Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation

Abstract

F plasmid replication during the Escherichia coli division cycle was investigated by using the membraneelution technique to produce cells labeled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The F plasmid replicated, like the minichromosome, during a restricted portion of the bacterial division cycle; i.e., F plasmid replication is cell-cycle specffic. The F plasmid replicated at a different time during the division cycle than a minichromosome present in the same cell. F plasmid replication coincided with doubling in the rate of enzyme synthesis from a plasmid-encoded gene. When the cell cycle age of replication of the F plasmid was determined over a range of growth rates, the cell size at which the F plasmid replicated followed the same rules as did replication of the bacterial chromosome-initiation occurred when a constant mass per origin was achieved-except that the initiation mass per origin for the F plasmid was different from that for the chromosome origin. In contrast, the high-copy mini-R6K plasmid replicated throughout the division cycle.Plasmids are stably inherited, independently replicating circles of DNA. As they are stably maintained in a culture, their replication must be attuned to the rate of cell growth and division. For low-copy plasmids, the F plasmid being an example, there must be at least one round of replication per division cycle. This requirement, coupled with a specific segregation mechanism, would enable low-copy plasmids to be stably maintained with few plasmid-free segregants. One way of coupling plasmid replication to the rate of cell growth and division would be to have replication of all plasmids occur at one specific time during the division cycle. The minichromosome, as well as the bacterial chromosome, replicates in a cell cycle-specific manner. Even though there are many copies of the minichromosome per cell, the minichromosomes replicate at the same time as the chromosomal origin initiates replication (13).The most recent results obtained by using the membraneelution method and quantitative autoradiography to measure F plasmid replication indicate that the F plasmid replicates throughout the division cycle (14); i.e., F plasmid replication is cell-cycle independent. Mini-F plasmids containing only one of several F plasmid replication origins (oriS) were also shown to replicate throughout the division cycle. These results are in sharp contrast to the large body of earlier work that had consistently shown a doubling in the ability of a cell to synthesize plasmid-encoded ,-galactosidase at a specific time during the division cycle (3,6,8,9,17,22). These enzyme experiments indicated that the F plasmid replicates in a cell-cycle-specific manner. How can the random replication of the F plasmid determined by autoradiography be reconciled with cell-cycle-specific replication determined by enzyme induction?We reinvestigated the problem of F plasmid replication during the division cycle by using the membr...

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

“…Specifically, interconversion between dimers and monomers as well as the amount of RepE must play critical roles in plasmid replication. At present, the question of how the control of RepE conversion (monomerization) and activation can be related to mini-F plasmid replication is not known, and further work is required to elucidate mechanisms that control the plasmid copy number, possibly coupled with that of the host cell cycle (38,39).…”

Nonrandom F-plasmid replication in Escherichia coli K-12

“…Again, the interreplication time of the chromosome shows a high degree of precision, whereas that of plasmid Rl DISCUSSION The nonrandom replication of F replicons described here is consistent with previous work on F'lac which used pulse induction of ,B-galactosidase by IPTG (3, 4, 17). This approach has been explicitly validated by control experiments using a minichromosome containing the lac gene (6). In recent years, some experiments have proposed, in contrast to the IPTG results, that F plasmids replicate randomly during the division cycle (1, 5, 10, 15).…”

“…However, more recently, a variety of methods have been used to suggest that replication of the same plasmid would be evenly spread throughout the cell division cycle (1,5,10,15). Recently, Keasling et al (6) used the membrane elution method and direct scintillation counting to determine the cell cycle replication pattern of the F plasmid. They found that the F plasmid replicated in a cell cycle-specific manner at a point midway between initiations of chromosomes or minichromosomes in the same cell.…”

“…The F plasmid replicates independently of the chromosome such that there is less than one copy per chromosomal origin (2,6,12,14). Early experiments with F'lac indicated that replication was confined to the final stage of the division cycle, coincident with termination of chromosome replication (3,4,17).…”

Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis

“…Fis binding to the numerous sites in the ␥ origin early in the cell cycle might block binding, which is essential for ␥-origin function (60). While studies have shown that an R6K replicon producing wt containing the ␥ and ␤ origins replicates continuously throughout the cell cycle (43,44), it remains unknown if replication of the isolated ␥ origin driven by copy-up is affected by the cell cycle and concomitant variations in Fis levels.…”