Plasmids
Plasmids are double stranded DNA molecules that can replicate independent of the chromosome. Most plasmids are circular although some species of bacteria, such as the causative agent of Lyme's disease Borrelia burgdorferi, have linear plasmids. In order for a plasmid to replicate it must contain a region of DNA that functions as a replicon.
Replicons and ori's
The terms replicon and ori are often used as synonyms.
Replicons contain the information necessary to begin and end the replication of DNA. Within each replicon the synthesis of new DNA (replication) begins at a defined location called origin of replication (oriV or ori). The origin of replication is usually about 300 bp long and is recognized by specific proteins that initiate replication. These proteins may be chromosomally or plasmid encoded. All DNA polymerases require a primer, a short chain of single stranded DNA or RNA, before replication can begin because they lack the ability to synthesize new nucleotide chains. In most cases this primer is a short chain of RNA nucleotides that is produced by a RNA polymerase. This primer hybridizes to the single stranded DNA at the oriV and is then extended by a DNA polymerase in the 5' to 3' direction.
Replication can be either unidirectional with only one replication fork proceeding from the oriV or bidirectional with two replication forks proceeding from the oriV in opposite directions.
Plasmids with the same replicon belong to the same incompatibility group and cannot coexist in the same cell.
Plasmid Incompatibility groups
Plasmids that cannot coexist in the same cell are said to be incompatible. Every plasmid belongs to one incompatibility group (inc). There are more than 30 known incompatibility groups and probably many more that have yet to be discovered.
Plasmids belonging to the same incompatibility group interefere with the stable inheritance of each other so that in any given cell one or the other plasmid is disproportinately represented. Incompatibility commonly arises when two plasmids replicate by the same mechanism. Plasmids that replicate by the same mechanism are incompatible with one another and belong to the same incompatibility group but they are compatible with plasmids that replicate by other mechanisms.
Plasmids belonging to different incompatibility groups can coexist in the same cell.; ie they are compatible with each other.
pMB1 / ColE1 incompatibility group
Many of the plasmids currently in use today carry a replicon from the naturally occurring plasmid pMB1 or plasmid ColE1. These plasmids were independently isolated (1) but have extensive functional and structural homologies including the same origin of replication and thus the same mode of replication. Plasmids with the pMB1 or ColE1 replicon are maintained at 15-30 copies per cell. The pMB1 replicon, carried by pBR322 and its derivatives, is incompatible with the ColE1 replicon because of their near identity.
The pMB1 and ColE1 replicons are compatible with the p15A replicon, carried by pACYC184 and pACYC177, the CloDF13 replicon, the ColK replicon, and the RSF1030 replicon and others.
The pMB1 / ColE1 replicon consists of the origin of replication, oriV, and three genes encoding RNA II, RNA I, and Rop. RNA I and Rop keep plasmid copy numbers between 15-30 copies per cell by negatively regulating DNA replication.
RNA II is the primer required for DNA replication. The RNA II primer is processed from a longer pre-primer transcript ~500 nucleotides long by RNAse H after a stable RNA-DNA hybrid is formed near oriV. The formation of a stable RNA-DNA hybrid is essential for processing fo the pre-primer by RNAse H.
RNA I negatively controls plasmid copy number by preventing the cleavage of RNA II from pre-primer. RNA I is ~100 nucleotides long and the same DNA that encodes pre-primer also encodes RNA I on the opposite strand. RNA I interferes with the formation of the stable pre-primer RNA-DNA hybrid thus preventing RNase H from cleaving RNA II from the pre-primer.
In addition to RNA I, plasmid copy number is also negatively regulated by the product of the rop (repressor of primer) gene. The rop gene encodes a 63 amino acid polypeptide, Rop, which is completely conserved in pMB1 and ColE1. Rop acts in trans to repress transcription of the RNA II primer (1).
Mutations or deletions that prevent RNA I or Rop function result in dramatically higher plasmid copy numbers as the result of higher plasmid replication. For example pUC plasmids have a single nucleotide substitution that prevent RNA I function, resulting in 500-700 copies per cell.
FI incompatibility group
The F plasmid is a member of the FI incompatibility group. The copy number of F is only about 1 per cell.
FII incompatibility group
Members of this incompatibility group include R1, R6-5, and R100 (plasmid R100 is also referred to as NR1 or R222) (2). These plasmids have a low copy number of 2-5 plasmids per cell.
pSC101 is a derivative of R6-5 and is positively controlled by its own protein.
X incompatibility group
Plasmids with the R6K replicon belong to the X incompatibility group. The require the product of pir for replication.
p15a incompatibility group
10-12 copies per cell. Plasmids with the p15a replicon include pACYC184 and pACYC177 and their derivatives.
References
1. G. Cesareni, M. A. Muesing, B. Polisky, PNAS 1982, v.79 p.6613-6317.
2. J. R. Scott, Microbiological Reviews 1984, v. 48(1) p. 1-23.