Characterization of a mini-ColC1 plasmid

Abstract

An in vitro constructed plasmid, pVH15, consisting of the entire genome of the plasmid ColE1, the tryptophan operon of Escherichia coli, and regions of the bacteriophage PHI80pt190, spontaneously gave rise in E. coli to a mini-ColE1 plasmid consisting of approximately one-half of the ColE1 genome and a small segment of phi80pt190 DNA. This mini-ColE1 plasmid, designated pVH51, has a molecular weight of approximately 2.1 X 10(6) and possesses a single EcoRI restriction site. Heteroduplex analyses showed that about 90% of the pVH51 plasmid hybridizes to about 50% of the ColE1 plasmid. Phenotypically, pVH51 did not produce colicin E1 but conferred immunity to this colicin. The number of mini-ColE1 plasmid molecules per cell was maintained at a four- to fivefold higher level than normal ColE1. A mini-ColE1 hybrid plasmid, designated pML21 and consisting of pVH51 and the kan fragment of plasmid pSC105 inserted at the EcoRI restriction site of mini-ColE1, was maintained at a lower copy number level than pVH51. As in the case of normal ColE1, both pVH51 and pML21 continued to replicate in the presence of chloramphenicol. The promotion of conjugal transfer of pVH51 and pML21 by a self-transmissible plasmid was greatly reduced compared with normal ColE1.