Replication of the R6K plasmid during the Escherichia coli cell cycle

Abstract

The cell-cycle replication pattern of the R6K plasmid has been investigated by using the membrane-elution technique to produce cells labeUled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The high-copy plasmid R6K replicates exponentially in a cell-cycle-independent manner. A mini-R6K plasmid deleted for the onra origin of replication also replicates, exponentially in a cell-cycle--independent manner. Plasmid R6K is a relatively large, high-copy, conjugative resistance plasmid with three replication origins (7). In vivo, replication initiates from orio about 50% of the time, from oril3 about 35% of the time, and from ori-y about 15% of the time (1). R6K is unique in that most large, self-transmissible plasmids exist at low copy number and are DNA polymerase I independent, whereas most high-copy plasmids are small, nonconjugative, and DNA polymerase I dependent (6).Recently, we showed that the low-copy F plasmid and P1 prophage replicate in a cell-cycle-specific manner and that replication occurs when a constant mass per origin is achieved (4, 5). In contrast, the high-copy ColEl-type plasmids replicate in a cell-cycle-independent manner (9, 14). Considering copy number alone, one might expect R6K to replicate in a cell-cycle-independent manner. Taking into account structural and functional characteristics of the plasmid, one might expect R6K to replicate in a cell-cyclespecific manner. The replication pattern has not been studied previously. In order to solve this dilemma, we have now investigated the replication patterns of the R6K plasmid and mini-R6K plasmid pMF26 (deleted for oria) by using the membrane-elution technique and quantitative determination of label incorporated into plasmid DNA.Escherichia coli B/r F65 (provided by Alan Leonard) containing the minichromosome pAL49 (7.6 kb, Kanr; provided by A. C. Leonard) and the R6K plasmid (Ampr Strr; provided by D. R. Helinski) or the mini-R6K plasmid pMF26 (Ampr; provided by D. R. Helinski) was grown for six to nine generations in 100 ml of C medium (3) supplemented with the amino acids histidine (42 ,ug/ml), proline (46 ,g/ml), and thymine (20 jig/ml) and with glucose (0.4%) or succinate (0.4%) at 37°C in order to obtain exponential growth prior to starting a membrane-elution experiment. The exponentially growing cells (100 ml, 108 cells per ml) were labelled with[3H]methyl thymidine (60 to 80 Ci/mmol, 10 RCi/ml) for 5% of the doubling time and filtered onto a nitrocellulose membrane at the end of the labelling period. The cells were washed with medium of the same composition containing cold thymidine (100 ,ug/ml). The membrane apparatus was inverted, and newborn cells were eluted from the membrane with prewarmed C medium pumped at a rate of 2 ml/min in * Corresponding author. t Present address: Department of Chemical Engineering, University of California, Berkeley, CA 94720. a 37°C incubator. Fractions were collected for an interval of time equivalent to the labelling period. Ce...

A Monte Carlo simulation of plasmid replication during the bacterial division cycle

“…Because a culture of cells is not an amorphous mass of cytoplasm but a distribution of cells at various points of the cell cycle, and because many plasmids are known to attach to the cell membrane either loosely or to perform functions like partition and replication, such models should be used with caution. The results of membrane-elution experiments for high-copy plasmid replication during the cell cycle (Keasling et al, 1991(Keasling et al, , 1992bLeonard and Helmstetter, 1988;Zeuthen et al, 1972) and models, such as the one presented here, justify the use of models that do not account for cell-cycle phenomena.…”

Replication of prophage P1 is cell-cycle specific

“…That replication of the P1 prophage is altered by the presence of the minichromosome and the mini-R6K plasmid is unlikely. The timing of chromosome replication initiation (or the cell size at initiation of chromosome replication) is unaffected by the presence of the minichromosome (14), the F plasmid (10), and/or the R6K plasmid (11). Furthermore, the F plasmid and the R6K plasmid have the same replication patterns when they are alone in a cell as they do when they are present with the minichromosome in a cell (10,11).…”

“…The replication pattern of the high-copy plasmids has been consistently found to be cell-cycle independent (10,11,15,21), whereas the replication pattern of the low-copy plasmids (F, P1, and Rl) has been controversial. Recently, the contradictory reports of F plasmid replication were resolved by using the membrane-elution technique and quantitative measurement of label incorporated into plasmid DNA in cells grown at different rates (10).…”

“…Thus, it is a good indicator for the time when chromosome replication is initiated. The mini-R6K plasmid was chosen as a control for cell-cycle-independent replication because this high-copy plasmid replicates in a cell-cycleindependent manner (10,11). The data in Fig.…”

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.…”