Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control

A novel method was devised to measure the number of plasmids in individual Escherichia coti cells. With this method, involving measurement of plasmid- driven expression of the green fluorescent protein gene by how cytometry, t he copy number distribution of a number of different plasmids was measured. Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide d istribution, suggesting that there is no copy number control for minichromo somes. When the selection pressure (kanamycin concentration) for minichromo somes was increased, the copy number of minichromosomes was also increased. At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected. This is evidence that there is no negative e lement for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome. However, higher copy numbers led to integration of the minichromosomes at the chromosomal o riC and to initiation asynchrony of the host chromosome. At a minichromosom e copy number of similar to 30, the cell's capacity for synchronous initiat ion is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.