REPLICATION, INCOMPATIBILITY, AND STABILITY FUNCTIONS OF R PLASMID NRI

Abstract

ABSTRACT The incompatibility ( inc ), copy number control ( cop ), and origin of replication ( ori ) of the 90 kilobase ( kb ) R plasmid NR1 (FII incompatibility group) and its copy mutant pRR12 have been located on two contiguous PstI fragments of size 1.1 kb ( inc, cop ) and 1.6 kb ( ori ). The cop mutant pRR12 is compatible ( inc ) with NR1, but is incompatible with other cop 12 derivatives. Evi-dence is presented that the incompatibility of inc FII plasmid derivatives is a manifestation of plasmid copy number control. Hybrid plasmids have been constructed which contain the Pst I 1.1 kb fragment of NR1 ligated to the Pst I 1.6 kb fragment of pRR12 (and vice versa). Copy number control, the ability to exclude an incompatible resident plasmid by transformation, and the ability to be excluded from host cells by transformation with an incom-patible plasmid were determined solely by the source of the Pst I 1.1 kb fragment. These findings suggest that, if incompatibility is due to the in-teraction of a repressor with a receptor site on a plasmid, both the structural gene for the repre-sor and its receptor site are located on the Pst I 1.1 kb fragment. Using a nitrocellulose filter binding assay, RNA polymerase binding sites and transcription initiation sites were mapped on the Pst I 1.1 and 1.6 kb fragments. Plasmids have been construct-ed which contain two copies of the Pst I 1.1 kb ( inc. cop ) fragment. It has not been possible to construct plasmids containing two Pst I 1.6 kb ( ori ) fragments in cis . This suggests that some DNA structural feature prevents the stable maintenance of plasmids containing two ori fragments. NR1 plasmid derivatives have been isolated which are Inc − Cop − owing to a deletion or to an insertion of a Tn element outside of the replication genes. This suggests that plasmid regions adjacent to the replication genes may affect copy number and incom-patibility, perhaps owing to transcriptional read-through from the external region. Although recom-binant mi niplasmids derived from NR1 are capable of autonomous replication, these plasmids are not stably inherited unless they also harbor a stability (stb) function which is located in a region of NR1 distal from the replication genes, stb confers stability only jn cis, suggesting that it plays a structural role in plasmid inheritance, rather than providing a diffusible gene product, stb may function in the partitioning of plasmid molecules to daughter cells at cellular division. The r-determi-nants component of incFII R plasmid NR84 has been shown to be able to replicate autonomously when Proteus mirabilis cells harboring NR84 are cultured in high concentrations of ampicillin in order to ob-tain R plasmid drug resistance gene amplification.