Philina S. Lee* and Alan D. Grossman* Department of Biology, Building , Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Transcription

1 Blackwell Publishing LtdOxford, UKMMIMolecular Microbiology9-382X; Journal compilation 26 Blackwell Publishing Ltd? Original ArticleChromosome partitioning in B. subtilisp. S. Lee and A. D. Grossman Molecular Microbiology (26) 6(4), doi:1.1111/j x First published online 13 March 26 The chromosome partitioning proteins Soj (ParA) and SpoJ (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis Philina S. Lee* and Alan D. Grossman* Department of Biology, Building 68-3, Massachusetts Institute of Technology, Cambridge, MA 2139, USA. Summary Soj (ParA) and SpoJ (ParB) of Bacillus subtilis belong to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. Unlike most Par systems, for which intact copies of both para and parb are required for the Par system to function, inactivating soj does not cause a detectable chromosome partitioning phenotype whereas inactivating spoj leads to a 1-fold increase in the production of anucleate cells. This suggested either that Soj does not function like other ParA homologues, or that a cellular factor might compensate for the absence of soj. We found that inactivating smc, the gene encoding the structural maintenance of chromosomes (SMC) protein, unmasked a role for Soj in chromosome partitioning. A soj null mutation dramatically enhanced production of anucleate cells in an smc null mutant. To look for effects of a soj null on other phenotypes perturbed in a spoj null mutant, we analysed replication initiation and origin positioning in (soj-spoj)+, Dsoj, DspoJ and D(soj-spoJ) cells. All of the mutations caused increased initiation of replication and, to varying extents, affected origin positioning. Using a new assay to measure separation of the chromosomal origins, we found that inactivating soj, spoj or both led to a significant defect in separating replicated sister origins, such that the origins remain too close to be spatially resolved. Separation of a region outside the origin was not affected. These results indicate that there are probably factors helping to pair sister origin regions for part of the replication cycle, and that Soj Accepted 2 January, 26. *For correspondence. adg@mit.edu; philinalee@gmail.com; Tel. (+1) ; Fax (+1) Present address: Department of Biomedical Engineering; Boston University; 44 Cummington Street; Boston, MA 221, USA. and SpoJ may antagonize this pairing to contribute to timely separation of replicated origins. The effects of Dsoj, DspoJ and D(soj-spoJ) mutations on origin positioning, chromosome partitioning and replication initiation may be a secondary consequence of a defect in separating replicated origins. Introduction Dividing cells must partition their chromosomes accurately in order for daughter cells to receive a complete copy of the genome. The well-conserved Par system contributes to accurate partitioning of low-copy-number plasmids and chromosomes in diverse bacterial species (Hiraga, 2; Bignell and Thomas, 21; Surtees and Funnell, 23). The Par system is defined by two transacting factors and a cis-acting site: ParA, a Walker-type ATPase that binds DNA, ParB, a DNA-binding protein that interacts with ParA, and pars, the site bound by ParB. The molecular mechanism of how the Par system functions remains unknown, although studies of the plasmid Par systems led to models that the Par system may (1) attach pars-containing DNA to putative anchors at the cell quarters (Ogura and Hiraga, 1983; Watanabe et al., 1989; Kim and Wang, 1998; Rodionov et al., 1999; Yamaichi and Niki, 24), and or (2) pair plasmids prior to moving them in opposite directions (Austin and Abeles, 1983a; Funnell, 1988; Nordström and Austin, 1989; Youngren and Austin, 1997; Edgar et al., 21; Surtees and Funnell, 23). Less is known about the function of chromosomally encoded Par systems. It is likely that the Par system helps to partition replicated origins, as known or putative ParB binding sites are located near the origin region (Mohl and Gober, 1997; Lin and Grossman, 1998; Kim et al., 2). Partitioning of chromosomal origins probably involves a mechanism that separates them, as well as a mechanism that maintains their characteristic subcellular positions. In many bacteria, replicated origins separate soon after initiation of replication and move to opposite halves of the cell even as replication of distal regions continues (Jensen and Shapiro, 1999; Lemon and Grossman, 21; Li et al., 22). For the majority of the cell cycle, replicated origins occupy characteristic positions: at the cell quarters in Journal compilation 26 Blackwell Publishing Ltd

2 84 P. S. Lee and A. D. Grossman Bacillus subtilis (Lin et al., 1997; Webb et al., 1997; 1998; Sharpe and Errington, 1998; Lee et al., 23), at the cell poles in Caulobacter crescentus (Mohl and Gober, 1997; Figge et al., 23) and near the cell quarters or poles in Escherichia coli (Gordon et al., 1997; Niki et al., 2; Li et al., 22; Lau et al., 23) (E. coli is not known to have a chromosomally encoded Par system). In this study, we present data demonstrating that efficient separation of replicated chromosomal origins depends on the Par system in B. subtilis. Bacillus subtilis possesses chromosomally encoded homologues of ParA and ParB, called Soj and SpoJ respectively. SpoJ binds to at least eight pars sites spread over nearly 8 kb in the origin-proximal 2% of the chromosome (Lin and Grossman, 1998) and probably brings the pars sites together, forming a nucleoprotein complex that can be visualized as a focus using immunofluorescence microscopy or a green fluorescent protein (GFP) fusion to SpoJ (Lin et al., 1997; Sharpe and Errington, 1998; Teleman et al., 1998). The subcellular location of SpoJ foci reflects the position of chromosomal origins (Lin et al., 1997; Sharpe and Errington, 1998; Teleman et al., 1998). In the spoj mutant, chromosome partitioning occurs with reduced accuracy, and replicated origins are mispositioned such that they are often closer together than the cell quarters (Ireton et al., 1994; Lee et al., 23; Ogura et al., 23), leading to the hypothesis that SpoJ may contribute to separation of replicated origins and/or maintenance of replicated origins at the cell quarters. Soj and SpoJ probably do not function to recruit pars sites to the cell quarters, because they are not sufficient to recruit an array of pars sites inserted elsewhere in the chromosome to the cell quarters (Lee et al., 23). The spoj mutant is pleiotropic, leading also to asynchronous and early initiation of replication (Lee et al., 23; Ogura et al., 23), and a defect in spore formation. SpoJ is needed for efficient sporulation because it relieves Soj-mediated transcriptional repression of sporulation genes (Ireton et al., 1994; Cervin et al., 1998; Quisel et al., 1999; Quisel and Grossman, 2). The mechanism by which SpoJ regulates initiation of replication is not known. This study presents comprehensive phenotypic analyses of a soj mutant in comparison with spoj and (sojspoj) mutants. The results show that Soj and SpoJ contribute to accurate chromosome partitioning, origin separation and regulation of replication initiation. Although a soj null mutant does not have a detectable chromosome partitioning defect on its own (Ireton et al., 1994), inactivating soj in a background lacking smc, the gene encoding structural maintenance of chromosomes (SMC) protein, led to a nearly 1-fold increase in anucleate formation, a degree of enhancement similar to spoj and (soj-spoj) mutants. Thus, the presence of SMC, a chromosome compaction protein, normally masks the contribution of Soj to chromosome partitioning. The soj, spoj and (soj-spoj) mutants all showed overinitiation of replication and, to varying extents, defects in positioning the origin of replication. Using an assay to measure separation of chromosomal regions, we found that inactivating soj and/or spoj leads to defects in separating replicated origins, such that a significant proportion of cells contain replicated origins that have not separated enough to be spatially resolved. Separation of a region outside the origin was not affected. The finding that efficient origin separation requires a committed mechanism suggests that there may be origin region-specific processes or factors that hold sister origins together for part of the replication cycle, and that Soj and SpoJ function to overcome these factors. Results and discussion Inactivating soj enhances the chromosome partitioning defect of an smc null mutant While inactivating spoj leads to an approximately 1- fold increase in the production of anucleate cells, inactivating soj does not cause an appreciable chromosome partitioning defect on its own (Ireton et al., 1994). In contrast, stable maintenance of pars plasmids requires both soj and spoj (Lin and Grossman, 1998). For other plasmids and chromosomes encoding Par homologues, inactivating para or parb leads to similar partitioning defects (Austin and Abeles, 1983a,b; Ogura and Hiraga, 1983; Mohl and Gober, 1997; Easter and Gober, 22; Figge et al., 23). In light of these findings, it seemed likely that Soj would contribute to chromosome partitioning during vegetative growth, but its contribution could be masked by other factors. As a spoj null mutation strongly enhances the chromosome partitioning defect of a null mutation in the smc gene (Britton et al., 1998; Britton and Grossman, 1999), we tested whether a soj null mutation caused a similar enhancement. SMC is a DNA-binding protein that contributes to chromosome compaction and organization (Hirano, 1998; 22; Lindow et al., 22a). A soj null mutation significantly enhanced the chromosome partitioning defect of an smc null mutant (Table 1). While most of the nucleoids in the smc single mutant had regular size and spacing, the soj smc double mutant had dramatically perturbed nucleoid morphology (Fig. 1), with irregular nucleoid size, anucleate and cut cells (in which the division septum bisects the nucleoid). The soj single mutant appeared similar to wild-type cells (data not shown). A soj mutation enhanced the partitioning defect of an smc mutant just as dramatically as a spoj mutation did. A soj mutant did not have a detectable chromo-

3 Chromosome partitioning in B. subtilis 8 Table 1. Chromosome partitioning defects in various mutants. Strain a Relevant genotype % anucleate b % cut c % anucleate + cut Total cells d AG174 Wild type <.6 <.6 <.6 16 SV132 soj <.4 <.4 < AG1468 spoj RB3 smc PSL21 soj smc RB41 spoj smc PSL68 (soj-spoj) smc PSL64 scpa PSL62 soj scpa PSL642 scpb PSL64 soj scpb a. Indicated strains were inoculated from light lawns on minimal plates into defined minimal glucose medium at 3 C. Samples were taken for microscopy during exponential growth. The cell membrane was stained with FM4-64 and the nucleoid was stained with DAPI. b. Cells devoid of any visible DAPI staining were scored as anucleate. c. Cells containing a nucleoid bisected by the division septum were scored as cut. d. The total number of cells analysed. some partitioning defect on its own, while a spoj mutant had.3% anucleate and.2% cut cells (Table 1). An smc single mutant had 2.1% anucleate and 2.% cut cells, and combining this mutation with soj, spoj or (soj-spoj) led to similar, strong levels of enhancement with production of 18 19% anucleates and 8 12% cut cells (Table 1). The chromosome partitioning defect of the smc mutant was lower than previously reported (Britton et al., 1998; Moriya et al., 1998). This was due to differences in the growth media and method of culture inoculation (see Experimental procedures). In B. subtilis, SMC can form a complex with two other proteins, ScpA and ScpB (Lindow et al., 22b; Soppa et al., 22; Volkov et al., 23). Inactivating soj enhanced anucleate production in the scpa or scpb mutants by nearly 1-fold, similar to the enhancement in the smc mutant (Table 1). Our results indicate that the synthetic chromosome partitioning defects are due mainly to loss of the SMC ScpA ScpB complex. The smc mutant background uncovers a role for Soj in chromosome partitioning. The finding that spoj, soj and (soj-spoj) mutations enhanced the chromosome partitioning defect of an smc null mutation to a similar extent indicates that Soj and SpoJ probably perform chromosome partitioning functions in the same pathway, analogous to ParA and ParB from other organisms (Austin and Abeles, 1983a,b; Ogura and Hiraga, 1983; Mohl and Gober, 1997; Easter and Gober, 22; Figge et al., 23). We suspect that Soj may help SpoJ bring the pars sites together, forming an organized nucleoprotein complex that compacts the origin region. Consistent with this model, inactivating soj can cause foci of SpoJ GFP to mislocalize as many smaller, fragmented foci (Marston and Errington, 1999). ParA from other organisms appears to modulate the size of ParB nucleoprotein complexes on pars-containing DNA (Mohl and Gober, 1997; Bouet and Funnell, 1999; Lemonnier et al., 2; Figge et al., 23), indicating that ParA proteins may generally regulate ParB binding and/or higher-order interactions between ParB molecules bound at the partition complex. To account for why a soj mutant does not have a detectable partitioning defect on its own, we propose that SMC-mediated chromosome compaction could also help to bring pars sites closer together. This model was motivated in part by the A * B * * * * * * * Fig. 1. Inactivating soj enhances the partitioning defect of an smc null mutant. The smc mutant and smc soj double mutant (RB3 and PSL21 respectively) were grown at 3 C in defined minimal glucose medium and samples were taken for microscopy during exponential growth. Membranes were stained red with FM4-64 and DNA was stained blue with DAPI. A. smc null mutant cells. B. smc soj double mutant cells showing dramatic defects in nucleoid partitioning. Asterisks and arrows indicate examples of anucleate cells and cut cells respectively.

4 86 P. S. Lee and A. D. Grossman finding that nucleated cells of an smc null mutant appear to have a defect in assembling SpoJ GFP foci (Britton et al., 1998). Inactivating soj, spoj or both leads to overinitiation of replication and production of elongated cells After uncovering a role for Soj in chromosome partitioning, we decided to explore whether a soj null mutant has additional phenotypes that are also perturbed in a spoj null mutant. As a spoj mutant overinitiates replication and produces elongated cells (Lee et al., 23; Ogura et al., 23), we examined the effects of soj on these phenotypes. Overreplication was monitored by measuring the DNA to protein ratio and cell lengths of wild-type, soj, spoj and (soj-spoj) cells. The DNA to protein ratio is a standard assay for perturbations in DNA replication, and overinitiation of replication leads to an increase in the DNA to protein ratio. All of the mutant strains had a DNA to protein ratio 4 % higher than that of wild-type cells, consistent with overinitiation of replication (Table 2). We propose that Soj and SpoJ affect initiation of replication by regulating accessibility of the origin to initiation factors. Formation of a nucleoprotein structure that compacts the origin region could exclude initiation factors from binding until a putative signal opens the structure. As a soj mutation causes overinitiation of replication, but not a significant defect in chromosome partitioning, perhaps cellular factors such as SMC can compensate for Soj s role in partitioning but not in replication initiation. Furthermore, the similar overreplication phenotype caused by spoj mutations is not likely to be the cause of its partitioning defect. We measured cell length in wild-type, soj and (sojspoj) cells. The average cell length was 2% longer in (soj-spoj) cells, similar to spoj mutants (Lee et al., 23), although the soj mutant alone had no detectable defect. Average cell lengths in wild-type, soj and (sojspoj) cells were 2.78 ±.1 µm, 2.82 ±.9 µm and Table 2. DNA to protein ratios in wild-type and mutant cells. Strain a Relevant genotype Normalized DNA to protein ratio b AG174 Wild type 1. SV132 soj 1.4 ±.2 AG1468 spoj 1. ±.2 AG1 (soj-spoj) 1.4 ±.2 a. Indicated strains were inoculated from light lawns on minimal glucose plates into defined minimal glucose medium and grown at 3 C. Samples were collected during exponential growth at an OD 6 of.4.6. Nucleic acid and protein were extracted and assayed for DNA and protein content as described in Experimental procedures. b. Experimentally determined ratios from four experiments, normalized to the ratio from wild type done in parallel. Ratios are followed by the 9% confidence interval for the mean. The average ratio for wild-type cells was.3 ± ±.14 µm respectively (results ± 9% confidence intervals, > 2 cells scored). The cell division delay of spoj and (soj-spoj) cells cannot explain the increased DNA to protein ratio, because cell growth, DNA replication and segregation appear to proceed normally even under conditions where cell division is blocked (Dai and Lutkenhaus, 1991; Huls et al., 1999; Kawai et al., 23). Although a soj null mutation did not affect cell length on its own, the soj smc double mutant had significantly elongated cells compared with the smc null mutant alone (data not shown). However, because the double mutant had many cells with long, unsegregated nucleoids, nucleoid occlusion would have inevitably led to longer cell lengths and it would be difficult to conclude whether there were additional effects on the division apparatus. The proper subcellular positioning of SpoJ and Soj depends on cell division proteins like DivIB, FtsZ, PBP and MinD (Marston and Errington, 1999; Autret and Errington, 23; Real et al., 2). Perhaps the cell division delay could reflect a stimulatory effect of SpoJ on the division apparatus. Alternatively, defects in chromosome partitioning may elicit a corresponding delay in cell division through an unidentified regulatory mechanism. Our observations that the (soj-spoj) double mutant has a higher DNA to protein ratio and elongated cells similar to a spoj single mutant differ from a report that inactivating soj partially suppresses the overinitiation and cell division phenotypes of a spoj null mutant (Ogura et al., 23). From these results, the authors concluded that Soj may have an activity opposite to SpoJ. This discrepancy could reflect differences in the strain backgrounds or growth media, and warrants further investigation, because the results lead to different predictions about the function of Soj. Inactivating soj, spoj or both perturbs the number of foci per cell of the origin, 27 and terminus regions of the chromosome Previously, it was shown that inactivating spoj perturbs the number of foci per cell of several chromosomal regions: the origin-proximal 39 region, the 27 region, and the terminus-proximal 181 region (Lee et al., 23). We visualized these regions in soj and (soj-spoj) mutants using LacI GFP bound to an array of lac operators inserted in the chromosome (Gordon et al., 1997; Webb et al., 1997). The majority of wild-type cells (8.9%) had two foci of the origin region, and 1.3% had more than two foci (Table 3) (Lee et al., 23). Inactivating soj, spoj or both led to a higher proportion of cells with more than two foci of the origin region (3.1%, 3.4% and 27.% respectively) (Table 3). These results are consistent with overinitiation of replication, demonstrated in the

5 Chromosome partitioning in B. subtilis 87 Table 3. Number of foci per cell of several chromosomal regions during exponential growth in wild-type and mutant cells. Insertion a Relevant genotype % of cells with indicated number of foci b Average > 4 Total cells c no. of foci per cell f Average no. of foci relative to wild type g 39 Wild type d soj < spoj e (soj-spoj) < Wild type d soj < < spoj e (soj-spoj) Wild type <.1 <.1 14 d soj <.2 < spoj <. 222 e (soj-spoj) a. An array of lac operators was inserted in the indicated region of the chromosome and visualized with LacI GFP or LacI CFP. b. The percentage of cells with the indicated number of foci of LacI GFP (or LacI CFP) was determined for cells growing exponentially in defined minimal glucose medium at 3 C. Cells with no foci were mostly anucleates. c. The total number of cells analysed for each strain. d. Combined data from newly repeated and previously published results (Lee et al., 23). e. Previously published results (Lee et al., 23) shown for comparison. f. Average number of foci per cell (a) was calculated for each strain using the data showing percentage of cells with indicated number of foci, and the formula: a = *(% cells with foci) + 1 *(% cells with 1 focus) + 2 *(% cells with 2 foci) + 3 *(% cells with 3 foci) + 4 *(% cells with 4 foci) + *(% cells with > 4 foci). g. Average number of foci per cell, normalized to the wild-type strain for that chromosomal region (b) was calculated for each strain using the formula: b = a wild type or mutant /a wild type. previous section, which would increase the number of origins per cell. Surprisingly, the mutants also had a higher proportion of cells with one focus of the origin: 3.1% of wild-type cells had a single focus, compared with 6.3%, 1.3% and 8.7% in soj, spoj and (soj-spoj) mutants respectively (Table 3). One caveat of using this method to count the number of chromosomal regions is that duplicated regions of the chromosome must be separated enough to be spatially resolved in order to be counted as two copies. If there were a defect in separating a particular chromosomal region, then this would lower the number of resolvable foci, leading to an underestimate of the copy number. The increased proportion of cells with one focus of the origin may be due to a defect in separating replicated sister origins. Because the mutants do not lead to an increased proportion of cells with a single focus of the 27 and 181 regions, the separation defect may be specific to the origin region. We present data supporting this hypothesis below. To determine whether the net effect of the mutations was to increase or decrease the number of origins, we calculated the average number of origin foci per cell: the wild-type strain had 2.21 foci per cell on average, while the soj, spoj and (soj-spoj) mutants had 2.42, 2.31 and 2.3 foci per cell respectively (Table 3). Thus, the mutants had 4 9% more foci of the origin than wild-type cells (Table 3). This effect was more pronounced for the 27 and 181 regions, for which the mutants had 8 31% more foci of the origin than wild-type cells (Table 3). These data indicate that the mutants, on average, have increased chromosome copy number relative to wild-type cells. These data also indicate that the mutations increase the apparent copy numbers of the 27 and 181 to a greater extent than the copy number of the origin region in the mutant strains, consistent with a defect in separating replicated origins but not other regions of the chromosome (see below). Effects of soj and spoj on positioning separated sister origin regions Previously, we showed that duplicated foci of the origin are closer together in a spoj null. To determine whether Soj affects positioning of separated sister origins, we measured origin positioning in wild-type, soj, spoj and (soj-spoj) mutant cells with two spatially resolved foci of the origin. We found that duplicated foci of the origin were positioned normally at the cell quarters in the soj mutant, but were closer together in (soj-spoj) cells, similar to the spoj mutant (Lee et al., 23) (Fig. 2, Table 4). Origins were visualized using LacI GFP bound to an array of lac operators inserted at 39 (near oric). Average distance from a focus to the nearest pole was 26.2 ± 1.3% of cell length in the soj mutant, statistically indistinguishable from positioning at 26.1 ± 1.% of cell length in wild-type cells (± 9% confidence interval for the mean) (Table 4). The (soj-spoj) mutant had foci positioned at 31.2 ± 1.8% of cell length, similar to 3.1 ± 1.% in the spoj mutant (Table 4).

6 88 P. S. Lee and A. D. Grossman Cell length (µm) A wild type B soj n = 33 n = C spoj 2 3 D (soj-spoj) Fig. 2. Subcellular locations of duplicated foci of the origin. The origin region was visualized using LacI GFP bound to an array of lac operators integrated at the 39 region of the chromosome. Strains were grown in defined minimal glucose medium at 3 C and cells with two separated foci of the origin were analysed. The distance from each focus to the same cell pole was measured from images of live cells in exponential growth (Experimental procedures) and is plotted on the x-axis; cell length is plotted on the y-axis. Cell poles and the midcell positions are indicated by solid diagonal lines. Cell quarters are indicated by grey diagonal lines. The number of cells analysed (n) is indicated in the lower right-hand corner of each panel. One focus is indicated with open circles and the other with crosses. A. Wild type (DCL696); B. soj (PSL41); C. spoj (DCL7); D. (sojspoj) (PSL37). Data in panels A and C were previously published (Lee et al., 23) and are included for comparison n = 222 n = Pole to focus distance (µm) Table 4. Subcellular positioning of replicated sister origins in cells with two foci of the origin region. a Strain Genotype Focus position b Interfocal distance c Total cells d DCL696 e Wild type 26.1 ± ± PSL41 soj 26.2 ± ± DCL7 e spoj 3.1 ± ± PSL37 (soj-spoj) 31.2 ± ± a. Indicated strains were grown at 3 C in defined minimal glucose medium and samples were taken for microscopy during exponential growth. LacI GFP was used to visualize lac operator arrays integrated at the 39 region of the chromosome. b. The distance from each focus to the nearest cell pole was measured in cells with two foci, and is presented as an average percentage of cell length ± the 9% confidence interval for the mean. c. The distance between each focus was measured in cells with two foci, and is presented as an average percentage of cell length ± the 9% confidence interval. d. The total number of cells with two foci that were analysed. e. Previously published results (Lee et al., 23) shown for comparison. In spoj and (soj-spoj) cells, the foci of the origins were positioned closer together than in wild-type cells (Fig. 3, Table 4): the average interfocal distance was 39.7 ± 1.8% of cell length in spoj (Lee et al., 23) and 37.6 ± 1.9% of cell length in (soj-spoj), compared with 47.8 ± 1.2% in wild-type cells (Table 4). The average interfocal distance in the soj mutant was 47. ± 1.3%, indistinguishable from wild-type cells (Table 4). Our results indicate that the detection of defects in positioning duplicated, spatially resolved origins, correlates with defects in chromosome partitioning. The effects of spoj and (soj-spoj) mutants on positioning of duplicated foci of the origin could be due to a defect in separating replicated origins and/or a defect in maintaining the position of replicated origins at the cell quarters. Below, we present results indicating that the spoj and (soj-spoj) mutants, and, to a lesser extent, the soj mutant, have defects in separating replicated origins. Effects of soj and spoj on positioning a single focus of the origin region The soj, spoj and (soj-spoj) mutations perturbed focus positioning in cells with a single visible focus of the origin region (Fig. 4). Cultures were grown with succinate instead of glucose as a carbon source, resulting in a slower doubling time and more cells with a single focus of the origin. The majority of wild-type cells had a focus positioned between 3% and 6% of cell length, and only 12.6% of cells had a focus outside this central region (Fig. 4). In the soj, spoj and (soj-spoj) mutants, 41.9%, 26.6% and 2.% of cells had a focus outside this region respectively (Fig. 4). The mispositioning of the origin region in these mutants could be indicative of a role for Soj and

7 Chromosome partitioning in B. subtilis 89 Interfocal distance (µm) A wild type n = B soj n = C spoj n = 222 D soj-spoj n = Fig. 3. Relationship between cell length and interfocal distance. The distance between the two origin-region foci in each cell (interfocal distance) was determined from the data in Fig. 2 and is plotted as a function of cell length. A. Wild type (DCL696); B. soj (PSL41); C. spoj (DCL7); D. (soj-spoj) (PSL37). The number of cells analysed (n) is indicated in the upper right-hand corner of each panel. An ellipse was drawn around the distribution of wild-type cells (A) and superimposed on the corresponding plots in panels B, C and D. The interfocal distances in most of the spoj and (soj-spoj) mutant cells were within this ellipse; a subset ( 1%) fell below and to the right of the wild-type distribution. These cells had replicated origins that were closer together than the origins in wild-type cells of similar length. Panels A and C were previously published (Lee et al., 23) and are included for comparison Cell length (µm) SpoJ in positioning a single origin at or near midcell. Alternatively, some of the mutant cells that appear to have a single origin might actually contain two replicated sister origins that remained close together (paired) and moved away from midcell. The experiments in the next section were designed to distinguish between these possibilities. Inactivating soj and/or spoj impaired separation of replicated DNA in the origin region To determine whether the mutant cells had replicated origins that were too close together to be spatially resolved, we visualized the origin and a nearby distal region simultaneously using two markers: LacI CFP (cyan fluorescent protein) bound to an array of lac operators inserted at the 39 region of the chromosome, and TetR YFP (yellow fluorescent protein) bound to an array of tet operators inserted at the 34 region of the chromosome (located 164 kb distal to the 39 region) (Fig. A). Cells that have two foci of the distal region must have initiated replication whether or not the replicated origins are spatially resolvable. In general, cells with fewer foci of the origin region than the distal region must contain replicated origins that have not separated. We found that inactivating soj, spoj or both caused a defect in origin separation: 3.2% of wild-type cells had fewer foci of the origin region than the distal region, compared with 13.7% in soj, 2.3% in spoj and 21.6% in (soj-spoj) mutants respectively (Table ). In addition, each of the mutants had an increase in the proportion of cells with more foci of the origin region than the distal region (Table ). Overinitiation of replication in these mutants could potentially increase the relative copy number of the origin region to the 34 region. Alternatively, there could also be a defect in separating the 34 region of the chromosome, which lies inside the range of the origin-proximal pars sites (Fig. A). Although soj, spoj and (soj-spoj) mutants have defects in separating replicated origins, most of the origins Table. Comparison of relative numbers of foci of an origin-proximal (39 ) and origin-distal (34 ) region of the chromosome. a Strain b Genotype %P < D %P = D %P > D Total cells c PSL438 Wild type PSL392 soj PSL44 spoj PSL39 (soj-spoj) a. The number of foci of the origin-proximal marker P (39 ) and origin-distal marker D (34 ) was determined in individual cells and the percentage of cells with the indicated relationship is shown. b. Indicated strains were grown at 3 C in defined minimal succinate medium and samples were taken during exponential growth for microscopic analysis. c. Total number of cells scored.

8 86 P. S. Lee and A. D. Grossman A C % Cells % Cells E % Cells G % Cells Wild type soj spoj (soj-spoj) 12.6% 41.9% 26.6% 2.% B D F Cell length (µm) Cell length (µm) Cell length (µm) Cell length (µm) H n = 111 n = 1 n = 124 n = 112 Fig. 4. Subcellular location of the origin region in cells with a single visible focus. The origin region was visualized using LacI GFP bound to an array of lac operator integrated at the 39 region of the chromosome. Strains were grown in defined minimal succinate medium at 3 C and cells with a single visible focus of the origin were analysed. The distance from the focus to a cell pole was determined and is presented as a percentage of cell length (panels A, C, E, G) or plotted on the x-axis compared with cell length on the y-axis (panels B, D, F, H) as in Fig. 2. For panels A, C, E and G, the percentage of cells with a focus in each % increment of cell length ( %, 1%, etc. of cell length) was calculated and plotted as a histogram. The length increments from to 3% and 6 1% are highlighted in grey, and the proportion of cells with a focus in these ranges is indicated in the upper right-hand corner of each histogram. A and B. Wild type (DCL696). C and D. soj (PSL41). E and F. spoj (DCL7). G and H. (soj-spoj) (PSL37) Focus position as % of cell length Pole to focus distance (µm) must separate eventually, because the proportion of cells with an origin separation defect far outweighs the proportion of anucleate cells produced. This delay in origin separation could be due to several possible primary defects, including: (1) a defect that slows migration of replicated origins, and/or (2) a defect in releasing origins from the constraints of putative factors that could pair them or anchor them in close proximity. The spoj and double mutants had more severe origin separation defects than the soj single mutant, correlated with measurable chromosome partitioning defects and elongated cells that were not observed in the soj single mutant.

9 Chromosome partitioning in B. subtilis 861 A teto laco / LacI-CFP TetR-YFP 18 T teto laco Fig.. Schematic of B. subtilis chromosomes with insertions of laco and teto arrays. The B. subtilis chromosome is represented as the thick circle with the origin of replication (O) at 36 / and the terminus region (T) at approximately 18. The eight known SpoJ binding sites (Lin and Grossman, 1998) are indicated with small open boxes in the origin region and the presence of laci cfp and tetr yfp fusions is indicated. A. Strain with a laco array at 39 and a teto array 34 for measuring separation of replicated sister origins. B. Strain with a laco array at 316 and a teto array at 3 for measuring separation of replicated sister 316 regions. Inactivating soj and/or spoj has little effect on separation of replicated DNA outside the origin region To determine whether soj, spoj or (soj-spoj) mutations affected separation of chromosomal regions outside the origin region, we simultaneously visualized the 316 region, using LacI CFP bound to an array of lac operators, and the 3 region (located 187 kb distal), using TetR YFP bound to an array of tet operators (Fig. B). Both sites are located outside the origin-proximal region spanned by the known pars sites. The 316 region is replicated first. Cells that failed to separate the 316 region before the 3 region would have fewer foci of the 316 region than the 3 region. Inactivating soj, spoj or both did not substantially affect separation of the 316 region (Table 6). In wild-type cells, 1.4% had fewer foci of the 316 region than the 3 region, compared with.7% in soj, 3.1% in spoj and 2.8% in (soj-spoj) mutants (Table 6). The lack of an appreciable separation defect was not due to the fact that the 316 and 3 regions are slightly further apart than 39 and 34, because a significant defect in origin separation was also seen when 39 and 329 were visualized simultaneously (data not shown). Taken together, our results demonstrate that soj and spoj are required for efficient separation of replicated DNA in the origin region, and that these effects are specific to the origin region. Sister origins may be paired prior to separation The finding that replicated origins, but not other regions of the chromosome, remain closely associated in the absence of soj and/or spoj indicates that there may be B 36 / LacI-CFP TetR-YFP 18 T specific barriers to separating sister origin regions. Replicated origins might be paired and/or anchored at nearby sites by an unidentified factor, and Soj and SpoJ function could overcome this barrier to separation. The processes or factors that might promote pairing are not known, but could be related to the SMC complex, similar to MukBEF that appear to promote chromosome pairing in E. coli (Sunako et al., 21). Alternatively, it is formally possible that SpoJ and Soj pair the origins, as proposed for plasmid Par homologues, although the simple prediction from this model is that deletion of soj or spoj would lead to increased separation of replicated origins, rather than the observed decrease. To our knowledge, only one other factor has been identified so far that contributes specifically to separation of replicated origins. Function of the bacterial actin homologue MreB is required during a period early in the cell cycle for separation of replicated origins in C. crescentus (Gitai et al., 2). Soj and spoj mutations cause pleiotropic phenotypes, but most of these effects may be explained by a single function Inactivating soj and/or spoj perturbs many cellular processes (Table 7). It seems unlikely that Soj and SpoJ would have distinct functions for each of these processes. We speculate that a single function may be sufficient to account for most of the phenotypes we examined. A common feature was that the (soj-spoj) double mutant followed the behaviour of the spoj single mutant for phenotypes in chromosome partitioning (formation of anucleate cells), replication initiation (overinitiation), cell division (elongated cell lengths), origin separation and origin positioning. In other words, spoj was epistatic to soj. Given the epistasis relationship, it is likely that if Soj affects these processes, it probably does so by exerting effects on SpoJ, i.e. Soj SpoJ phenotype (Fig. 6A). The soj null either did not have a phenotype or it was not as Table 6. Comparison of relative numbers of foci of chromosomal regions (316 and 3 ) away from the origin. a Strain b Genotype %P < D %P = D %P > D Total cells c PSL82 Wild type PSL78 soj PSL8 spoj PSL76 (soj-spoj) a. The number of foci of the origin-proximal marker P (316 ) and origin-distal marker D (3 ) was determined in individual cells and the percentage of cells with the indicated relationship is shown. b. Indicated strains were grown at 3 C in defined minimal succinate medium and samples were taken during exponential growth for microscopic analysis. c. Total number of cells scored.

10 862 P. S. Lee and A. D. Grossman Table 7. Genetic interactions between soj and spoj mutants for several phenotypes. Phenotypic perturbation relative to soj+ spoj+ a Cellular phenotypes soj+ spoj+ soj spoj+ soj+ spoj soj spoj Epistasis b Chromosome partitioning spoj is epistatic Cell division Position of sister origin regions Origin separation + - Chromosome partitioning in an smc null background c All mutants similar Controlled replication initiation Sporulation soj is epistatic a. + indicates a phenotype similar to the soj+ spoj+ strain; - indicates a defect in the indicated phenotype and indicates a more severe defect. b. Epistasis refers to the genetic interaction between two genes, when one allele masks the phenotype of another (e.g. spoj is epistatic for cell division: the spoj null mutation leads to a cell division delay whether or not soj is present). c. Severity of phenotypes of the soj smc, spoj smc and (soj-spoj) smc mutants is considered relative to the smc single mutant, which is normalized to +. severe, with the exception of replication initiation, for which all of the mutants exhibited overinitiation to a similar extent. This could be due to partial compensation by another factor, such as SMC. We propose that a primary defect in separating replicated origins could lead to defects in origin positioning, delay cell division until origins separate, and produce anucleates in cases where the origins failed to separate. The Par homologues could plausibly affect these A Soj SpoJ Origin separation Chromosome partitioning, Origin positioning, Cell division SpoJ Fig. 6. Actual and proposed orders of interactions by which Soj and SpoJ regulate different cellular processes. Soj and SpoJ probably affect chromosome partitioning, separation and positioning of replicated origins, and cell division through a different order of interactions from the order by which they affect sporulation. A. Proposed order of interactions by which Soj and SpoJ could affect origin separation, chromosome partitioning, origin positioning and cell division. We propose that Soj helps SpoJ bring together pars sites by facilitating higher-order interactions between SpoJ molecules, forming an organized nucleoprotein structure at the origin region. This could plausibly contribute to origin separation by minimizing resistance or keeping sister origins untangled. Formation of such a structure could also regulate initiation of replication by limiting accessibility of the origin to initiation factors (not shown). In the absence of spoj, failure to form this structure properly would decrease the efficiency of origin separation, which could lead to origin positioning defects, delay cell division and produce anucleate cells in cases where the origins did not separate at all. Thus, we propose that the defects in chromosome partitioning, origin positioning and cell division may be a secondary consequence of the separation defect. In the absence of soj, other cellular factors such as SMC may compensate by helping to compact the chromosome, bringing pars sites closer together (not shown). B. Order of interactions by which SpoJ and Soj affect sporulation. SpoJ regulates sporulation via its effect on Soj, relieving Soj-mediated transcriptional repression of early sporulation genes. This order of interaction is distinct from the order proposed in part A. B Soj Sporulation phenotypes by performing a single molecular function: SpoJ could bind pars sites and bring them together through higher-order interactions between SpoJ dimers, and Soj could facilitate these higher-order interactions. Assembly of SpoJ into a nucleoprotein complex that brings distant pars sites into close proximity could presumably organize the origin region into a compacted structure. Origin compaction could facilitate origin separation by preventing entanglement of sister origins, and/or by minimizing resistance against them as they move through the cytoplasm. Compaction of chromosomes prior to segregation is an essential step in eukaryotic chromosome partitioning (Nasmyth and Haering, 2). We also found that Soj and SpoJ affect replication initiation. In this case, all of the mutants exhibit overinitiation of replication to a similar degree, so it is not clear whether Soj acts through SpoJ or vice versa. It may be possible that Soj and SpoJ regulate replication initiation by performing some function that is specific to initiation. Alternatively, it is also possible (and simpler) that a primary effect on origin compaction could regulate accessibility of initiation factors, as discussed in the initiation section. Whether other chromosomal or plasmid-encoded Par homologues affect replication initiation remains a provocative question. Where tested, plasmid-encoded Par systems do not appear to affect plasmid copy number substantially, and plasmid loss in the absence of the par system is not due to insufficient replication (Austin et al., 1982; Ogura and Hiraga, 1983). However, the assays used would not have been sensitive enough to detect the degree of overinitiation we observed for Soj and SpoJ. Soj and SpoJ perform at least one additional function: they are involved in transcriptional regulation during the initiation of sporulation (Table 7). Soj is a transcriptional repressor of early sporulation genes, and SpoJ relieves Soj-mediated repression by preventing Soj from binding to its target promoters (Quisel and Grossman, 2). This

11 Chromosome partitioning in B. subtilis 863 epistasis interaction differs from the previously discussed phenotypes in that soj is epistatic: the spoj null is sporulation-defective (Spo ) due to unopposed Soj activity, and a soj null is sporulation-proficient (Spo+), as is the double mutant. SpoJ affects initiation of sporulation via its effects on Soj, which is a different order of interactions from what we proposed for the phenotypes discussed earlier (Fig. 6B). SpoJ likely regulates Soj s repressor activity by influencing Soj s nucleotide-bound state. Several ParB homologues are known to alter the nucleotide-bound state of their cognate ParA s, either by stimulating nucleotide hydrolysis (Hu and Lutkenhaus, 21; Leonard et al., 2) or nucleotide exchange (Easter and Gober, 22; Figge et al., 23). Binding to ATP versus ADP regulates ParA function by controlling the ability of ParA proteins to bind target promoters, to interact with the partition complex or to associate with other partners (Schindelin et al., 1997; Bouet and Funnell, 1999; Lutkenhaus and Sundaramoorthy, 23; Leonard et al., 2). In addition to regulating Soj s function as a transcriptional repressor, we postulate that nucleotide binding probably also affects Soj s participation in the phenotypes examined here. Upon binding ATP, Soj from Thermus thermophilus forms a DNA-binding dimer that interacts with SpoJ and pars sites (Leonard et al., 2). Perhaps ATP binding activates B. subtilis Soj to interact with SpoJ bound to pars sites. We speculate that energy generated from SpoJ-stimulated ATP hydrolysis could be coupled to remodelling of nearby DNA protein complexes that hold sister origins together, enabling paired origins to separate. Soj and SpoJ also contribute to origin positioning in the forespore, a compartment formed in sporulating cells when the division septum forms very close to one of the cell poles (Sharpe and Errington, 1996; Wu and Errington, 22; Lee et al., 23). A soj null enhances the sporulation defect of a null mutation in raca, the gene encoding a DNA-binding protein that contributes to origin positioning in the forespore (Wu and Errington, 23; Ben-Yehuda et al., 2). RacA and Soj/SpoJ both likely contribute to assembling the origin region into a highly condensed structure during sporulation. Thus, the Soj-SpoJ system appears to have partial redundancy with RacA. Ironically, it appears that Soj has both inhibitory and stimulatory effects on sporulation. Effects of the Par system on plasmids and chromosomes The Par system has different phenotypic effects on plasmid and chromosome positioning. SpoJ is not sufficient to recruit pars sites inserted at ectopic positions in the chromosome to the cell quarters (Lee et al., 23), arguing against the model that SpoJ s primary function is to facilitate attachment of replicated origins to putative anchors at the cell quarters. In contrast, plasmid-encoded Par systems appear to be sufficient to recruit pars plasmids to the cell quarters, consistent with the anchoring model (Niki and Hiraga, 1997; 1999; Erdmann et al., 1999). Chromosomally encoded Par systems can stabilize plasmids containing the cognate pars site (Lin and Grossman, 1998; Godfrin-Estevenon et al., 22), and Soj and SpoJ can recruit pars plasmids to the cell quarters (Yamaichi and Niki, 2). One interpretation of these results is that the same Par system can perform different functions on plasmids and chromosomes. However, a more likely interpretation is that the Par system performs the same biochemical function on plasmids and chromosomes, and that plasmid recruitment to the cell quarters is a secondary effect of a Par-dependent function other than anchoring. The difference in phenotypic effects could reflect innate differences between plasmids and chromosomes, such as size. What similar partitioning function could the Par system perform on plasmids and chromosomes? In both cases, ParB could bind and bring together pars sites, forming a nucleoprotein structure that facilitates partitioning. Plasmid-encoded ParB appears to mediate plasmid pairing by bringing together pars sites on sister molecules (Funnell, 1988; Youngren and Austin, 1997; Edgar et al., 21). Recent work provides structural insight into how a plasmid-encoded ParB homologue can bring different pars sites together (Schumacher and Funnell, 2). Plasmid pairing could potentially facilitate co-ordinated plasmid separation in opposite directions. In the case of chromosomes, similar types of higher-order interactions might bring together pars sites that are located far apart on the same chromosome, organizing the origin region into a compacted structure that facilitates separation of replicated origins. The Par system is widely conserved and contributes to faithful chromosome and plasmid partitioning in diverse bacterial species (Yamaichi, 2). Par systems confer a significant survival advantage and affect the spread of plasmid-borne antibiotic resistance and virulence genes (Yamaichi, 2; Youngren et al., 2). Although Par systems have been intensely studied for over two decades, the molecular mechanism by which they affect segregation remains ill-defined. In addition, how Par homologues regulate other cellular processes like replication initiation, cell division and development, and whether other homologues have similar functions, remain the subject of intense investigation. Experimental procedures Media and growth conditions For all experiments, cultures were grown at 3 C in S7