Behavior of Sister Copies of Mini-F Plasmid after Synchronized Plasmid Replication in Escherichia coli Cells

Abstract

To clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F. Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sister oriC copies of the Escherichia coli chromosome.

We have recently demonstrated that sister copies of the oriC-proximal half of the Escherichia coli chromosome are cohesive with each other and that the cohesion is released almost all at once in the late period of chromosome replication (5, 15). The MukFEB complex (17, 18) participates in the sister chromosome cohesion (15). Sister chromosomes form two separated nucleoids after release from the cohesion. Sister copies of the oriC region are localized in the pole-proximal regions of each nucleoid, and sister copies of the replication-terminal region are localized near the constriction site before cell division (2, 11, 12, 13).

F plasmid DNA molecules are partitioned with fidelity to both daughter cells during cell division cycles owing to F plasmid's own partition system. The partition system, constituted of the plasmid genes sopA and sopB and the cis-acting plasmid region sopC, is essential for the stable maintenance of the plasmid during proliferation (9, 14; for a review, see reference 4). The sister copies of mini-F plasmid are replicated in the mid-cell position and migrate in opposite directions up to the 1/4 and 3/4 cellular positions without coupling but with cell elongation and the formation of tethered copies at the cellular positions, where they remain until the cell divides (11). Movement to these positions is not coupled with cell division. Single fluorescent foci representing mini-F plasmid DNA were seen to be localized in mid-cell, whereas pairs of fluorescent foci were seen to be localized at the 1/4 and 3/4 cellular positions within the nucleoid. In contrast, DNA molecules of a mini-F plasmid lacking the partition genes are randomly localized in cytosolic spaces of the cell poles. The partition system of mini-F is thus essential for the proper localization of plasmid molecules (4, 11).

Mini-F plasmid can be replicated at all stages of the division cycle, and the plasmid is segregated in a nonrandom fashion similar to that of the E. coli chromosome and minichromosomes (3, 8). Three models can be imagined concerning the separation and migration of replicated sister copies of mini-F plasmid. First, sister copies of mini-F are separated from each other and migrate from mid-cell to the 1/4 and 3/4 cellular positions immediately or shortly after replication, being independent of the mechanism of host sister chromosome cohesion. Second, sister copies of mini-F are cohesive with each other, and the cohesion is released at the same time that host sister chromosome cohesion is released by an unknown cellular signal. In the second model, a common cohesion mechanism might act for both mini-F and the E. coli chromosome. Third, sister copies of mini-F are cohesive for a long term, and then the cohesion is released independently of the host chromosome cohesion.

In this work, we analyzed the behavior of sister copies of mini-F after synchronized replication of mini-F by fluorescence in situ hybridization (FISH).

Kinetics of plasmid maintenance after temperature shift.

E. coli cells harboring a temperature-sensitive repE mutant mini-F plasmid were synchronized for replication of the mutant mini-F plasmid by temperature shifts as described below. We used E. coli cells harboring the mini-F plasmid pKP2375 (10) to synchronize the initiation of replication of the plasmid. As shown in Fig. 1A, pKP2375 carries the origin ori-2, the repE gene, and the sopABC genes (9, 14) but not the DNA segment controlling the postsegregation killing function of F plasmid (7). The plasmid has a mutated repE gene encoding a temperature-sensitive initiation protein essential for replication of the plasmid. The plasmid has the chloramphenicol resistance-conferring cat gene and the spectinomycin resistance-conferring spc gene. The plasmid was introduced into bacterial cells of strain YK1100 (a tryptophan-deficient mutant derived from W3110) (17) by transformation, resulting in strain KK346.

FIG. 1.

Structure of plasmids pKP2375 (A) and pOT100 (B). The filled regions are derived from F plasmid. ts, temperature sensitive.

An exponentially growing culture of KK346 at 30°C in L medium (6) was separated into three subcultures. One of them was further incubated at 30°C. The second subculture was transferred to the nonpermissive temperature of 42°C. The third one was transferred to 42°C for 90 min and then transferred back to 30°C. Samples were removed at intervals, diluted, and spread on L agar plates, which were then incubated overnight at 30°C. The colonies thus obtained were examined for chloramphenicol resistance by incubation on plates containing 5 μg of chloramphenicol/ml.

As shown in Fig. 2, in the first subculture, which was continuously incubated at 30°C, 100% of cells were resistant to chloramphenicol even after 240 min, indicating that the plasmid was stably maintained at 30°C. In contrast, in the second subculture, which was incubated continuously at 42°C, the cells tested retained the plasmid for the first 60 min after the temperature shift and thereafter plasmid-free cells appeared in the population (Fig. 2). The time at which 50% of the plasmid was lost was 30 min, corresponding to the doubling time at 42°C. These results suggest that the copy number of plasmid DNA had been reduced to 1 per cell in almost all cells after 60 min of growth at 42°C. Thereafter, plasmid-free cells were produced by cell divisions without plasmid replication. Fifty percent of cells had lost the plasmid after 90 min at 42°C (Fig. 2).

FIG. 2.

Kinetics of plasmid maintenance of the temperature-sensitive mini-F plasmid pKP2375 after temperature shift. Cells of E. coli trpC mutant strain KK346, which harbored the temperature-sensitive mini-F plasmid pKP2375, were exponentially grown at 30°C in L medium (6). The culture was separated into three subcultures. The first subculture was further incubated at 30°C (filled circles). The second subculture was transferred to 42°C (open circles). The third subculture was incubated at 42°C for 90 min and then transferred back to 30°C (triangles). One hundred colonies with each sample were analyzed for chloramphenicol resistance (Cm^r).

The third subculture was incubated at 42°C for 90 min and then transferred back to 30°C. The proportion of plasmid-bearing cells was reduced from 50 to 40% during the first 15 min after the temperature shift to 30°C. However, the proportion of plasmid-bearing cells became constant thereafter (Fig. 2). This result with the third subculture indicates that plasmid replication is restored after the transfer to 30°C and that sister copies are segregated into both daughter cells. We repeated the experiment and obtained similar results. Thus, the partition mechanism of mini-F acts normally under the experimental conditions.

Analysis of plasmid DNA molecules by FISH.

To examine the behavior of sister copies of plasmid DNA after replication, cells were removed at intervals from a culture that was synchronized for plasmid replication under the same experimental conditions as those of the third subculture (Fig. 3A) and analyzed by FISH according to the method of Niki and Hiraga (11). Plasmid DNA of pOT100 (Fig. 1B) was labeled with the fluorescent compound Cy3 (Amersham) and used as a probe for FISH analysis. Plasmid pOT100 was constructed by ligation of pKP2375 and the high-copy-number plasmid pBR322 (1, 16) after digestion with EcoRI and BamHI.

FIG. 3.

Kinetics of cells with fluorescent foci of mini-F in temperature shifts. (A) Exponentially growing cells of KK346 harboring pKP2375 in L medium at 30°C were transferred to 42°C for 90 min and then transferred back to 30°C (at zero time). Samples were removed at intervals and analyzed for mini-F DNA molecules by FISH. (B) Cells with one, two, three, and four fluorescent foci. (C) The vertical axis represents the proportions of cells with one, two, three, and four foci relative to the sum of cells with one or multiple foci. Filled circles, cells with a single focus; open circles, cells with two foci; filled triangles, cells with three foci; open triangles, cells with four foci. Approximately 200 cells were scored in each sample.

Forty percent of total cells had either one or two fluorescent foci after the incubation at 42°C for 90 min (at zero time), as expected. Among the cells with fluorescent foci, the majority (93%) had a single fluorescent focus while the remainder (7%) had two fluorescent foci (Fig. 3B). After the temperature shift to 30°C, the proportion of cells with a single focus immediately decreased and, concomitantly, the proportion of cells with two foci immediately increased (Fig. 3C). These results indicate that sister plasmid copies can be separated within 5 min after replication. Cells with three or four foci appeared after 15 min, and the proportions of these types of cells gradually increased (Fig. 3C), suggesting further replications of the plasmid. In cells with a single fluorescent focus, the focus was located in mid-cell (Fig. 4A). In cells with two fluorescent foci, these foci were located in the 1/4 and 3/4 cellular positions in cell length (Fig. 4B). These results indicate that the subcellular localization is normal, as described previously (11). If the cellular concentration of SopA and SopB was abnormally higher or lower than the normal level under the experimental conditions, mini-F plasmid DNA molecules were localized in cytosolic spaces of the cell poles or between two nucleoids and plasmid-free segregants appeared at high frequency upon cell divisions (11, 12, 14). The results shown in Fig. 2 and 4 demonstrate that mini-F plasmid is normally partitioned under the experimental conditions. Furthermore, we analyzed the amount of SopA and SopB proteins in cells growing under experimental conditions by Western blotting with anti-SopA and -SopB antibodies. The results show that the amount of these proteins per plasmid-bearing cell was nearly constant during the experiment (data not shown). This result is reasonable, because expression of the sopA-sopB operon is autoregulated by the SopA and SopB proteins (4, 9).

FIG. 4.

The intracellular localization of mini-F plasmid. (A) Cells with one fluorescent focus at zero time; (B) cells with two fluorescent foci at 5 min. The horizontal axis represents the subcellular position in the cell length in a cell pole.

Further discussion.

Sister oriC copies of the E. coli chromosome are cohesive for approximately 40 to 50 min at 30°C in temperature-sensitive dnaA mutant cells synchronized for initiation of chromosome replication (5). The present results show that sister copies of mini-F plasmid can be separated within 5 min after replication. The results eliminate model 3. Model 1 is consistent with the results. Since host cells grew randomly and/or asynchronously under the experimental conditions, there were cells of all stages of the division cycle when the culture was transferred back to 30°C. Therefore, model 2 cannot be eliminated completely by the present results.