TY - JOUR

T1 - Distribution of minichromosomes in individual Escherichia coli cells

T2 - implications for replication control

AU - Løbner-Olesen, Anders

PY - 1999

Y1 - 1999

N2 - A novel method was devised to measure the number of plasmids in individual Escherichia coli cells. With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the 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 distribution, suggesting that there is no copy number control for minichromosomes. When the selection pressure (kanamycin concentration) for minichromosomes 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 element 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 oriC and to initiation asynchrony of the host chromosome. At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.

AB - A novel method was devised to measure the number of plasmids in individual Escherichia coli cells. With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the 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 distribution, suggesting that there is no copy number control for minichromosomes. When the selection pressure (kanamycin concentration) for minichromosomes 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 element 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 oriC and to initiation asynchrony of the host chromosome. At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.

KW - Chromosomes, Bacterial/genetics

KW - DNA Replication

KW - Escherichia coli/genetics

KW - Genes, Reporter

KW - Green Fluorescent Proteins

KW - Kinetics

KW - Luminescent Proteins/biosynthesis

KW - Plasmids

KW - Polymerase Chain Reaction

KW - Recombinant Proteins/biosynthesis

KW - Replication Origin

KW - Restriction Mapping

U2 - 10.1093/emboj/18.6.1712

DO - 10.1093/emboj/18.6.1712

M3 - Journal article

C2 - 10075940

VL - 18

SP - 1712

EP - 1721

JO - E M B O Journal

JF - E M B O Journal

SN - 0261-4189

IS - 6

ER -