Multicopy Plasmids Affect Replisome Positioning in Bacillus subtilis

The DNA replication machinery, various regions of the chromosome,and some plasmids occupy characteristic subcellular positionsin bacterial cells. We visualized the location of a multicopyplasmid, pHP13, in living cells of Bacillus subtilis using anarray of lac operators and LacI-green fluorescent protein (GFP).In the majority of cells, plasmids appeared to be highly mobileand randomly distributed. In a small fraction of cells, thereappeared to be clusters of plasmids located predominantly ator near a cell pole. We also monitored the effects of the presenceof multicopy plasmids on the position of DNA polymerase usinga fusion of a subunit of DNA polymerase to GFP. Many of theplasmid-containing cells had extra foci of the replisome, andthese were often found at uncharacteristic locations in thecell. Some of the replisome foci were dynamic and highly mobile,similar to what was observed for the plasmid. In contrast, replisomefoci in plasmid-free cells were relatively stationary. Our resultsindicate that in B. subtilis, plasmid-associated replisomesare recruited to the subcellular position of the plasmid. Extendingthis notion to the chromosome, we postulated that the subcellularposition of the chromosomally associated replisome is establishedby the subcellular location of oriC at the time of initiationof replication.

INTRODUCTION

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Introduction

The DNA replication machinery and regions of chromosomal DNAoccupy characteristic positions within bacterial cells (reviewedin references 15, 17, 35, and 56). In Bacillus subtilis, theorigin of chromosomal replication, oriC, of the single circularchromosome is typically positioned at or near midcell in cellswith a single unreplicated origin and at or near the cell quarters(future midcell) in growing cells that contain a partly duplicatedchromosome (10, 31, 32, 36, 40, 57, 58, 61, 62). In Escherichiacoli, the location of sister oriC regions appears to be at thecell quarters or closer to the cell poles (14, 29, 38, 45, 47,54, 55).

Replication of the B. subtilis chromosome typically takes placein centralized replication factories. The replisome (the multiproteincomplex that includes the replicative polymerase, helicase,and associated proteins that are present at the replicationfork) is localized at or near midcell or at the cell quarterswhich will be midcell following division (33, 34). During replication,chromosomal DNA moves to the replication factory, is duplicated,and then moves away from the central factory (34). Replicationin E. coli also appears to take place at or near midcell orat positions that will be midcell (3, 8, 28, 49, 50, 54, 55).In contrast, in Caulobacter crescentus, the replication factoryis initially positioned at the stalked cell pole and graduallymoves to midcell as replication proceeds (24).

The mechanisms that establish and maintain the subcellular positionof the replisome are not known, nor is it known how the locationof oriC is established or maintained, although several genesand sites are known to contribute to positioning of the originregion (4, 13, 15, 17, 25, 32, 43, 66). Since origins and thereplication machinery must interact, at least initially duringthe initiation of replication, it is possible that either thereplisome recruits the origin or the origin recruits the replisometo a particular location.

Plasmids provide a good tool for investigating the requirementsfor positioning the replisome. Plasmids utilize much of thesame replication machinery as the cell chromosome, yet theyare physically separate. Many processes can contribute to plasmidstability, including decatenation and multimer resolution. Manyplasmids encode partitioning systems that contribute to thestability and subcellular positioning of the plasmid. For example,in E. coli, both the unit copy plasmid F and plasmid prophageP1 are found predominantly at midcell or the cell quarters,and this positioning depends on a functional partitioning systemnamed Par in P1 and Sop (stability of plasmids) in F (12, 14,19, 37, 44, 50).

The Par systems found on many plasmids and in many bacterialchromosomes contribute to plasmid and chromosome partitioning.They consist of ParA (an ATPase and DNA binding protein), ParB(a DNA binding protein), and parS (the binding site for ParB)(reviewed in references 1, 9, 15, and 17). The subcellular locationof ParB (7, 37) and SopB (18, 27) generally correlates withthe location of the plasmid and depends on the presence of cognatebinding sites (7, 18, 37). In the absence of the Par system,the plasmids appear in nucleoid-free regions of the cell, typicallynear the cell poles. An E. coli oriC-based plasmid is also foundin these regions, indicating that the chromosomal origin perse does not contain information to establish and maintain theposition at midcell and the cell quarters (46). However, theoriC-based plasmid is positioned at midcell and the cell quartersif it contains a plasmid Par system (46).

In B. subtilis, chromosomally encoded ParA (Soj) and ParB (Spo0J)contribute to positioning the oriC region of the chromosome,most likely by facilitating separation of sister origins andprobably not by recruiting parS sites to a specific subcellularlocation (31, 32). However, when placed on an otherwise unstableplasmid, chromosomal Par systems can stabilize the plasmid (11,39) and can even position plasmids to the midcell and cell quarters(65).

The subcellular location of multicopy plasmid RK2 and its ParBhomolog, KorB, has been visualized in E. coli, and both arefound predominantly at midcell and the cell quarters (2, 19,52, 53). RK2 is also found at these positions in two of itsother hosts, Pseudomonas aeruginosa and Vibrio cholerae, indicatingthat the mechanisms for determining subcellular positioningare conserved among these bacteria (19, 52, 53). In addition,the number of plasmid foci per cell was much less than the knownplasmid copy number, indicating that the plasmid molecules areprobably clustered (19, 52, 53).

Multicopy plasmid R1 is positioned at midcell and toward thecell poles, probably past the cell quarters (22, 41, 64). Asin the case of RK2, the number of visible plasmid foci was significantlyless than the known plasmid copy number, indicating that plasmidmolecules are probably clustered (22, 48, 63, 64). Mutationsin the R1 partitioning system result in altered positioning,and the plasmids are predominantly in nucleoid-free regions(22). The R1 partitioning system is not homologous to the type1 Par systems first described for P1 and F. Rather, R1 parMencodes an actin-like ATPase that forms helical filaments neededfor partitioning (41, 42, 59). ParR is a DNA binding proteinthat holds R1 plasmids together (23). ParM interacts with theParR-DNA complex to promote partitioning (41).

Several high-copy-number plasmids do not have known partitioningsystems. Derivatives of ColE1, like pUC19, are present in E.coli at levels of approximately 50 to 200 copies per cell. Asseen with the lower-copy-number plasmids R1 and RK2, there aremany fewer foci of pUC19 per cell than the known plasmid copynumber, indicating that the plasmids are largely clustered predominantlyat midcell or the cell quarters (53). In addition to these clusters,approximately 30% of cells appear to have plasmids rapidly movingthroughout the cytoplasm (53).

In all of the cases described above, it is not clear if theplasmids have any effect on assembly or the subcellular positioningof the replisome. In fact, since the plasmid foci are often,but not always, at positions known to be occupied by the replisome,it has been suggested that perhaps the plasmids must go to thereplisomes located at midcell and the cell quarters to be replicated(48, 52, 53). In this way, the position of the replisome wouldbe involved in determining the position of the plasmid. Alternatively,the plasmid could recruit the replisome, as has been suggestedfor partition-defective plasmids and oriC-based plasmids thatappear to localize randomly (46, 52). In addition, the presenceof a multicopy plasmid in C. crescentus affects the number ofreplisome foci per cell, and the replisome foci are found inpositions not normally associated with chromosomal replication(24). These findings indicate that perhaps the subcellular positionof the replisome is established by the position of the plasmidat the time of initiation of replication.

We visualized both the subcellular position of a multicopy plasmidin B. subtilis and the effects of plasmids on the positioningof the replisome, building on previous work that looked onlyat one or the other of these aspects. We found that in a majorityof cells, the plasmids are dynamically distributed throughoutthe cell. The presence of a multicopy plasmid altered the numberand position of foci of the replisome. In plasmid-containingcells, we observed dynamic and polar replisome foci that wereuncharacteristic of the chromosomal replisome pattern but reflectedthe dynamic or polar patterns of the plasmids. Our results indicatethat in B. subtilis, plasmids can influence the location ofthe replisome, likely by recruiting the replication machineryto the subcellular position of the plasmid. Applying this notionto the chromosome, we propose that the replisome position isestablished by the subcellular location of oriC at the timeof initiation of replication.

MATERIALS AND METHODS

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Materials and Methods

Strains and plasmids. All B. subtilis strains used were derived from strain JH642(trpC2 pheA1) (51) and are listed in Table 1. Genetic manipulationswere performed by using standard protocols (16).

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TABLE 1. B. subtilis strains used

pUB110. pUB110 was originally isolated from Staphylococcus aureus andhas been used extensively as a cloning vector in B. subtilis(5). It encodes resistance to kanamycin (or neomycin) and phleomycin(or bleomycin), replicates by the rolling circle mechanism,and exists in B. subtilis at a level of $~$ 50 copies per chromosome(reviewed in references 5 and 26).

pHP13. pHP13 has the replication functions of pTA1060, a naturallyoccurring cryptic plasmid in some Bacillus strains that replicatesby the rolling circle mechanism (5, 26). pHP13 is maintainedin B. subtilis at a level of approximately five copies per chromosomeand contains the selectable cat (chloramphenicol resistance)and mls (resistance to macrolide-lincosamide-streptogramin Bantibiotics) genes, the ColE1 origin of replication for E. coli,and some useful cloning sites (5).

pJW101 (pHP13-lacO). We constructed a derivative of pHP13, pJW101, that containsan array of lac operators, and we used LacI-green fluorescentprotein (GFP) or LacI-cyan fluorescent protein (CFP) to visualizethe subcellular location of this plasmid in living cells ofB. subtilis. Briefly, the approximately 5-kb EcoRI-HindIII fragmentfrom pLAU43 (29) containing an array of lac operators was ligatedinto pHP13. The ligation mixture was transformed into B. subtiliscells containing plasmid pJL52, a derivative of pHP13 that ismissing the cat gene such that incoming DNA containing pHP13sequences and cat can recombine onto the resident plasmid (30).Chloramphenicol-resistant transformants were selected, and theplasmid was purified and retransformed to generate cells containingpJW101.

Fusions to fluorescent proteins. Fusions of lacI to the mut2 allele of gfp or the wt allele ofcfp were used to visualize the subcellular locations of lacoperator arrays and have been described in detail previously(34). dnaX-gfpmut2 and dnaX-yfpmut2 (33, 34) were used to visualizethe tau subunit of the replisome. These fusions are functionalfusions that are present as single copies in the chromosomeat the endogenous dnaX locus. polC-gfp (catalytic subunit) wasalso used to visualize the replisome (33).

Media and growth conditions. For all experiments, cells were grown with vigorous shakingat 30°C in S7 defined minimal medium with MOPS (morpholinepropanesulfonicacid) buffer at a concentration of 50 mM rather than 100 mM(21, 60); the medium was supplemented with 0.1% glutamate, therequired amino acids, and either 1% glucose or 1.5% succinateas the carbon source. Antibiotics were used at standard concentrations(16) to maintain selection for plasmids or fusions, as needed.

Fluorescence microscopy. Cells were sampled during exponential growth (optical densityat 600 nm, 0.2 to 0.3) at 30°C in defined minimal mediumwith succinate or glucose as the carbon source. The vital membranedye FM4-64 was added to the culture at a concentration of 0.05µg/ml for 10 min before samples were taken. DAPI (4,6-diamidino-2-phenylindole)was added at a concentration of 0.1 µg/ml before cellswere placed on pads of 1% agarose in 1x T' base (16) with 1mM MgSO₄. Time-lapse images were obtained at room temperature.Fluorescence was viewed with a Nikon E800 microscope equippedwith a x100 differential interference contrast objective byusing appropriate filters. Images were obtained with a cooledcharge-coupled device camera (model C4742-95; Hamamatsu) andwere analyzed by using the Improvision OpenLab software.

RESULTS AND DISCUSSION

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Results and Discussion

Visualization of a multicopy plasmid in living cells. We visualized the multicopy plasmid pHP13 in live B. subtiliscells by using the lacO/LacI-GFP (or CFP) system (14, 34, 62).pHP13 is a 4.7-kb plasmid that undergoes rolling circle replication,does not have a known partitioning system, and is present atan average level of approximately five copies per chromosome(16, 26). We inserted an array of lac operators (lacO) intopHP13 to obtain plasmid pJW101, which was referred to as pHP13-lacO.A LacI-GFP fusion protein was constitutively expressed to allowvisualization of the subcellular location of the lacO-containingDNA.

We observed fluorescent foci (Fig. 1A and B) in 80% of the cells(228 of 286 cells observed) with pHP13-lacO expressing LacI-GFPduring exponential growth in defined minimal succinate medium.Succinate was used as a carbon source to support slow growth,which greatly simplified the bacterial cell cycle by minimizingthe amount of multifork replication. The appearance of fluorescentfoci was dependent on the presence of the lac operators in theplasmid. In cells with pHP13 (without the lacO array), we detectedfaint distributed fluorescence (Fig. 1A) but no defined fociin any of the 134 cells examined.

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FIG. 1. Visualization of pHP13-lacO and the replisome in live cells. Cells were grown in defined minimal succinate (A to C and E to G) or glucose (D) medium, and samples were removed during exponential growth and used for microscopy. DAPI staining of DNA is indicated by blue, FM4-64 vital membrane staining is indicated by red (except in panel E), and GFP (or YFP) staining is indicated by green. Differential interference contrast (DIC) light images are shown in some panels. (A) LacI-GFP in cells with pHP13-lacO (strain JDW55) or pHP13 (no lac operators; strain JDW46). (B) Examples of cells (four images) with relatively fixed, usually polar foci of LacI-GFP in cells with pHP13-lacO (strain JDW55). (C) Dynamic plasmid foci revealed by time-lapse microscopy of LacI-GFP in cells containing pHP13-lacO (strain JDW55). Images were captured at 0, 1.5, 3.0, 4.3, 5.8, 7.3, 8.8, 10.0, 11.6, and 13.1 s. (D) DnaX-GFP in cells with no plasmid (strain KPL382) and cells with pUB110 (strain MER9). (E) Visualization of pHP13-lacO with LacI-CFP (red) and the replisome with DnaX-YFP (green) in the same cells (strain JDW78). (F) Time-lapse microscopy of DnaX-GFP in plasmid-free cells (strain KPL382). Images were captured at 0, 2.0, 3.6, and 5.3 s. (G) Time-lapse microscopy of DnaX-GFP in cells containing pHP13 (strain MER5). Images were captured at 0, 2.5, 6.2, 9.5, and 12.3 s. The arrows indicate relatively stationary replisome foci that are most likely associated with chromosomal replication.

Of the plasmid-containing cells with fluorescent foci, 89% (203of 228 cells) had at least one highly dynamic focus (Fig. 1C).The positions of the foci were different in images capturedas little as 1 to 2 s apart. In addition, some of the dynamicfoci were distinct and others were more diffuse, perhaps dueto rapid movement during microscopic exposure or to movementin and out of the plane of focus. Of the 203 cells with at leastone dynamic plasmid focus, 15 also had an apparently stationaryfocus.

The other 11% of cells with visible plasmid foci (25 of 228cells) contained a single, apparently fixed focus (Fig. 1B).These foci appeared to be brighter and much less mobile thanthe dynamic foci and may have represented multiple plasmidsclustered together. Most appeared as a distinct focus near acell pole and sometimes near midcell, and others occupied amuch larger area of the cell (Fig. 1B). The visualization ofboth dynamic and apparently fixed foci of pHP13-lacO was similarto that observed for pUC-based plasmids in E. coli, althoughthe positions and proportions were quite different (53).

Cells with plasmids have more foci of the replisome. Cells with either pHP13 or another multicopy rolling circleplasmid, pUB110 (16, 26), had more replisome foci than plasmid-freecells (Fig. 2). We visualized the B. subtilis replication machineryusing a functional GFP fusion to dnaX (34), which encodes thetau subunit of the replicative DNA polymerase. During exponentialgrowth in defined minimal succinate medium, the vast majorityof plasmid-free cells had either one or two foci of the replisome(33) (Fig. 2A), and a minority of these cells ( $~$ 10%) had no focusof the replisome. Under these growth conditions, in cells containingpHP13 or pUB110 there was a marked increase in the proportionof cells with three or more foci of the replisome (Fig. 2A),and there was a decrease in the proportion of cells with nofoci of the replisome.

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FIG. 2. Increased numbers of replisome foci in plasmid-containing cells. Cells were grown in defined minimal succinate (A) or glucose (B) medium and sampled during exponential growth. Membranes were visualized with FM4-64, and the replisome was visualized with DnaX-GFP. The percentages of cells with different numbers of replisome foci are shown for strains with no plasmid (strain KPL382), pHP13 (strain MER5), and pUB110 (strain MER9). n is the total number of cells examined.

At higher growth rates in defined glucose medium, there wasalso an increase in the proportion of plasmid-containing cellswith three or more replisome foci compared to plasmid-free cells(Fig. 1D and 2B). As expected under these growth conditions(33), >95% of plasmid-free cells had at least one focus ofthe replisome (Fig. 2B).

An increase in replisome foci due to the presence of pHP13 orpUB110 was also observed in cells expressing a GFP fusion toPolC, the catalytic subunit of the replicative DNA polymerase(data not shown), similar to the effects of plasmids on DnaX-GFPfoci.

Altered positioning of replisome foci in plasmid-containing cells. We measured the position of the replisome during exponentialgrowth in minimal succinate medium (slow growth) in cells witha single replisome focus. During slow growth without a plasmid,a significant fraction of cells have no visible replisome foci,and many have only a single focus (33) (Fig. 2A). In cells witha single focus, the replisome was located predominantly at ornear midcell (Fig. 3A). Approximately 10% of these cells hada focus in the polar one-third of the cell (Fig. 3D).

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FIG. 3. Subcellular position of replisome foci is altered in plasmid-containing cells. Cells were grown in defined minimal succinate medium, sampled during exponential growth, and processed for microscopy as described in the legend to Fig. 2. In cells with a single replisome focus, the distance from the focus to the nearest cell pole (a) and cell length were measured. This created a systematic bias such that in every cell, a was less than one-half the cell length. To remove this bias, a random number generator was used to assign each focus a $~$ 50% chance of remaining as scored and a $~$ 50% chance of recalculating a as the distance from the focus to the far pole. Panels A to C are plots of cell length versus the position of the replisome as a fraction of the cell length. The vertical line at 0.5 represents midcell. The dotted lines represent one-third of the cell length. n is the total number of cells with a single focus that were measured. (A) Position of DnaX-GFP in cells with no plasmid (strain KPL382). (B) Position of DnaX-GFP in cells with pHP13 (strain MER5). (C) Position of DnaX-GFP in cells with pUB110 (strain MER9). (D) Data from panels A to C tabulated to indicate the percentage of cells with a replisome focus in a polar or central third of the cell. Solid bars, DnaX-GFP in cells with no plasmid (strain KPL382); gray bars, DnaX-GFP in cells with pHP13 (strain MER5); open bars, DnaX-GFP in cells with pUB110 (strain MER9).

The localization pattern of the replisome was notably differentin plasmid-containing cells than in plasmid-free cells (Fig.3). The majority of cells with pHP13 or pUB110 still had a replisomefocus at or near midcell, as in the case of plasmid-free cells(Fig. 3). However, there were many other plasmid-containingcells with a replisome focus located away from midcell and nearera cell pole (Fig. 3B, C, and D). In pHP13-containing cells witha single replisome focus, approximately 30% had a focus in thepolar one-third of the cell (Fig. 3D). We suspect that in theplasmid-containing cells, the centrally located replisome fociare probably associated with chromosomal DNA replication, andthe more polar foci are probably associated with plasmids.

Colocalization of plasmid and replisome foci. We found that some of the polar replisome foci colocalized withplasmid DNA. We visualized the replisome (DnaX-yellow fluorescentprotein [YFP]) and plasmids (pHP13-lacO/LacI-CFP) in the samecells (Fig. 1E). Of the plasmid-containing cells with a polarfocus of the replisome, 89% (85 of 96 cells observed) had aplasmid focus that appeared to colocalize with the polar replisomefocus. These findings strongly indicate that the replisome fociare associated with plasmids at the cell poles.

It should be noted that many plasmid foci, polar or nonpolar,were not visibly associated with a replisome focus. Most cellswith a polar replisome focus also had multiple, nonpolar plasmidfoci that were not visibly associated with a replisome. In addition,in some cells with a polar plasmid focus, there was not visiblecolocalization of this focus with the replisome. These resultswere expected since the small size of the plasmids used allowedreplication of these plasmids to occur in seconds.

Dynamic replisome foci are found in plasmid-containing cells. We found that plasmid-containing cells had highly dynamic replisomefoci (Fig. 1G), similar to the dynamic plasmid localizationdescribed above (Fig. 1C). We visualized the replisome withDnaX-GFP by time-lapse microscopy, taking several exposures1 to 2 s apart (Fig. 1F and G). In plasmid-free cells (60 of63 foci observed), the replisome foci appeared either to beimmobile or to move very small distances, almost as if theywere jiggling (Fig. 1F). Many of these foci appeared to be asingle focus that split into two foci and then reverted backto one focus (data not shown). In none of the 60 cells did thefoci move appreciable distances through the cell.

In contrast, many cells harboring pHP13 (50 of 77 cells observed)had dynamic replisome foci whose subcellular positions changed.Some of these foci appeared and disappeared during the $~$ 12-stime course. Others appeared to move as much as half a celllength (Fig. 1G). This could reflect actual movement of thereplisome or rapid assembly and disassembly of the replisomeon the plasmids. This dynamic behavior was similar to that observedfor the plasmid itself (Fig. 1C). Cells with pUB110 also hadmany moving replisome foci (data not shown), indicating thatthe effects on the replisome were not limited to pHP13.

Plasmid positioning. As described above, the subcellular positions of many plasmids,including unit copy plasmids F and P1, moderate-copy-numberplasmids RK2 and R1, and high-copy-number plasmid pUC19, havebeen visualized in gram-negative bacteria (reviewed in reference52). The plasmids are located predominantly at or near midcellor the cell quarters, and multicopy plasmids appear to be clustered.For many plasmids the proper positioning depends on a ParA/ParBsystem. In the absence of one of these systems, proper plasmidpositioning is lost. It is not known how Par systems positionplasmids at specific subcellular locations, but they could establishthe plasmid position and/or help maintain the position onceit is established by some other means. Since the ColE1-basedplasmid pUC19 does not have a Par system and yet still is foundpredominantly at midcell and the cell quarters, it has beensuggested that positioning of some plasmids might be determinedby the location of the replisome (48, 52, 53).

Based on our findings for B. subtilis with pHP13 and the effectson the replisome, we favor a model in which the subcellularposition of plasmids is not determined by the prior positionof the replisome. Rather, it seems that the position of a plasmidis determined by some other means (often a Par system) and thatthe replicon (plasmid) can recruit the replisome. Of course,different mechanisms might be used by different plasmids andin different organisms, and it would be difficult to rule outany possible contribution of the replisome to plasmid positioning.

Establishment of replisome position. It is clear from several studies that the active replicationmachinery occupies characteristic subcellular locations in avariety of bacterial species (3, 6, 8, 20, 24, 28, 33, 34, 49,50, 54, 55). It is not clear how the position of the replisomeis established or maintained. Our results indicate that in B.subtilis, plasmids pHP13 and pUB110 influence the position ofthe replisome. Consistent with previous work (33, 34), we foundthat in plasmid-free cells, the replisomes responsible for chromosomalreplication are at the characteristic subcellular positionsand are relatively stationary. In plasmid-containing cells,we also observed replisomes with properties characteristic ofthe replisomes in plasmid-free cells. In addition, plasmid-containingcells had replisome foci at uncharacteristic positions in thecell. Many of these replisome foci were highly dynamic, similarto the dynamic properties of plasmid pHP13. The dynamic natureof the replisome indicates either that the foci move rapidlyor that they assemble, disassemble, and reassemble quite rapidly.In either case, the results indicate that the plasmid originrecruits the replisome to the location of the plasmid. Thesefindings are consistent with observations that in C. crescentusthe number and positions of replisome foci are altered in plasmid-containingcells (24). Extending this notion to the chromosome, we suspectthat the subcellular position of the replisome is establishedby the prior positioning of the oriC region at the time of initiationof replication. Alternatively, the replisome, or a subunit ofthe replisome, could recruit the chromosomal origin to a specificlocation, and the plasmid-associated replisomes might then befree to move because most of the time the specific locationwould be occupied.

Maintenance of replisome position. It is not known what maintains the localization of the relativelystationary chromosome-associated replisomes. It is possiblethat some part of the replication machinery is anchored to thecell membrane and thus is relatively immobile. However, thereis some small movement of the replication machinery in plasmid-freecells, indicating that if there is an anchor, it is not absolutelyfixed in place.

If the replisome is anchored, then the apparent differencesbetween the plasmid- and chromosome-associated replisomes couldbe due to the time that it takes to replicate each DNA molecule.Since the plasmids should be replicated in much less time (theyare much smaller than the chromosome), the dynamic replisomefoci could simply represent disassembly of the replisome fromone plasmid and reassembly on the same or another plasmid. Alternatively,since some of the proteins involved in plasmid replication aredifferent from the proteins needed for chromosomal replication,it is possible that the chromosome-associated replisomes areanchored and the plasmid-associated replisomes are not.

In addition to these possibilities, we suspect that the differentmechanisms of chromosomal replication and plasmid replicationcould contribute to the relatively stationary versus dynamicproperties of the replisome. The plasmids used here replicateby the rolling circle mechanism, in which one DNA strand issynthesized first, followed by uncoupled synthesis of the secondstrand (26). We postulate that maintenance of the relativelystationary chromosome-associated replisome might be due in partto bidirectional replication. Bidirectional replication of thechromosome involves two replication forks pointed in oppositedirections and two opposing replicative helicases, one at eachreplication fork. It is possible that the force generated bythe replisome at one fork is opposed by the force exerted bythe replisome at the opposite fork, resulting in a relativelystationary factory. In contrast, the dynamic properties of theplasmid-associated replisomes might be a consequence of theirunidirectional rolling circle replication.

Conclusions. The presence of multicopy rolling circle plasmids in B. subtiliscells causes the appearance of extra assemblies of the replisomewith properties different from those of chromosome-associatedreplisomes. The dynamic nature of the plasmid-associated replisomescould indicate that maintenance of the position of the chromosome-associatedreplisome might be due to bidirectional replication and notdue to anchoring of the replisome to a subcellular structure.Further studies with plasmids that replicate by different mechanismsshould help to test this. More importantly, our results indicatethat the subcellular position of the replisome is probably establishedby the prior subcellular position of the origin from which replicationinitiates.

ACKNOWLEDGMENTS

We are grateful to D. Sherratt for the gift of pLAU43, the membersof the Grossman lab for their helpful insights and comments,and C. Lee for comments on the manuscript.

This work was supported in part by Public Health Service grantGM41934 (to A.D.G.). J.D.W. was supported in part by a DamonRunyon Cancer Research Foundation postdoctoral fellowship; M.E.R.was supported in part by an HHMI predoctoral fellowship; andM.M.B. was supported in part by a Jane Coffin Child postdoctoralfellowship.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 253-1515. Fax: (617) 253-2643. E-mail: adg{at}mit.edu.

REFERENCES

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