Cloning of replication, incompatibility, and stability functions of R plasmid NR1

Abstract

The region of R plasmid NR1 that is capable of mediating autonomous replication was cloned by using EcoRI, SalI, and PstI restriction endonucleases. The only EcoRI fragment capable of mediating autonomous replication in either a pol+ or a polA host was fragment B. SalI fragment E joined in native orientation with the part of SalI fragment C that overlapped with EcoRI fragment B, and also two contiguous PstI fragments of sizes 1.6 and 1.1 kilobases from EcoRI fragment B-mediated autonomous replication. When these individual SalI fragments were cloned onto plasmid pBR313 or the individual PstI fragments were cloned onto plasmid pBR322, none of these single fragments could rescue the replication of the ColE1-like vectors in a polA host, even in the presence of a compatible "helper" plasmid derived from a copy mutant of NR1. In contrast to the results reported for closely related R plasmid R6, EcoRI fragment A of NR1 could not rescue the replication of ColE1 derivative RSF2124 in a polA(Am) mutant or in a polA(Ts) mutant at the restrictive temperature. Although capable of autonomous replication, EcoRI fragment B of NR1 (or smaller replicator fragments cloned from it by using other restriction enzymes) was not stably inherited in the absence of selection for the recombinant plasmid. When EcoRI fragment B was ligated to EcoRI fragment A of NR1, the recombinant plasmid was stable. Thus, EcoRI fragment A contained a stability (stb) function. The stb function did not act in trans since EcoRI fragment B was not stably inherited when a ColE1 derivative (RSF2124) ligated to EcoRI fragment A was present in the same cell. A cointegrate plasmid consisting of EcoRI fragment B of NR1 ligated to RSF2124 was also not stably inherited, whereas only EcoRI fragment B was unstable when both RSF2124 and EcoRI fragment B coexisted as autonomous plasmids in the same cell. The incompatibility gene of NR1 was shown to be located within the region of overlap between SalI fragment E and the PstI 1.1-kilobase fragment. A copy mutant of NR1 (called pRR12) was found to have greatly reduced incompatibility with NR1; this Inc- phenotype is cis dominant.

Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3‐dioxygenase activity in free and immobilized Escherichia coli cells

“…Consequently, the genetic make up of the plasmid and the host strain type together with culture conditions have a great importance in the relationship between the plasmid copy number and the growth rate. (4), generation 15(5), generation 17 (7), generation 20 (8).…”

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two‐component system regulator

“…Mechanisms mediating the stable inheritance of plasmids in actively dividing bacterial cultures have been studied for more than two decades, and multiple plasmid-encoded genes and cis-acting partition (par ) loci implicated in this process have been found on a variety of extrachromosomal replicons, including pSC101, NR1, R1, F, and R6K (Meacock and Cohen, 1980;Miki et al, 1980;Nordström et al, 1980;Ogura and Hiraga, 1983;Roberts et al, 1990). Plasmid partitioning is also affected by host-encoded genes that influence the ability of replicating plasmid DNA molecules to form concatamers (i.e.…”

Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters.

“…The oligocopy pSC101 plasmid encodes a cis-acting genetic determinant termed par that accomplishes stable inheritance of the plasmid in populations of actively dividing bacterial cells in the absence of selective pressure (Meacock and Cohen, 1980). Regions functionally analogous to the pSC101 par locus have been identified on other bacterial plasmids, including NRI (Miki et al, 1980), RI (Nordstrom et al, 1980), F (Ogura and Hiraga, 1983) and P1 (Austin and Abeles, 1983), and on the yeast plasmid pSR1 (Jearnpipatkul et al, 1987). In contrast to certain of these other partition loci, the par locus of pSC101 does not include a proteincoding sequence (Miller et al, 1983).…”