|Home | About | Journals | Submit | Contact Us | Français|
The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.
The DNA replication machinery and regions of chromosomal DNA occupy characteristic positions within bacterial cells (reviewed in references 15, 17, 35, and 56). In Bacillus subtilis, the origin of chromosomal replication, oriC, of the single circular chromosome is typically positioned at or near midcell in cells with a single unreplicated origin and at or near the cell quarters (future midcell) in growing cells that contain a partly duplicated chromosome (10, 31, 32, 36, 40, 57, 58, 61, 62). In Escherichia coli, the location of sister oriC regions appears to be at the cell quarters or closer to the cell poles (14, 29, 38, 45, 47, 54, 55).
Replication of the B. subtilis chromosome typically takes place in centralized replication factories. The replisome (the multiprotein complex that includes the replicative polymerase, helicase, and associated proteins that are present at the replication fork) is localized at or near midcell or at the cell quarters which 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). Replication in E. coli also appears to take place at or near midcell or at positions that will be midcell (3, 8, 28, 49, 50, 54, 55). In contrast, in Caulobacter crescentus, the replication factory is initially positioned at the stalked cell pole and gradually moves to midcell as replication proceeds (24).
The mechanisms that establish and maintain the subcellular position of the replisome are not known, nor is it known how the location of oriC is established or maintained, although several genes and sites are known to contribute to positioning of the origin region (4, 13, 15, 17, 25, 32, 43, 66). Since origins and the replication machinery must interact, at least initially during the initiation of replication, it is possible that either the replisome recruits the origin or the origin recruits the replisome to a particular location.
Plasmids provide a good tool for investigating the requirements for positioning the replisome. Plasmids utilize much of the same replication machinery as the cell chromosome, yet they are physically separate. Many processes can contribute to plasmid stability, including decatenation and multimer resolution. Many plasmids encode partitioning systems that contribute to the stability and subcellular positioning of the plasmid. For example, in E. coli, both the unit copy plasmid F and plasmid prophage P1 are found predominantly at midcell or the cell quarters, and this positioning depends on a functional partitioning system named 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 bacterial chromosomes 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 location of ParB (7, 37) and SopB (18, 27) generally correlates with the location of the plasmid and depends on the presence of cognate binding sites (7, 18, 37). In the absence of the Par system, the plasmids appear in nucleoid-free regions of the cell, typically near the cell poles. An E. coli oriC-based plasmid is also found in these regions, indicating that the chromosomal origin per se does not contain information to establish and maintain the position at midcell and the cell quarters (46). However, the oriC-based plasmid is positioned at midcell and the cell quarters if 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 and probably not by recruiting parS sites to a specific subcellular location (31, 32). However, when placed on an otherwise unstable plasmid, 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 ParB homolog, KorB, has been visualized in E. coli, and both are found predominantly at midcell and the cell quarters (2, 19, 52, 53). RK2 is also found at these positions in two of its other hosts, Pseudomonas aeruginosa and Vibrio cholerae, indicating that the mechanisms for determining subcellular positioning are conserved among these bacteria (19, 52, 53). In addition, the number of plasmid foci per cell was much less than the known plasmid copy number, indicating that the plasmid molecules are probably clustered (19, 52, 53).
Multicopy plasmid R1 is positioned at midcell and toward the cell poles, probably past the cell quarters (22, 41, 64). As in the case of RK2, the number of visible plasmid foci was significantly less than the known plasmid copy number, indicating that plasmid molecules are probably clustered (22, 48, 63, 64). Mutations in 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 type 1 Par systems first described for P1 and F. Rather, R1 parM encodes an actin-like ATPase that forms helical filaments needed for partitioning (41, 42, 59). ParR is a DNA binding protein that holds R1 plasmids together (23). ParM interacts with the ParR-DNA complex to promote partitioning (41).
Several high-copy-number plasmids do not have known partitioning systems. Derivatives of ColE1, like pUC19, are present in E. coli at levels of approximately 50 to 200 copies per cell. As seen with the lower-copy-number plasmids R1 and RK2, there are many fewer foci of pUC19 per cell than the known plasmid copy number, indicating that the plasmids are largely clustered predominantly at midcell or the cell quarters (53). In addition to these clusters, approximately 30% of cells appear to have plasmids rapidly moving throughout the cytoplasm (53).
In all of the cases described above, it is not clear if the plasmids have any effect on assembly or the subcellular positioning of 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 the replisomes located at midcell and the cell quarters to be replicated (48, 52, 53). In this way, the position of the replisome would be involved in determining the position of the plasmid. Alternatively, the plasmid could recruit the replisome, as has been suggested for partition-defective plasmids and oriC-based plasmids that appear to localize randomly (46, 52). In addition, the presence of a multicopy plasmid in C. crescentus affects the number of replisome foci per cell, and the replisome foci are found in positions not normally associated with chromosomal replication (24). These findings indicate that perhaps the subcellular position of the replisome is established by the position of the plasmid at the time of initiation of replication.
We visualized both the subcellular position of a multicopy plasmid in B. subtilis and the effects of plasmids on the positioning of the replisome, building on previous work that looked only at one or the other of these aspects. We found that in a majority of cells, the plasmids are dynamically distributed throughout the cell. The presence of a multicopy plasmid altered the number and position of foci of the replisome. In plasmid-containing cells, we observed dynamic and polar replisome foci that were uncharacteristic of the chromosomal replisome pattern but reflected the dynamic or polar patterns of the plasmids. Our results indicate that in B. subtilis, plasmids can influence the location of the replisome, likely by recruiting the replication machinery to the subcellular position of the plasmid. Applying this notion to the chromosome, we propose that the replisome position is established by the subcellular location of oriC at the time of initiation of replication.
pUB110 was originally isolated from Staphylococcus aureus and has 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 has the replication functions of pTA1060, a naturally occurring cryptic plasmid in some Bacillus strains that replicates by the rolling circle mechanism (5, 26). pHP13 is maintained in B. subtilis at a level of approximately five copies per chromosome and contains the selectable cat (chloramphenicol resistance) and mls (resistance to macrolide-lincosamide-streptogramin B antibiotics) genes, the ColE1 origin of replication for E. coli, and some useful cloning sites (5).
We constructed a derivative of pHP13, pJW101, that contains an array of lac operators, and we used LacI-green fluorescent protein (GFP) or LacI-cyan fluorescent protein (CFP) to visualize the subcellular location of this plasmid in living cells of B. subtilis. Briefly, the approximately 5-kb EcoRI-HindIII fragment from pLAU43 (29) containing an array of lac operators was ligated into pHP13. The ligation mixture was transformed into B. subtilis cells containing plasmid pJL52, a derivative of pHP13 that is missing the cat gene such that incoming DNA containing pHP13 sequences and cat can recombine onto the resident plasmid (30). Chloramphenicol-resistant transformants were selected, and the plasmid was purified and retransformed to generate cells containing pJW101.
Fusions of lacI to the mut2 allele of gfp or the wt allele of cfp were used to visualize the subcellular locations of lac operator arrays and have been described in detail previously (34). dnaX-gfpmut2 and dnaX-yfpmut2 (33, 34) were used to visualize the tau subunit of the replisome. These fusions are functional fusions that are present as single copies in the chromosome at the endogenous dnaX locus. polC-gfp (catalytic subunit) was also used to visualize the replisome (33).
For all experiments, cells were grown with vigorous shaking at 30°C in S7 defined minimal medium with MOPS (morpholinepropanesulfonic acid) buffer at a concentration of 50 mM rather than 100 mM (21, 60); the medium was supplemented with 0.1% glutamate, the required amino acids, and either 1% glucose or 1.5% succinate as the carbon source. Antibiotics were used at standard concentrations (16) to maintain selection for plasmids or fusions, as needed.
Cells were sampled during exponential growth (optical density at 600 nm, 0.2 to 0.3) at 30°C in defined minimal medium with succinate or glucose as the carbon source. The vital membrane dye FM4-64 was added to the culture at a concentration of 0.05 μg/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 cells were placed on pads of 1% agarose in 1× T′ base (16) with 1 mM MgSO4. Time-lapse images were obtained at room temperature. Fluorescence was viewed with a Nikon E800 microscope equipped with a ×100 differential interference contrast objective by using appropriate filters. Images were obtained with a cooled charge-coupled device camera (model C4742-95; Hamamatsu) and were analyzed by using the Improvision OpenLab software.
We visualized the multicopy plasmid pHP13 in live B. subtilis cells 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 at an average level of approximately five copies per chromosome (16, 26). We inserted an array of lac operators (lacO) into pHP13 to obtain plasmid pJW101, which was referred to as pHP13-lacO. A LacI-GFP fusion protein was constitutively expressed to allow visualization of the subcellular location of the lacO-containing DNA.
We observed fluorescent foci (Fig. 1A and B) in 80% of the cells (228 of 286 cells observed) with pHP13-lacO expressing LacI-GFP during 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 minimizing the amount of multifork replication. The appearance of fluorescent foci was dependent on the presence of the lac operators in the plasmid. In cells with pHP13 (without the lacO array), we detected faint distributed fluorescence (Fig. (Fig.1A)1A) but no defined foci in any of the 134 cells examined.
Of the plasmid-containing cells with fluorescent foci, 89% (203 of 228 cells) had at least one highly dynamic focus (Fig. (Fig.1C).1C). The positions of the foci were different in images captured as little as 1 to 2 s apart. In addition, some of the dynamic foci were distinct and others were more diffuse, perhaps due to rapid movement during microscopic exposure or to movement in and out of the plane of focus. Of the 203 cells with at least one dynamic plasmid focus, 15 also had an apparently stationary focus.
The other 11% of cells with visible plasmid foci (25 of 228 cells) contained a single, apparently fixed focus (Fig. (Fig.1B).1B). These foci appeared to be brighter and much less mobile than the dynamic foci and may have represented multiple plasmids clustered together. Most appeared as a distinct focus near a cell pole and sometimes near midcell, and others occupied a much larger area of the cell (Fig. (Fig.1B).1B). The visualization of both dynamic and apparently fixed foci of pHP13-lacO was similar to that observed for pUC-based plasmids in E. coli, although the positions and proportions were quite different (53).
Cells with either pHP13 or another multicopy rolling circle plasmid, pUB110 (16, 26), had more replisome foci than plasmid-free cells (Fig. (Fig.2).2). We visualized the B. subtilis replication machinery using a functional GFP fusion to dnaX (34), which encodes the tau subunit of the replicative DNA polymerase. During exponential growth in defined minimal succinate medium, the vast majority of plasmid-free cells had either one or two foci of the replisome (33) (Fig. (Fig.2A),2A), and a minority of these cells (~10%) had no focus of the replisome. Under these growth conditions, in cells containing pHP13 or pUB110 there was a marked increase in the proportion of cells with three or more foci of the replisome (Fig. (Fig.2A),2A), and there was a decrease in the proportion of cells with no foci of the replisome.
At higher growth rates in defined glucose medium, there was also an increase in the proportion of plasmid-containing cells with three or more replisome foci compared to plasmid-free cells (Fig. (Fig.1D1D and and2B).2B). As expected under these growth conditions (33), >95% of plasmid-free cells had at least one focus of the replisome (Fig. (Fig.2B2B).
An increase in replisome foci due to the presence of pHP13 or pUB110 was also observed in cells expressing a GFP fusion to PolC, the catalytic subunit of the replicative DNA polymerase (data not shown), similar to the effects of plasmids on DnaX-GFP foci.
We measured the position of the replisome during exponential growth in minimal succinate medium (slow growth) in cells with a 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. (Fig.2A).2A). In cells with a single focus, the replisome was located predominantly at or near midcell (Fig. (Fig.3A).3A). Approximately 10% of these cells had a focus in the polar one-third of the cell (Fig. (Fig.3D3D).
The localization pattern of the replisome was notably different in plasmid-containing cells than in plasmid-free cells (Fig. (Fig.3).3). The majority of cells with pHP13 or pUB110 still had a replisome focus at or near midcell, as in the case of plasmid-free cells (Fig. (Fig.3).3). However, there were many other plasmid-containing cells with a replisome focus located away from midcell and nearer a cell pole (Fig. 3B, C, and D). In pHP13-containing cells with a single replisome focus, approximately 30% had a focus in the polar one-third of the cell (Fig. (Fig.3D).3D). We suspect that in the plasmid-containing cells, the centrally located replisome foci are probably associated with chromosomal DNA replication, and the more polar foci are probably associated with plasmids.
We found that some of the polar replisome foci colocalized with plasmid DNA. We visualized the replisome (DnaX-yellow fluorescent protein [YFP]) and plasmids (pHP13-lacO/LacI-CFP) in the same cells (Fig. (Fig.1E).1E). Of the plasmid-containing cells with a polar focus of the replisome, 89% (85 of 96 cells observed) had a plasmid focus that appeared to colocalize with the polar replisome focus. These findings strongly indicate that the replisome foci are 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 cells with a polar replisome focus also had multiple, nonpolar plasmid foci that were not visibly associated with a replisome. In addition, in some cells with a polar plasmid focus, there was not visible colocalization of this focus with the replisome. These results were expected since the small size of the plasmids used allowed replication of these plasmids to occur in seconds.
We found that plasmid-containing cells had highly dynamic replisome foci (Fig. (Fig.1G),1G), similar to the dynamic plasmid localization described above (Fig. (Fig.1C).1C). We visualized the replisome with DnaX-GFP by time-lapse microscopy, taking several exposures 1 to 2 s apart (Fig. 1F and G). In plasmid-free cells (60 of 63 foci observed), the replisome foci appeared either to be immobile or to move very small distances, almost as if they were jiggling (Fig. (Fig.1F).1F). Many of these foci appeared to be a single focus that split into two foci and then reverted back to one focus (data not shown). In none of the 60 cells did the foci 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-s time course. Others appeared to move as much as half a cell length (Fig. (Fig.1G).1G). This could reflect actual movement of the replisome or rapid assembly and disassembly of the replisome on the plasmids. This dynamic behavior was similar to that observed for the plasmid itself (Fig. (Fig.1C).1C). Cells with pUB110 also had many moving replisome foci (data not shown), indicating that the effects on the replisome were not limited to pHP13.
As described above, the subcellular positions of many plasmids, including unit copy plasmids F and P1, moderate-copy-number plasmids RK2 and R1, and high-copy-number plasmid pUC19, have been visualized in gram-negative bacteria (reviewed in reference 52). The plasmids are located predominantly at or near midcell or the cell quarters, and multicopy plasmids appear to be clustered. For many plasmids the proper positioning depends on a ParA/ParB system. In the absence of one of these systems, proper plasmid positioning is lost. It is not known how Par systems position plasmids at specific subcellular locations, but they could establish the plasmid position and/or help maintain the position once it is established by some other means. Since the ColE1-based plasmid pUC19 does not have a Par system and yet still is found predominantly at midcell and the cell quarters, it has been suggested that positioning of some plasmids might be determined by the location of the replisome (48, 52, 53).
Based on our findings for B. subtilis with pHP13 and the effects on the replisome, we favor a model in which the subcellular position of plasmids is not determined by the prior position of the replisome. Rather, it seems that the position of a plasmid is determined by some other means (often a Par system) and that the replicon (plasmid) can recruit the replisome. Of course, different mechanisms might be used by different plasmids and in different organisms, and it would be difficult to rule out any possible contribution of the replisome to plasmid positioning.
It is clear from several studies that the active replication machinery occupies characteristic subcellular locations in a variety of bacterial species (3, 6, 8, 20, 24, 28, 33, 34, 49, 50, 54, 55). It is not clear how the position of the replisome is established or maintained. Our results indicate that in B. subtilis, plasmids pHP13 and pUB110 influence the position of the replisome. Consistent with previous work (33, 34), we found that in plasmid-free cells, the replisomes responsible for chromosomal replication are at the characteristic subcellular positions and are relatively stationary. In plasmid-containing cells, we also observed replisomes with properties characteristic of the replisomes in plasmid-free cells. In addition, plasmid-containing cells had replisome foci at uncharacteristic positions in the cell. Many of these replisome foci were highly dynamic, similar to the dynamic properties of plasmid pHP13. The dynamic nature of the replisome indicates either that the foci move rapidly or that they assemble, disassemble, and reassemble quite rapidly. In either case, the results indicate that the plasmid origin recruits the replisome to the location of the plasmid. These findings are consistent with observations that in C. crescentus the number and positions of replisome foci are altered in plasmid-containing cells (24). Extending this notion to the chromosome, we suspect that the subcellular position of the replisome is established by the prior positioning of the oriC region at the time of initiation of replication. Alternatively, the replisome, or a subunit of the replisome, could recruit the chromosomal origin to a specific location, and the plasmid-associated replisomes might then be free to move because most of the time the specific location would be occupied.
It is not known what maintains the localization of the relatively stationary chromosome-associated replisomes. It is possible that some part of the replication machinery is anchored to the cell membrane and thus is relatively immobile. However, there is some small movement of the replication machinery in plasmid-free cells, indicating that if there is an anchor, it is not absolutely fixed in place.
If the replisome is anchored, then the apparent differences between the plasmid- and chromosome-associated replisomes could be due to the time that it takes to replicate each DNA molecule. Since the plasmids should be replicated in much less time (they are much smaller than the chromosome), the dynamic replisome foci could simply represent disassembly of the replisome from one plasmid and reassembly on the same or another plasmid. Alternatively, since some of the proteins involved in plasmid replication are different from the proteins needed for chromosomal replication, it is possible that the chromosome-associated replisomes are anchored and the plasmid-associated replisomes are not.
In addition to these possibilities, we suspect that the different mechanisms of chromosomal replication and plasmid replication could contribute to the relatively stationary versus dynamic properties of the replisome. The plasmids used here replicate by the rolling circle mechanism, in which one DNA strand is synthesized first, followed by uncoupled synthesis of the second strand (26). We postulate that maintenance of the relatively stationary chromosome-associated replisome might be due in part to bidirectional replication. Bidirectional replication of the chromosome involves two replication forks pointed in opposite directions and two opposing replicative helicases, one at each replication fork. It is possible that the force generated by the replisome at one fork is opposed by the force exerted by the replisome at the opposite fork, resulting in a relatively stationary factory. In contrast, the dynamic properties of the plasmid-associated replisomes might be a consequence of their unidirectional rolling circle replication.
The presence of multicopy rolling circle plasmids in B. subtilis cells causes the appearance of extra assemblies of the replisome with properties different from those of chromosome-associated replisomes. The dynamic nature of the plasmid-associated replisomes could indicate that maintenance of the position of the chromosome-associated replisome might be due to bidirectional replication and not due to anchoring of the replisome to a subcellular structure. Further studies with plasmids that replicate by different mechanisms should help to test this. More importantly, our results indicate that the subcellular position of the replisome is probably established by the prior subcellular position of the origin from which replication initiates.
We are grateful to D. Sherratt for the gift of pLAU43, the members of 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 grant GM41934 (to A.D.G.). J.D.W. was supported in part by a Damon Runyon Cancer Research Foundation postdoctoral fellowship; M.E.R. was supported in part by an HHMI predoctoral fellowship; and M.M.B. was supported in part by a Jane Coffin Child postdoctoral fellowship.