Replication of ColE2 and ColE3 plasmids: The regions sufficient for autonomous replication

Abstract

We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions. A 1.3 kb segment of each plasmid is sufficient for autonomous replication. Plasmids carrying this segment retain the replication properties of the original plasmid. The 1.3 kb segment consists of three functional portions. Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid. Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter. The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid. The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s). The action of the trans-acting factor(s) on the origin is plasmid specific. The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.

Comparative analysis of the replicon regions of eleven ColE2-related plasmids

“…The Escherichia coli K-12 strains used are RRlbtuB (4, 36), JM109 (79), AGlrecAJ (17), and AKlOlrecA::TnlO (57a), a derivative of JM103 (38). Other bacterial strains have been described elsewhere (19,70,80). Plasmids used are pUC118 and pUC119 (74) and the 17 ColE plasmids listed in Table 1.…”

“…Plasmids used are pUC118 and pUC119 (74) and the 17 ColE plasmids listed in Table 1. Other plasmids have been described elsewhere (19,72,80,81) (19), and each plasmid is distinguished by a suffix representing the original plasmid; e.g., pEC22-CA42 derived from ColE2-CA42. A derivative of pNT51incl,2 (72) carrying the incA or incB region of each plasmid (pTI51A or pTI51B series, respectively) was constructed by replacing the BamHI-Eco47III or BamHI-EcoRV fragment with an appropriate fragment of each plasmid of the pEC series: the 0.6-kb BamHI-HincII fragments of pEC22-CA42, pEC52, pEC72, and pEC92; the 0.7-kb BamHI-HincII fragment of pEC22imm; the 0.6-kb BglII-HincII fragment of pEC42-CT9; and the 1.1-kb BamHI-ClaI fragments of pEC62-CT14 and pEC82 for the pTI51A series; and the 1.8-kb BamHI-HincII fragments of pEC22-CA42, pEC52, pEC72, and pEC92; the 0.8-kb BamHIHincII fragment of pEC22imm; the 1.7-kb BamHI-HincII fragment of pEC42-CT9; and the 1.3-kb BamHI-ClaI fragments of pEC62-CT14 and pEC82 for the pTI51B series.…”

“…Restriction analyses (8,32,76,77) (Fig. 1) have suggested that each of these ColE2-related plasmids contains a region which shows a certain structural similarity to the region of ColE2-P9 or ColE3-CA38 containing the 1.3-kb segments sufficient for autonomous replication (19). The fragment of each ColE2-related plasmid containing the corresponding region ligated to the fragment containing a chloramphenicol resistance gene as described in Materials and Methods results in an autonomously replicating plasmid (data not shown).…”

“…Plasmids ColE3-CA38, ColE7-K317, and ColE8-J belong to one incompatibility group (11,39), and plasmids ColE5-099, ColE6-CT14, and ColE9-J belong to another incompatibility group (11). Plasmids ColE2-P9 and ColE3-CA38 require plasmid-coded Rep proteins (19,22) and host DNA polymerase I (25,66) for plasmid replication. The Rep protein binds to the origin in a plasmid-specific manner (23) and synthesizes a unique primer RNA (ppApGpA) for initiation of the leading-strand DNA synthesis by DNA polymerase I at the origin (68, 69; see Fig.…”

Specific Interaction between the Initiator Protein (Rep) and Origin of Plasmid ColE2-P9

“…It is present at about 10 to 15 copies per host chromosome (1,12). The initiator protein (Rep; 35 kDa) of plasmid ColE2 is the only plasmid-specified trans-acting factor required for the initiation of plasmid replication (17,18,37).…”