Plasmid partition and incompatibility the focus shifts

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1 Molecular Microbiology (2007) 65(6), doi: /j x First published online 21 August 2007 MicroReview Plasmid partition and incompatibility the focus shifts Jean-Yves Bouet, 1 Kurt Nordström 2 and David Lane 1 * 1 Laboratoire de Microbiologie et Génétique Moléculaires, Centre National de Recherche Scientifique, Campus Université Paul Sabatier, 118 route de Narbonne, Toulouse, France. 2 Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, PO Box S , Sweden. Summary The mitotic apparatus that a plasmid uses to ensure its stable inheritance responds to the appearance of an additional copy of the plasmid s centromere by segregating it from the pre-existing copies: if the new copy arises by replication of the plasmid the result is partition, if it arrives on a different plasmid the result is incompatibility. Incompatibility thus serves as a probe of the partition mechanism. Coupling of distinct plasmids via their shared centromeres to form mixed pairs has been the favoured explanation for centromere-based incompatibility, because it supports a long-standing assumption that pairing of plasmid replicas is a prerequisite for their partition into daughter cells. Recent results from molecular genetic and fluorescence microscopy studies challenge this mixed pairing model. Partition incompatibility is seen to result from various processes, including titration, randomized positioning and a form of mixed pairing that is based on co-activation of the same partition event rather than direct contact between partition complexes. The perspectives thus opened onto the partition mechanism confirm the continuing utility of incompatibility as an approach to understanding bacterial mitosis. The results considered are compatible with the view that direct pairing of plasmids is not essential to plasmid partition. Introduction It was recognized early in the history of plasmid biology that a plasmid is not entirely welcome when it enters a cell Accepted 18 July, *For correspondence. dave@ibcg. biotoul.fr; Tel. (+33) ; Fax (+33) that already harbours a plasmid of the same ancestry (Scaife and Gross, 1962). Entering and resident plasmids segregate from each other over succeeding generations to form single-plasmid lines, which are then stable. Two such plasmids are said to be incompatible. Incompatibility is the failure of mechanisms suited for propagation of one plasmid to cope when confronted with two. In the case of replication the relationship is direct; the elements that restrict the frequency of replication of one plasmid iterons, antisense RNAs, etc. continue to act in the presence of a second plasmid so that neither plasmid replicates often enough to be stable. This approach provided some of the seminal findings on which our understanding of replication control was built. The basis of partition incompatibility has been less obvious. Early observations that a particular plasmid sequence could both stabilize an unstable vector and destabilize the plasmid from which it originated (e.g. Austin, 1984) reinforced the view that partition and incompatibility are two facets of the same molecular mechanism, and bolstered confidence... that by understanding one, we will understand the other, as Austin and Nordstrom (1990) concluded the last widely disseminated review of the topic. Since then we have learnt much more about the actors in partition, their interactions and their disposition in the cell, even if incompatibility data have been complementary rather than essential to this progress. Nevertheless, the essence of the partition mechanism remains elusive, and incompatibility may still prove to be the perturbation that yields crucial mechanistic insights. This view has motivated two recent investigations; the results challenge one of the central tenets of current partition models, and prompt this review. Partition Partition is an active process, functionally equivalent to mitosis, needed by low-copy-number plasmids to ensure regular distribution of their copies to daughter cells; random diffusion is inadequate for the task. All such plasmids possess a specific partition module comprising three elements (Fig. 1A): a cis-acting, centromere-like site consisting of an array of sequence repeats (generically named pars, after the well-studied partition system of Journal compilation 2007 Blackwell Publishing Ltd

2 1406 J.-Y. Bouet, K. Nordström and D. Lane A B sop (F) par2 (pb171) a parc1 e sopa sopb para parb parc2 b ParB ParA centromere d sopc c Fig. 1. A. Partition loci of plasmids F and pb171 used in incompatibility studies. The 43 bp units in the sopc centromere which carry inverted repeats recognized by SopB protein are shown as V; the related 6 bp tandem repeats in the parc (C1, C2) centromere recognized by ParB are shown as slanted lines. Blocks in the sopab promoter are 7 bp motifs bound by SopA repressor. Scale of locus components is approximate. B. Plasmid partition, a general view. ParA protein is shown as migrating en masse through the cell (a c), then briefly polymerizing to push plasmid replicas apart (d) before dissociating to resume oscillatory migration. The underlying mechanism is unknown. ParB extends the specific complex it forms on the centromere by binding non-specifically to flanking DNA, thus enlarging the partition complex. This representation is slanted towards partition systems based on Walker-box ATPases such as Sop and Par2; actin-like ParAs are not known to oscillate. plasmid P1), and two genes, one coding for an ATPase (ParA), the other for the centromere-binding protein (ParB). Most ParA proteins have the characteristic motifs of the Walker-box ATPase superfamily, while a minority are actin-like ATPases. In both, the integrity of the ATP binding site is essential for partition function. We can be reasonably sure of certain aspects of the partition mechanism (Fig. 1B). The event that sets partition in motion is the assembly (or re-assembly) of ParB on pars to form a partition complex on each plasmid replica (Austin and Abeles, 1983; Ogura and Hiraga, 1983). The complex may enlarge itself by recruiting further ParB molecules which bind non-specifically to neighbouring DNA and spread over it (Lynch and Wang, 1995; Rodionov et al., 1999). The ParA ATPase recognizes its plasmid by interacting specifically with ParB protein in the complex (Hirano et al., 1998; Bouet and Funnell, 1999). The interaction prompts ParA to form metastable assemblies that push or drag plasmid copies towards each pole and place them in defined regions of the two cell-halves. The clearest picture to emerge so far is that of the actin-like ParA system of plasmid R1. Its partition complex appears to stabilize otherwise unstable protofilaments of the ATPase (ParM) and to focus insertion of further ParM molecules, thus allowing the filaments to lengthen and push the attached plasmids towards the cell poles (Moller-Jensen et al., 2003; Garner et al., 2004). Other aspects remain obscure. How spreading of ParB contributes to partition is unclear; preventing partition complexes from spreading was found to reduce mini-p1 plasmid stability only slightly (Rodionov and Yarmolinsky, 2004). At least some of the ParA ATPases (the Walkerbox class) appear to migrate en masse back and forth over the nucleoid region a few times per cell cycle (Ebersbach and Gerdes, 2001); how is this activity related to the bidirectional segregation of plasmid copies? These ParAs generally position their plasmids in the central region of the daughter cells-to-be (Erdmann et al., 1999); how is this specific placement achieved? A major gap in our knowledge is the role of plasmid pairing. It has generally been assumed that prior to segregation, plasmid siblings are paired via their partition complexes, and that segregation itself begins with active splitting of the pair by ParA. The intuitive appeal of this model is hard to resist, and not only because the pervasive image of paired metaphase chromosomes suggests a pleasing unity across the eukaryote prokaryote divide. A paired configuration allows direct orientation of each plasmid copy relative to the other, helping to ensure that segregation is bidirectional. By acting as the substrate for ParA, paired plasmids would constitute a key element of the partition mechanism. Moreover, plasmid R1 partition complexes were shown by electron microscopy and ligation kinetics to pair in vitro (Jensen et al., 1998), and two mini-p1 partition complexes on the same plasmid molecule were shown by a supercoil-trap assay to pair in vivo (Edgar et al., 2001). A possible difficulty with centromere pairing as a necessary step in partition is the observation that a plasmid carrying two identical par loci is stable (Austin, 1984). A centromere in such a plasmid might be

3 Partition-based incompatibility 1407 expected to pair more readily with its twin in cis than with a copy on another molecule, resulting in a block to interplasmid pairing and destabilization of the plasmid. The same problem could beset plasmids with naturally dispersed centromere sites, such as N15 and RK2. Explanations can be imagined spreading of ParB might effectively rigidify the DNA and so hinder interactions in cis, or, as Treptow et al. (1994) suggested, proximity of just-replicated centromeres might favour pairing of sibs over internal pairing but the issue has yet to be settled. What has been missing is an assay applicable under normal in vivo conditions that would allow the role of pairing to be defined and the pairing process to be manipulated experimentally. Incompatibility has appeared to be a useful substitute for such an assay. A B A + Autoregulation Titration model B + C Partition incompatibility The term usually pertains to the reciprocal instability of plasmids carrying the same centromere, but it has also been extended to instability caused by an excess of one or the other partition protein (Ogura and Hiraga, 1983; Kusukawa et al., 1987). The importance of avoiding an imbalance or excess of ParA and/or ParB proteins is indicated by the operon arrangement of the para and parb genes and the autoregulation of their transcription by one or both proteins. Destabilization of mini-f by an excess of its partition ATPase (SopA) has been correlated with removal of SopB from DNA flanking the SopB sopc partition complex, giving rise to the suggestion that this reflects a role for SopA in breaking paired complexes (Lemonnier et al., 2000). However, an excess of the similar ATPase of plasmid P1 did not disrupt P1 partition complexes (Edgar et al., 2006), leaving the significance of excess partition ATPase effects unclear. In the case of ParB proteins, very high levels led to loss so precipitous as to suggest sequestration of plasmids in clumps held together by ParB ParB interactions (Kusukawa et al., 1987; Funnell, 1988). In contrast, SopB at about five times the in vivo concentration led to instability by promoting plasmid dimerization (Bouet et al., 2006). It was suggested that increased spreading prolongs contact between paired sibling plasmids and so raises the chance of crossing over between them. Destabilization of plasmid R1 by an excess of its ParR protein, which doubles as centromere binder and repressor, was suggested to result from a deficit of the ATPase (ParM) created by overrepression (Dam and Gerdes, 1994). The potential of these approaches for providing insights into partition protein interactions has yet to be realized. An excess of centromeres is closer to the essential partition process: incompatibility and partition are both responses to the appearance of a second centromere, on a distinct plasmid in the first case, by replication in the Fig. 2. A. Autoregulation of sopab transcription. Binding of SopA repressor to the promoter region is strengthened (thickened arrow) by SopB co-repressor. Centromere sites enhance co-repression, both in cis and in trans, so that additional centromeres reduce Sop protein concentrations. In contrast, ParB of the Par2 system directly autoregulates its operon, without involving ParA and with no known enhancement by parc repeats. In this case, additional parc repeats are expected to derepress parab transcription and increase ParAB protein levels. Data for the similar autoregulatory system of plasmid R1 (Dam and Gerdes, 1994) confirm this effect, although derepression was weak. B. In systems such as Sop, where the ParB centromere-binding protein is not the primary repressor, additional centromeres do not necessarily derepress partition protein synthesis. Instead the centromeres can deprive plasmids of their ParB protein by titration. Titration is not expected to work as an incompatibility mechanism in the Par2 system. second. Accordingly, an early explanation of incompatibility was derived directly from the replicon model of Jacob et al. (1963): plasmids bearing the same centromere (and hence partition complex) compete for a very limited number of specific cellular sites present in each daughter cell, leaving unattached plasmids to disperse at random and to be lost frequently. This proposal was (and remains) unlikely given the large number of known incompatibility specificities and the lack of supporting evidence. The observation that fluorescently labelled plasmids are usually seen in the cell quarter regions stirred the ghost of this model with frequent references to tethering, but again no evidence for specific host factors has appeared. An alternative suggestion, that additional centromeres deprive plasmids of the ParB needed for partition by competing for the protein (Lane et al., 1987), was rendered implausible by the finding that par gene expression is autoregulated (Fig. 2A) and should thus compensate for any deficit. The discovery that additional centromeres actually reinforce repression of Par protein production rather than relieve it (Yates et al., 1999; Hao and Yarmolinsky, 2002) has rehabilitated this model to some extent, as detailed later.

4 1408 J.-Y. Bouet, K. Nordström and D. Lane partition incompatibility{ Pairing model Fig. 3. The mixed pairing model of incompatibility is based on participation of an introduced plasmid (orange circles) in the normal paring-based mechanism (top) that partitions the resident plasmid. Random orientation of heterologous pairs leads to frequent missegregation, seen as incompatibility (bottom). introduce a graded incompatibility stimulus into cells containing mini-f plasmids. Using multicopy vectors, they found that the greater the fraction of centromere units borne on the vector the higher the rate of mini-f loss, up to a limit corresponding to loss of partition-defective mini-f. The results were consistent with increasing randomness of mini-f segregation according to any of the models proposed competition of centromeres for host sites, for SopB protein or for each other. However, the relationship did not hold for sopc on low-copy vectors (mini-p1, chromosome), which exhibited incompatibility weaker than expected on the basis of the number of additional SopB binding sites. This suggested that partition complex pairing based on SopB binding capacity is not the only level at which incompatibility acts. A I A more robust model is based on the idea that partition complexes of a given specificity pair with each other, and are acted upon by their cognate partition apparatus, regardless of the replicon on which they are situated. Two incompatible plasmids are proposed to occupy a pool where they expose their centromeres or centromere protein complexes to each other, allowing formation of mixed as well as kindred pairs. Mixed pairs are oriented in either direction with equal probability so that the partition apparatus segregates their plasmids randomly. Plasmid loss ensues (Fig. 3). There is good evidence that partition complexes can pair plasmids, as noted above. On the other hand, how the proximity of new replicas is overcome to allow access to a competing partition complex, the essence of pooling, is unclear. Furthermore, the preferential pairing of sibling centromeres suggested as an explanation for dicentric plasmid stability, discussed above, would imply that pooling of partition complexes is limited. Nevertheless, the incompatibility behaviour and ParB affinities of full and shortened centromeres were fully consistent with mixed pairing (Funnell, 1991), and this has maintained its status as the preferred model (e.g. Funnell, 2005). B mp1 sop+ Δpar mp1 sop+ Δpar mp1 sop+ par+ mp1 sop+ Δpar repa* I mfδsop mfsop+ mf - 43bp mfsop+ x 5 mfsop+ Problems with mixed pairing Bouet et al. (2005) undertook a systematic analysis of incompatibility exerted by the sopc centromere of the plasmid F. Like many plasmid centromeres, sopc consists of a tandem array of sites for binding its protein (Fig. 1A), in this case SopB. A single SopB-binding unit suffices to stabilize a mini-f containing it (Biek and Shi, 1994) but exerts weaker incompatibility than sopc itself (Lane et al., 1987; Fig. 4A). Bouet et al. exploited this difference to Fig. 4. Observed incompatibility reactions. A. Selection for a centromere-bearing plasmid (entering arrows) causes incompatibility (I) with a resident plasmid which is excluded at a greater (solid arrows) or lesser (dotted arrows) rate. On the incoming multicopy plasmid the reduced centromere (filled ovoid) excludes a low-copy-number plasmid with the full centromere (filled circle) weakly, but on the low-copy resident plasmid it is the more strongly excluded. B. Degree of exclusion of mini-f plasmids resulting from deletion of the sop locus or from incompatibility with mini-p1 sopc + Dpar, mini-p1 sopc + par + or mini-p1 sopc + Dpar repam5; the repa mutant allele (E184G) eliminates handcuffing, resulting in a fivefold copy-up phenotype.

5 Partition-based incompatibility 1409 How else it might do so was indicated by the further demonstration that a supplement of SopB protein restored to wild-type mini-f most of the stability lost upon introduction of sopc on a psc101 vector. As discussed above, the possibility that extra centromeres might deprive plasmids of their binding protein had not been seriously considered as an incompatibility mechanism because it had been envisaged as simple titration, readily reversed by autoregulated par gene expression. But because sopc in fact acts as a co-repressor of sopab transcription (Yates et al., 1999), extra copies of it reduce SopB concentration. They presumably titrate the protein as well, as a chromosomally located sopc that did not significantly reduce SopB concentration did reduce the amount of SopB bound to mini-f. In any case, the net effect of sopc on multicopy vectors was to denude mini-f of most of its SopB protein and to destabilize it proportionally (Fig. 2B). Conversely, deficiency in SopB binding and loss of mini-f were largely suppressed by supplying more SopB. Incompatibility is most simply explained in this case as the result of an induced deficit in SopB that leaves mini-f molecules unable to engage in partition. The weakness of a single sopc unit in imposing and resisting incompatibility is also consistent with reduced ability to compete for SopB protein. Although stabilization by added SopB was incomplete, leaving a possible minor role for other mechanisms, these experiments cast doubt on mixed pairing as the primary mechanism of incompatibility provoked by centromeres on multicopy plasmids. Very different behaviour came to light when Bouet et al. examined incompatibility exerted by sopc on a low-copynumber vector, mini-p1, under equivalent conditions. As multicopy plasmids do not carry active partition loci, equivalence entails removal of the low-copy-number plasmid s own partition locus (parabs in this case). Incompatibility between mini-f and the par-deleted mini-p1-sopc was unexpectedly strong, and reciprocal, each plasmid being lost much faster than the random rate when the other was selected for (Fig. 4B). Moreover, a supplement of SopB had no effect, and mini-f with a single centromere unit was less sensitive to exclusion than wild-type mini-f. Clearly, a different incompatibility mechanism was operating. It was not obviously based on mixed pairing, which would lead to loss rates no higher than random, or on over-repression of sopab transcription as copy numbers were low and a mini-p1 carrying sopab in addition to sopc showed the same strong incompatibility. Some characteristic of the mini-p1 plasmid not shared by multicopy plasmids must be responsible. Bouet et al. found that reinsertion of the parabs locus into mini-p1-sopc + -Dpar largely suppressed incompatibility with mini-f (Fig. 4B), as if the second partition system enabled each plasmid to segregate independently with almost no interference from their shared sopc centromere. This weakness contrasts with the stronger incompatibility seen by Funnell (1991) for the analogous P1 ParABS, and may signal a difference between the two partition systems. Bouet et al. also observed that raising mini-p1-sopc copy number by introduction of a replication control mutation sharply reduced incompatibility with mini-f, to that of the equivalent multicopy plasmid (Fig. 4B); moreover, this residual plasmid loss was heavily reduced by additional SopB, as if the incompatibility mechanism had switched to the multicopy mode. This result is not easily accounted for by any of the models so far proposed. It seems counterintuitive for incompatibility to decline as the copy number of the destabilizing plasmid increases. A possible explanation lies in the nature of the copy number mutation and the phenomenon of plasmid clustering. The high rates of mini-p1-sopc and mini-f loss corresponded to random loss of a number of segregating units about half that of the measured copy number, as if the plasmids were segregating as clusters of two molecules rather than as individuals. Now clustering of plasmid molecules has often been invoked to explain the disparity between known plasmid copy number and the number of foci seen by fluorescence microscopy (Gordon et al., 1997; Weitao et al., 2000; Pogliano et al., 2001). The phenomenon is not understood. The partition apparatus is not needed, as par-minus plasmids cluster too. Interactions that could play a part include the packing of similarly conformed DNA molecules (Reich et al., 1994) and in trans handcuffing by replication control proteins (Pal and Chattoraj, 1988). The mutation introduced into mini-p1-sopc + -Dpar by Bouet et al. is known to abolish handcuffing and to raise mini-p1 copy number. If handcuffing were needed for clustering, another consequence of the mutation would be the dispersal of mini-p1-sopc + -Dpar molecules. The mutant plasmid indeed forms more visible foci than its parent (J. Rech and D. Lane, unpubl. data). Even if elevated copy number alone contributes substantially to this multiplication of foci, the result is a scattering of SopB binding sites that could explain why the incompatibility behaviour mimics that of multicopy sopc vectors. From the perspective of clustered mini-plasmids, we can envisage a form of mixed pairing being responsible for strong incompatibility. The effective partition complex in a cluster might be not the single complex but an ensemble, possibly fused into a massive complex by spreading of the centromere-binding protein. The combined complex would stimulate more strongly than a single-partition complex the dynamic behaviour of ParA, creating a larger ParA assembly that more readily accesses a similar complex on an incompatible plasmid. Thus the balance between splitting a cluster to partition siblings and pushing apart two entire clusters, including

6 1410 J.-Y. Bouet, K. Nordström and D. Lane incompatible ones, would shift in favour of the latter. A single centromere unit makes a less extensive complex and would compete less well than sopc for the centromere on mini-p1, rendering the mini-f carrying it less susceptible to exclusion, as observed. In fact it is often as clusters that plasmids are seen to segregate (Pogliano et al., 2001; Gordon et al., 2004), and foci of fluorescently tagged versions of the mini-f and mini-p1-sopc plasmids used by Bouet et al. are two- to threefold less numerous than plasmid copy number (J. Rech, unpubl. results). If a combined complex can serve as a partition substrate, the conceptual problem of requiring a complex on a single incompatible plasmid to gain access to and compete with sibling complexes in a plasmid cluster in order to exert its incompatibility would be largely resolved. The lack of plasmid visualization data prevented Bouet et al. from drawing firmer conclusions, but their results clearly pointed to the need for a wider view of the nature of the partition substrate or for a model based on interaction between incompatible plasmids at a different point in the partition pathway. The latter was not long coming. A B Random positioning model Mid-cell competition An alternative model Ebersbach et al. (2005) used fluorescence tagging to visualize incompatible plasmids, and arrived at the radical conclusion that mixed pairing makes little if any contribution to incompatibility. They based their study on the plasmid pb171 Par2 system (Fig. 1A), a Walker-box ATPase partition module analogous to Sop, and used mini-f and mini-r1 (lacking their own par loci) as vectors of par2. The par2 centromere also consists of tandemly arrayed binding sites but unlike sopc these are in two groups, upstream (parc1) and downstream (parc2)ofthe pb171 parab operon. If mixed pairing contributes significantly to incompatibility, and provided plasmid pairs persist for an appreciable time prior to being segregated, foci composed of both plasmids should be seen at a higher frequency than that due to random co-positioning. In fact the opposite was observed. When present in the same cell, the unrelated mini-f and mini-r1-par2 plasmids formed an average of two to three foci each with typical positioning at approximately quarter and mid-cell points; fortuitously coincident foci constituted 8% of the total. In cells containing the incompatible plasmid pair, mini-f-par2 and mini-r1-par2, fewer than 1% of the foci coincided. This result indicates that partition complexes interact to repel each other rather than to cohere, as the frequency of coincidence was much lower than chance encounter. Ebersbach et al. also found that the foci of both plasmids were distributed throughout the cells rather than localized at the quarter points. This suggested to them that incompatibility results from randomized positioning over the length of the cell rather than from random Fig. 5. Random positioning of incompatible plasmids. A. Sibling plasmids are initially paired, segregated and positioned along the cell. As fresh replicas are generated during the cell cycle, the positioning mechanism continues to be activated and to reposition plasmids two at a time, leading to asymmetric plasmid distribution and often to the absence of a plasmid from one cell-half. B. A large partition complex is more likely than a smaller one to activate and stabilize the positioning process. The plasmid with the larger centromere (circle) is thus more likely than that with the smaller one (ovoid) to be chosen for positioning and to be placed at the centres of the cell-halves; plasmids with smaller centromeres are more likely to wander and be lost. Note that repositioning will tend to moderate a preference for positioning plasmids with large centromeres/partition complexes. pairing (Fig. 5A). The disturbed positioning would often result in cells having all copies of a given plasmid in one cell-half at the moment of division so that the other half becomes a negative segregant for this plasmid, and indeed a substantial fraction of the population was seen to display such asymmetric distributions.

7 Partition-based incompatibility 1411 Notably, the relative incompatibility of different centromere sequences was correlated with, and perhaps determined by, their ability to occupy the mid-cell position. The reduced centromeres parc1 and parc2, like a single sopc unit in relation to the full sopc, excluded mini-r1- par2 more weakly than did par2 itself, and also occupied the cell centre less frequently. By occupying the centre a plasmid maximizes the chances that its replicas will segregate to both daughter cells, and by pushing an incompatible plasmid polewards it will reduce the chances of that plasmid s replicas doing so. The plasmid at mid-cell thus tends to be inherited at the expense of the more polar, incompatible plasmid (Fig. 5B). Although it is not known why mid-cell is preferentially occupied by the most partition-competent plasmid, the observation is consistent with the proposal of randomized distribution of plasmid copies as a basis for incompatibility. Ebersbach et al. (2006) have recently published evidence that pb171 par2 can segregate plasmids in this manner in the absence of incompatibility. This model in no way weakens the assertion that centromere-based incompatibility is a direct reflection of the partition mechanism, but it does shift the focus away from the centromere itself, as a component of partition complexes which pair plasmids, and towards the active segregation event mediated by ParA. Effectively, incompatibility is based not on direct interactions between centromere-bound ParB/SopB molecules but on partition complexes being pushed apart by an elongating bundle of metastable ParA/SopA filaments (the repulsive force inferred above). A significant difference from the mixed pairing model is that non-central positioning of incompatible plasmids can cause their replicas to segregate entirely within a cell-half rather than across the midline. Implicit in the model is the idea that initial segregation of plasmid copies can be followed by repositioning in response to the presence of any other plasmid with the same partition complex (Fig. 5A), as pointed out in other terms by Ebersbach et al. (2006). A corollary is that plasmid replication can also occur far from mid-cell, as partition is expected to succeed it closely (Onogi et al., 2002). Bacillus subtilis plasmids have been reported to replicate far from mid-cell (Wang et al., 2004), and the highly excentric positioning of Escherichia coli par-minus plasmids (Niki and Hiraga, 1997), as well as unpublished results from the Nordstöm laboratory, suggests that E. coli plasmids can do too. Random positioning versus the data The emphasis on positioning helps explain some otherwise puzzling observations. The reason for Bouet et al. s observation that reinsertion of the P1 par locus into mini- P1-sopC + -Dpar suppressed strong incompatibility with mini-f was not obvious. We can now see that in cells where the Sop system initially places all copies (or clusters) of mini-p1-sopc + -par + in the same cell-half the Par system can subsequently displace one of them to the other cell-half and thus abolish a potential missegregation. The very weak incompatibility exhibited by sopc on the chromosome (attl) might well result from its relatively rapid passage through mid-cell in the course of its own replication and segregation. Reduced occupation of mid-cell also provides an alternative view of the reduced incompatibility properties of shortened centromeres such as the single sopc unit. Removal of the single-unit mini-f from mid-cell could be a factor in the observed reduction of intense incompatibility with mini- P1-sopC + -Dpar experienced by wild-type mini-f (Fig. 4B). The model is not easily applied to all situations however. It does not by itself explain the (theoretical) problem of stability of dicentric plasmids. It does not supplant titration of centromere-binding protein as an explanation of incompatibility due to centromeres on multicopy vectors, because its effects should not be sensitive to a supplement of the protein. Moreover, according to random positioning, a low-copy-number plasmid whose centromere is also present on a multicopy vector would initially occupy a few positions among numerous partition complexes throughout the cell. The plasmid could soon find itself and its replicas trapped in one half of a cell and be lost far faster than the random rate observed. No evidence for this conjecture has been reported, possibly because the relevant experiments have been performed not with the full locus but with multicopy vector-borne centromeres that dissipate the positioning effect by titrating the centromere-binding protein. The experiment with the full locus could prove a useful test of the model. In the meantime, random positioning remains relevant mainly to incompatibility of two low-copy-number plasmids. As shown by Bouet et al. the incompatibility can be severe enough to indicate that the plasmids segregate as clusters. This implies that the effective partition substrate recognized by the positioning apparatus could be composed of two or more individual partition complexes further enlarged by spreading. With this modest extension, the random positioning model can explain stability of dicentric plasmids, because pairing of two partition complexes on the same molecule will create an enlarged partition substrate that, with its twin on the other plasmid replica, will activate partition in the same way as would paired complexes on single-centromere plasmid siblings. Conclusions Instead of the one viable model of centromere-based incompatibility, mixed pairing, we now have three. It would be wrong to consider them as mutually exclusive alternatives however. Titration of the centromere-binding protein

8 1412 J.-Y. Bouet, K. Nordström and D. Lane Indirect pairing model Fig. 6. Indirect pairing. Partition does not necessarily start by breaking the ParB ParB interactions responsible for pairing partition complexes on sibling plasmids. Instead, ParA can be activated by a ParB DNA entity that consists of either an isolated ParB pars complex, a paired complex (as shown) or an ensemble in a plasmid cluster. Partition ensues when the activated ParA encounters another ParB DNA entity, which may never have been in contact with the activating one. Thus, plasmids can be segregated as either clusters, pairs or individuals. applies only when the centromere is carried by a multicopy vector, and although it reveals quantitative aspects of interactions between partition proteins and sites it has not told us much about the partition mechanism itself. The power of the mixed-pairing model for explaining incompatibility is seen to have limits but the essential nature of the model participation of two incompatible plasmids in the same segregation event remains relevant to partition. The random partitioning model does not entirely negate mixed pairing, only the way we have been used to seeing it. In the form proposed by Ebersbach et al. the model focused on positioning of sibling plasmids. However, repositioning implies further interaction with incompatible plasmids. In effect, the model allows plasmids, as individuals or clusters, to constitute an analogue of the classic mixed pair by attaching independently to opposite ends of the ParA positioning apparatus rather than directly to each other (Fig. 6). The extended nature of budding ParA filaments might raise the chances of contacting cognate partition complexes on distinct plasmids. We suggest that this extension of the random partitioning model and the variable geometry of plasmid positioning could both contribute to the missegregation seen as incompatibility. Perspectives Positioning of plasmid replicas seems to be a function of the size and persistence of the partition complexes they present to ParA. Partition complexes consist of two parts, the specific ParB-parS core and the ParB DNA complexes that spread from it. The robustness of the former portion is presumably proportional to the number of binding sites it contains, while the latter portion depends on non-specific ParB DNA links that are intrinsically unstable, as pointed out elsewhere (Funnell, 2005), and are likely to be highly variable in extent. The productive interaction with the partition ATPase that underlies positioning is expected to be sensitive to this factor a large, longer-lived complex is more likely than a small, fragile one to activate ParA and to be mobilized by it. In the context of incompatibility, the more robust the partition complex the more likely its plasmids are to be positioned at the quarter positions and thus to occupy mid-cell in the next cell cycle (Fig. 5B), as seen by the incompatibility behaviour of subcentromeres (parc1/c2, single-unit sopc) in comparison with that of the full parc and sopc. By affecting the likelihood of activating ParA, stochastic variations in partition complex size may well contribute to the randomness of positioning. A mechanism for the activation of SopA filamentation based on nonspecific binding of SopB to DNA has been proposed recently (Bouet et al., 2007). Clustering of plasmids provides an opportunity to change partition complex size in the other direction, by creating a combination of individual complexes that strongly activate ParA. Whether this opportunity is actually exploited remains to be demonstrated. A combinedcomplex partition substrate could allow molecules with internally paired complexes, such as dimers, to be partitioned in this form rather than pose topological problems for the partition apparatus. It would also be consistent with several time-lapse observations of clusters segregating as such. It could contribute to the severity of incompatibility of clustered low-copy-number plasmids, although reduction in partitionable unit number may well be the main cause of this extreme incompatibility. Unfortunately too little is known about the causes and perpetuation of the clustered state for insights into incompatibility to be readily gleaned from it. An attempt to solve the central riddle posed by clustering that visibly clustered parminus plasmids generally segregate at the rate of randomly diffusing single molecules (Nordstrom and Gerdes, 2003) proposed that individual plasmids dissociate from the cluster to replicate at mid-cell where the copies then segregate at random before rejoining their cluster or seeding another one. However, this is difficult to reconcile with evidence from incompatibility experiments and microscopy that the partitionable units may be clusters, and recent work in the Nordström laboratory has indicated

9 Partition-based incompatibility 1413 that replication takes place in clusters rather than at midcell (B. Singh, K. Nordström and S. Dasgupta, unpublished). For the moment, the riddle remains. If we can do without direct pairing of partition complexes for incompatibility, as suggested by the random positioning model, can we dispense with it for partition? Demonstration that partition complexes can pair is not proof that partition needs them to. This slightly heretical notion acquired respectability upon publication of in vitro evidence that simple proximity of partition complexes suffices to stabilize protofilaments of the actin-like ParM and allow their extension (Garner et al., 2004). Although certain Walker-box ParAs express their dynamic potential differently from ParM, by oscillating over the nucleoid region rather than forming a metastable rod, we suggest that the same mechanistic principle applies: that assembly of ParA activated by a partition complex on one replica will be stabilized and extended if it finds the (usually nearby) sibling complex. The resulting polymerization partitions the plasmids. Segregation and redistribution of incompatible plasmids (random positioning) would use the same process. A test of the mixed pairing model of incompatibility has led to discovery of random positioning as a property of a partition system. A survey of plasmid incompatibility has revealed the influence of replication mode and plasmid clustering on partition. Will studies of incompatibility continue to further our understanding of partition? In any case we do not fully comprehend either yet, and it would be foolhardy to neglect incompatibility until we do. 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