TY - JOUR
T1 - Distribution of minichromosomes in individual Escherichia coli cells
T2 - implications for replication control
AU - Løbner-Olesen, A
PY - 1999/3/15
Y1 - 1999/3/15
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
SN - 0261-4189
VL - 18
SP - 1712
EP - 1721
JO - E M B O Journal
JF - E M B O Journal
IS - 6
ER -