PubMed · 6259723
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R P Novick. 1980. Plasmids.. https://doi.org/10.1038/scientificamerican1280-102
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Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.
A number of Escherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained in Pseudomonas stutzeri ATCC 17588. A restrictionless mutant of P. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With the E. coli cloning vector pHSG298, transformation frequencies of up to 2 x 10(7) transformants/micrograms DNA were achieved. This frequency is comparable to that obtained for CaCl2-mediated transformation of E. coli; thus, direct cloning of DNA into P. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G + C genera in cases in which E. coli is not a suitable heterologous cloning host.
A survey of colicins in the ECOR reference collection of Escherichia coli is presented. Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E. coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts. Multiple representatives of two Col plasmids, low-molecular-mass ColE1 plasmids and high-molecular-mass, conjugative ColIa plasmids were isolated from the ECOR collection and were examined with a combination of restriction fragment and Southern analysis. These data suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa plasmids represent a family of distinct plasmid lineages united by the presence of the colicin Ia operon.