enough (in practice >10 per bacterium), an active segregation system becomes unnecessary, because even a statistical distribution of plasmids to daughter cells will result in the loss of plasmids at frequencies <10 ^V6.

Plasmids are maintained in bacterial populations with very low rates of loss (<10 ^V7 per cell division is typical, even for a single Vcopy plasmid). The systems that control plasmid segregation can be identified by mutations that increase the frequency of loss, but that do not act upon replication itself. Several types of mechanism are used to ensure the survival of a plasmid in a bacterial population. It is common for a plasmid to carry several systems, often of different types, all acting independently to ensure its survival. Some of these systems act indirectly, while others are concerned directly with regulating the partition event. However, in terms of evolution, all serve the same purpose: to help ensure perpetuation of the plasmid to the maximum number of progeny bacteria:

Figure 12.31 Intermolecular recombination merges monomers into dimers, and intramolecular recombination releases individual units from oligomers.

Because the multiple copies of a plasmid in a bacterium consist of the same DNA sequences, they are able to recombine. Figure 12.31 demonstrates the consequences. A single intermolecular recombination event between two circles generates a dimeric circle; further recombination can generate higher multimeric forms. Such an event reduces the number of physically segregating units. In the extreme case of a single-copy plasmid that has just replicated, formation of a dimer by recombination means that the cell only has one unit to segregate, and the plasmid therefore must inevitably be lost from one daughter cell. To counteract this effect, plasmids often have site-specific recombination systems that act upon particular sequences to sponsor an intramolecular recombination that restores the monomeric condition. Mutations in these systems increase plasmid loss, and therefore have a phenotype that is similar to partition mutants. (The bacterial chromosome itself may have a similar system to deal with the consequences of recombination occurring between homologous sequences in the daughter chromosomes produced by a replication cycle.)

Figure 12.32 Plasmids may ensure that bacteria cannot live without them by synthesizing a long-lived killer and a short-lived antidote.

Addiction systems, operating on the basis that "we hang together or we hang separately," ensure that a bacterium carrying a plasmid can survive only so long as it retains the plasmid. There are several ways to ensure that a cell dies if it is "cured" of a plasmid, all sharing the principle illustrated in Figure 12.32 that the plasmid produces both a poison and an antidote. The poison is a killer substance that is relatively stable, whereas the antidote consists of a substance that blocks killer action, but is relatively short lived. When the plasmid is lost, the antidote decays, and then the killer substance causes death of the cell. So bacteria that lose the plasmid inevitably die, and the population is condemned to retain the plasmid indefinitely. These systems take various forms. One specified by the F plasmid consists of killer and blocking proteins. The plasmid R1 has a killer that is the mRNA for a toxic protein, while the antidote is a small antisense RNA that prevents expression of the mRNA.

Figure 12.30 A common segregation system consists of genes parA and parB and the target site parS.

True partition systems act upon duplicate DNA molecules to ensure that they reside on either side of the septum at cell division. Probably all low copy number plasmids have such a system. Systems that have been characterized for the plasmids F, P1, and R1 have the generally similar organization depicted in Figure 12.30. There are two trans-acting proteins and a single cis-acting site. In the cases of both P1 and F, the smaller of the two proteins binds to the cis-acting site. In spite of their overall similarities, there are no significant sequence homologies between the corresponding genes or cis-acting sites (for review see Hiraga, 1992).

How does a true partition system segregate replica plasmids to different daughter cells? We may imagine two general types of system:

Figure 12.26 Attachment of bacterial DNA to the membrane could provide a mechanism for segregation.

• Extrinsic structures to which the plasmids must bind are limiting. The model for a single-copy plasmid is the same as illustrated previously in Figure 12.26. The cell must contain only two sites, and each copy of the plasmid binds to one. The sites could be membrane Vbound or could simply constitute regions of the bacterial chromosome itself. A disadvantage of this type of model is that many different types of plasmids are found in bacterial populations, which makes it necessary to suppose that there are separate and exclusive pairs of sites for each type of plasmid.
  
• Segregation is an intrinsic process, in which the replica plasmids pass through some stage equivalent to chromosome pairing. Following association of plasmids into pairs, a partition mechanism moves one member of each pair into opposite daughter cells. The difficulty remains that the nature of this mechanism is not easy to envisage, and it is still necessary to explain how the cell can cope with partition of multiple types of plasmids.

Except for the fact that proteins required for partition are coded by the plasmid and bind to cis-acting sites, we know little about the mechanism of segregation. The importance to the plasmid of ensuring that all daughter cells gain replica plasmids is emphasized by the existence of multiple, independent systems in individual plasmids that ensure proper partition.

Reviews

Hiraga, S. (1992). Chromosome and plasmid partition in E. coli. Ann. Rev. Biochem 61, 283-306.