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

Abstract

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~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.

Single-cell Analysis of λ Immunity Regulation

“…16,17 Once protein synthesis was blocked (Materials and Methods), we observed no change in Measurement of phage release and ability to complement a temperature-sensitive repressor are described in Materials and Methods.…”

Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures

“…Because the pBR322 origin requires DNA polymerase I to replicate, colonies arise only when plasmid oriC is functional. If the plasmid oriC harbors a mutation that makes initiation less efficient, then it will not be able to compete effectively with the chromosomal copy of oriC for initiation factors, and the plasmid will not be capable of transforming cells (25,26). In the competition assay, plasmids harboring oriC 1,2,4/R5M were not able to transform polA strains, indicating that the function of the mutant oriC was impaired sufficiently to prevent it from competing with WT chromosomal oriC (transformation efficiencies were less than 0.1% of those obtained by using WT oriC plasmids).…”

Single-Copy Green Fluorescent Protein Gene FusionsAllow Accurate Measurement of Salmonella Gene Expression InVitro and during Infection of MammalianCells

“…Such fusions have proven to be useful for identifying bacterial genes induced during infection of eukaryotic cells or animal hosts and have allowed some assessment of bacterial gene expression levels in such complex environments. However, the variation in plasmid copy number that can occur in individual bacterial cells makes plasmidborne gene fusions unsuitable for measuring differences in gene expression between single bacteria (34,35). Furthermore, plasmid-borne GFP fusions are useful only for reporting the induction of genes; any reduction in fluorescence may simply reflect plasmid loss, because it is not possible to guarantee that plasmids are maintained in every bacterial cell in plant or animal hosts.…”