The F sex factor plasmid of Escherichia coli contains a pair of genes, ccdA and ccdB, whose protein gene products are involved in an unusual feature of plasmid maintenance. The CcdB protein is a cytotoxin that becomes activated when the F plasmid is lost, thereby killing the F- segregant cells. In F+ cells, the CcdA protein protects against the lethal effects of CcdB. In the present study we show that ccdA and ccdB expressions are negatively autoregulated at the level of transcription. Genetic studies showed that repression required at least ccdB; ccdA alone was without effect, and ccdB alone was not examined because it is lethal. Ccd-operator complexes were purified and contained a mixture of both CcdA and CcdB proteins; however, we could not conclude from our results whether CcdA was necessary for DNA binding or autorepression. By using restriction fragments of the promoter-operator region, we obtained results indicating that at least two DNA-binding sites existed for the Ccd protein(s). Subsequent footprinting of the binding sites showed protection over about a 113-base-pair region encompassing the putative promoter-operator and the beginning of the ccdA gene.

In Escherichia coli, concentrations of a mini-F plasmid with two origins of replication (oriV and oriS) were 50% lower in fast-growing cells than in slow-growing cells. By contrast, a mini-F plasmid deleted for oriV maintained a uniform concentration in both fast- and slow-growing cells, and in this behavior the plasmid mimicked the control by the host of chromosomal origin (oriC) concentration.

Determination of plasmid concentration (plasmids/unit volume) is a tedious, time-consuming undertaking when classical procedures are used. Measurement of relative plasmid concentration (plasmids/chromosome equivalent) is quicker but quite inaccurate for very small, low copy number plasmids such as mini-F. Also, one cannot compare validly the plasmid/chromosome equivalents at different growth rates because the amount of chromosome per unit cell volume varies. Therefore, we have devised a method that simultaneously avoids both of these difficulties. We have done this by adapting the Grunstein-Hogness technique [Grunstein, M. and Hogness, D. (1975) Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. U.S.A. 72 3961–3965] of in situ colony lysis with known cell volumes applied to a filter membrane with the aid of a filtration manifold. The amount of specific gene sequence was then determined by hybridization with a radioactive probe. We have succesfully used the technique to measure the concentration of mini-F and native F plasmids that vary in size from 5.0 to 95.0 kb. This method offers several advantages over currently employed techniques. These include: sensitivity, speed, and cost and equipment that is required.

There are DnaA protein-binding sites in at least one F origin of replication, and only potentially leaky dnaA(Ts) mutations had ever been used in previous studies indicating that F replication was independent of the dnaA gene product. Here we show that an Escherichia coli dnaA::Tn10 host which does not make a dnaA gene product cannot sustain autonomous or integrated F plasmid maintenance.