Plasmid replication functions: two distinct segments of plasmid R1, RepA and RepD, express incompatibility and are capable of autonomous replication

Abstract

The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.

AT-rich region and repeated sequences – the essential elements of replication origins of bacterial replicons

“…The in vitro helix opening assays with KMnO 4 probing and P1, S1 or mung bean nucleases were also performed to find the site, where helix melting occurs and where the replication complex is exactly formed Mukhopadhyay et al, 1993;Krause et al, 1997;Speck and Messer, 2001;Ozaki et al, 2006). Analogous biochemical analyses were applied to determine the plasmids' replication origins, for instance the ColEI (Tomizawa et al, 1977), P1 (Abeles, 1986;Abeles et al, 1990;Wickner et al, 1990;Papp et al, 1993;Park et al, 1998, F (Eichenlaub et al, 1977;Zzaman et al, 2004), R6K (Kunnimalaiyaan et al, 2004), RK2 Doran et al, 1998) and other (Danbara et al, 1980;Diaz & Staudenbauer, 1982;Kuzminov et al, 1997;Diaz-Lopez et al, 2003;Schvartzman et al 2010). For identification of the archaeal and eukaryotic origins, where the recognition of the replication start point is much more difficult, other methods, such as marker frequency analysis utilizing whole-genome DNA microarrays and replication initiation point analyses (Gerbi & Bielinsky, 1997;Bielinsky & Gerbi, 1998, 1999Abdurashidova et al, 2000;Gomez & Antequera, 1999;Romero & Lee, 2008), have been applied.…”

Isolation of the kanamycin resistance region (Tn2350) of plasmid R1drd-19 as an autonomous replicon

“…2). Another segment capable of autonomous replication, recently described by Danbara et al, carries a function that has been called RepD (14). These authors locate the RepD function between ISlb and oriV of Rldrd-19.…”

“…It seems, th'erefore, that a potential site for Rldrd-19 replication exists within the Tn2350 transposon. The origin (or origins) of replication used by Rldrd-19 has not been mapped directly on the parent plasmid, but two segments of Rldrd-19 capable of autonomous replication have been described (14,28).…”

Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1

“…The incompatibility phenotype of the copB gene was analyzed by construction of the strains carrying wild-type copB chimeric plasmids and Rl plasmids (wild-type or copB mutants). In addition, we used plasmid R100 as a test plasmid, since this plasmid has been claimed to be more sensitive than Rl in such tests (9). Moreover, it was recently claimed by Burger et al (6) that the Rl copB gene, when cloned on multicopy plasmids, exerts incompatibility against R100.…”