Direct evidence for active segregation of oriC regions ofthe Bacillus subtilis chromosome and co-localizationwith the Spo0J partitioning protein

Peter J. Lewis * and Jeffery ErringtonSir William Dunn School of Pathology, University ofOxford, South Parks Road, Oxford, OX1 3RE, UK.

Summary

We have developed methods for labelling regions ofthe Bacillus subtilis chromosome with the nucleotideanalogue 5-bromodeoxyuridine (BrdU) and for sub-cellular visualization of the labelled DNA. Exami-nation of oriC -labelled chromosomes in outgrowingspores has provided direct evidence for active segre-gation of sister chromosomes. Co-immunodetectionof Spo0J and BrdU-labelled DNA has directly con-firmed the expected close association between thischromosome partitioning protein and the oriC regionof the chromosome. The results provide further sup-port for the notion that bacterial cells use an activemitotic-like mechanism to segregate their chromo-somes.

Introduction

During the cell cycle the bacterial chromosome must bereplicated and the resultant daughter chromosomes accu-rately segregated (partitioned) into the progeny cells.Surprisingly little is known about the mechanism of parti-tioning, despite many decades of research (reviewed byWake and Errington, 1995). There are no clear homo-logues of proteins involved in eukaryotic mitosis, and noobvious spindle-like apparatus has been detected. Formany years, it was supposed that chromosome segrega-tion was effected by association of the replicating chromo-somes with cell envelope attachment sites, which draw thechromosomes apart during cell growth. In accordancewith such models, careful measurements on growing cellsof Escherichia coli (van Helvoort and Woldringh, 1994)and Bacillus subtilis (M. E. Sharpe, P. M. Hauser, R.Sharpe and J. Errington, in preparation) have revealedthat the nucleoid expands more or less continuously dur-ing growth. However, in experiments in which the nucleoid

is collapsed by inhibition of protein synthesis, there are indi-cations for an underlying discontinuous movement (Hiragaet al., 1990; Begg and Donachie, 1991). In support of theexistence of a more active chromosome partitioning appa-ratus, a gene required for accurate partitioning in E. coli,mukB, was found to encode a myosin-like protein (Nikiet al., 1991; 1992). Moreover, during sporulation in B. sub-tilis, aspects of the phenotype of mutants affected in thespoIIIE gene suggested that the chromosome has a speci-fic orientation, with the oriC regions located close to thecell poles (Wu and Errington, 1994; L. J. Wu and J. Erring-ton, unpublished). This orientation of the B. subtilis chromo-some was recently confirmed and extended to vegetativecells by use of a green fluorescent protein (GFP) deriva-tive that would bind to specific sites in either the oriC orterC regions of the chromosome (Webb et al., 1997).

Mutations in the spo0J gene of B. subtilis affect the polarorientation of the chromosome at the onset of sporulation(Sharpe and Errington, 1996) and produce a mild partition-ing defect in vegetative cells (Ireton et al., 1994). Thesephenotypes and the similarity of Spo0J to the ParB familyof proteins needed for partitioning of stable low copy num-ber plasmids (Hoch, 1993), suggested that spo0J might beinvolved in an active chromosome partitioning mechan-ism. Support for this idea has recently been obtained byexamination of the subcellular localization of the Spo0Jprotein during growth and sporulation (Glaser et al., 1997;Lin et al., 1997). Thus, the Spo0J protein forms discretefoci closely associated with the nucleoid. The number offoci closely parallels the number of copies of oriC per cellat a range of different growth rates (Glaser et al., 1997).Examination of the behaviour of the foci in living cells indi-cated that the foci duplicate at about the same time as oriCreplicates and that the foci then move apart towards oppo-site poles of the cell (Glaser et al., 1997). These resultsstrongly point to the existence of a mitotic-like mechanismthat actively segregates the products of a round of chro-mosome replication to daughter cells at division.

We have now devised a means of directly labelling theB. subtilis chromosome either at oriC or at some distancefrom the origin. We show that the labelled oriC sequencesmove apart relatively early in the DNA replication cycle,and that the Spo0J partitioning protein is closely associ-ated with these sequences, even when DNA replication

Molecular Microbiology (1997) 25(5), 945–954

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Received 21 May, 1997; revised 15 July, 1997; accepted 15 July,1997. *For correspondence. E-mail: plewis@worf.molbiol.ox.ac.uk;Tel. (1865) 275582; Fax (1865) 275556.

m

is inhibited. The results reinforce the likelihood of therebeing an active, mitotic-like partitioning mechanism in bac-teria based on movement apart of newly replicated sequ-ences in or near oriC.

Results

Specific labelling of the oriC region of the B. subtilischromosome and co-detection of Spo0J protein

To localize specific regions of the B. subtilis chromosomein situ, we attempted to adapt methods used to labeleukaryotic chromosomes with the thymine analogue 5-bromo-28-deoxyuridine (BrdU) (Dolbeare et al., 1990; Dol-beare, 1995). Incorporation of BrdU into the DNA wasfacilitated by use of a thymine-requiring mutant of B. sub-tilis. To detect the BrdU-substituted DNA, it was neces-sary to partially hydrolyse the DNA by treatment withHCl. In preliminary experiments, samples were treatedwith 0.5, 1.5, 2.5 and 4 M HCl for 5, 15, 30 and 60 minbefore incubation with anti-BrdU antibodies and second-ary fluorescent antibody. Treatment with 4 M HCl for60 min was found to give the most reproducible and brightsignals (data not shown).

To co-detect the Spo0J chromosome partitioning pro-tein, we initially attempted to use FITC-conjugated sec-ondary antibodies, as described by Glaser et al. (1997),but no signal was detected. We were able to overcomethis problem by using Cy3-conjugated secondary anti-bodies. Presumably, FITC is sensitive to HCl treatment,whereas Cy3 is not. Thus, to detect both antigens thecells were first stained for Spo0J by use of Cy3-conjugatedsecondary antibodies, and then gently fixed. The prepara-tions were then treated with HCl and stained for BrdU withFITC-conjugated secondary antibodies (see Experimentalprocedures).

Labelling and subcellular localization of the oriCregion of the chromosome in outgrowing spores

To specifically label the oriC region of the chromosome,spores of the thy-A strain were incubated in germination

medium devoid of thymine or BrdU for approximately2.5 h. After this time, almost all of the spores had germi-nated and begun to outgrow as judged by phase contrastmicroscopy. BrdU was then added to allow DNA replica-tion to initiate and to label the nascent DNA (BrdU is incor-porated in place of thymine). Labelling was terminatedafter 10 min by addition of a large excess of thymine. Thecells were then allowed to grow on in the presence of thy-mine only. About half of the cells showed BrdU stainingunder these conditions. Presumably, the remaining cellshad failed either to initiate DNA replication during the per-iod of labelling or to incorporate sufficient BrdU to bedetected.

Table 1 shows that from T10 (the end of the labelling per-iod) through to T60, the cells increased in length (i.e. theygrew), and that growth was accompanied, as expected, byincreases in both relative DNA content and nucleoid length(the latter measurements were performed on a parallelsample of cells because the HCl treatment needed tovisualize that the BrdU resulted in degradation of theDNA). In accordance with a DNA replication (‘C’) time ofabout 55 min (Ephrati-Elizur and Borenstein, 1971; Dunnet al., 1978; Hauser and Errington, 1995; M. E. Sharpeet al., preparation), and the likelihood of some cells havinginitiated second rounds of DNA replication by T60 (see alsobelow), the overall increase in DNA content was just overtwofold. These data were thus consistent with the popula-tion of cells undergoing a fairly synchronous round of DNAreplication.

A 10 min exposure to BrdU should result in a segmentcorresponding to at most one-fifth of the chromosomebeing labelled, centred approximately on oriC. Owing tosemiconservative DNA replication, the BrdU would beincorporated into one strand of each daughter DNA duplex.As chromosome replication was initiated by the addition ofBrdU, two oriC-labelled foci would be expected, and at celldivision these signals should segregate into the daughtercells. During the next round of DNA replication, the labelledand unlabelled strands of each duplex should segregate togive two labelled and two unlabelled copies of oriC. Imagesof the BrdU staining patterns of representative cells from

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Table 1. Cell cycle parameters, BrdU and Spo0J foci, in outgrowing cells, and the effects of HPUra treatment.

DNA contentTime of HPUra Cell length per nucleoid Nucleoid length BrdU foci Separation of Spo0J foci BrdU co-localizationsample from time (mm) (×106)a (mm) per cellb BrdU foci (mm)b per cellb with Spo0J (%)b

T10 – 3.0 6 1.0 0.67 6 0.24 1.1 6 0.44 1.2 0.77 6 0.29 1.2 90T30 – 4.3 6 1.4 0.90 6 0.26 1.8 6 0.46 1.4 1.2 6 0.46 1.6 80T60 – 5.6 6 2.5 1.5 6 0.76 2.7 6 0.83 1.2 2.3 6 1.1 4.0 100T60 T10 5.2 6 1.4 0.74 6 0.28 1.9 6 1.3 1.5 1.0 6 0.70 1.5 100T60 T30 5.8 6 1.8 1.0 6 0.46 2.7 6 1.2 1.7 1.6 6 1.0 1.7 93

a. Arbitary units.b. About 50% of total cells examined had a clear BrdU signal. Only cells with clear BrdU and Spo0J signals were counted (>60 for each time point;about 30% of cells with a BrdU signal).

946 P. J. Lewis and J. Errington

samples taken at various times are shown in Fig. 1 (greenchannels; B, F, K and O). Numbers of BrdU foci per cell atdifferent time points are summarized in Table 2. In gen-eral, the results were in accordance with expectation.Immediately after the BrdU labelling (T10) about half ofthe cells showed a BrdU signal (Table 2), mostly in theform of a single well-defined focus near mid-cell (Fig.1B). In a few cells the signal was elongated (Fig. 2A) orthere were two discrete but closely juxtaposed foci (notshown). We interpret these cells as having a pair oflabelled segments that were beginning to separate. Laterin the DNA replication cycle (T50), a similar proportion ofthe cells showed label (Table 2), but now the majority ofcells showed two discrete foci, as expected for two segre-gating oriC regions moving away from each other (Fig. 1Fand K). When the population of cells had undergoneseveral mass doublings (T120), the elongated cell chainstypically still contained a total of two BrdU foci. Thesehad generally segregated into sister cells (Fig. 1O), sothat most labelled cells now only contained one focus(Table 2).

The detection of discrete pairs of BrdU foci after only10 min suggested that the newly replicated sister copiesof oriC begin to separate very early in the replicationcycle. To confirm this, we measured the distance betweenpairs of BrdU foci during the first round of replication(Table 1), and representative images are shown in Fig.2A–C. Initially (T10), the separation between the still rarepairs of BrdU foci was small (0.77 mm). After 1 h (T60),pairs of foci were much further apart (2.3 mm; Fig. 2C),as expected for segregating copies of oriC. Most interest-ingly, however, at an intermediate time, well before the firstround of DNA replication should have been completed(T30), the foci were significantly more separated than atT10 (1.2 mm; Fig. 2B). This strongly suggests that sistercopies of oriC begin to segregate early in the replicationcycle.

Co-localization of SpoOJ and oriC region labels

The above results were consistent with the BrdU focirepresenting oriC regions of the chromosome, labelled dur-ing the first round of DNA replication and then stably seg-regated and inherited. The staining procedure describedabove allowed us to test directly whether the Spo0J chro-mosome-partitioning protein was indeed associated withthe oriC region of the chromosome, as previous resultshad suggested (Glaser et al., 1997). In Fig. 1, the redchannels show immunodetection of the Spo0J proteinstained with Cy3. In these doubly stained HCl-treated pre-parations, the SpoOJ foci were not as clear and preciselyoutlined as in our previous single immunostained images(Glaser et al., 1997), but the general distribution of thefoci was still visible and consistent with the previous results.

An additional complication arose from autofluorescence ofthe residual spore coats that were often attached to oneend of the outgrowing cell (see phase-contrast images inFigs 1 and 2). However, control experiments showedthat this autofluorescence was restricted to the spore resi-dues and did not occur in the outgrowing cells (data notshown).

In accordance with our previous observations (Glaser etal., 1997), the Spo0J protein formed discrete foci thatappeared to increase in number in parallel with rounds ofDNA replication, and later to become equipartitionedeither side of future division sites. Arrows are included inFig. 1 (P and T) to indicate the positions of Spo0J foci.As a result of the acid treatment, the foci were slightlyless clear, and our interpretation of their positions waspartly based on our previous experience observingSpo0J behaviour (Glaser et al., 1997) and on the appear-ance of parallel samples of cells that were immunostainedwithout HCl treatment. Note that some of the foci are rela-tively elongated; presumably these are in the process ofduplicating (see Glaser et al., 1997).

Overlays of the red and green images in Fig. 1 allowedthe possibility of co-localization of BrdU-substituted DNAand SpoOJ foci to be assessed. At the earliest timepoint, 10 min after the initiation of chromosome replication(t10), cells such as the one shown in Fig. 1 (A-D) usuallycontained a single focus of SpoOJ and of BrdU-substitutedDNA that co-localized. Table 3 shows that 90% of the BrdUfoci co-localized with a SpoOJ focus at this time, confirm-ing their close association at the onset of DNA replication.

At later time points, the Spo0J foci increased in number,as expected (Fig. 1, G, L and P; Table 3). The cells shownin Fig. 1 (E-M) (taken at T50) each contained four Spo0Jfoci, as was expected if a second round of DNA replicationhad been initiated. As noted previously, the SpoOJ focitended to be grouped in pairs, with the newly separatedfoci lying closer together (Glaser et al., 1997). The imagesclearly show that the BrdU foci in these cells still co-loca-lized with Spo0J foci. Moreover, the BrdU foci were alwaysassociated with well separated Spo0J foci, rather than withclosely paired (and thus newly separated) Spo0J foci. Car-toon representations of the probable topology of the chro-mosomes and the positioning of Spo0J foci in these cellsare shown alongside the images. Interestingly, in one ofthe cells (Fig. 1E–H) the BrdU foci co-localized with thefirst and third SpoOJ foci (left to right), whereas in theother cell shown (Fig. 1J–M) the co-localization was withthe first and fourth SpoOJ foci. We did not detect enoughcells of this class to be able to quantify these two statesaccurately, but the fact that both classes were readilydetectable is important because it suggests that segrega-tion of sister oriC regions does not follow a rigid linearpattern.

At T120 the cells contained multiple SpoOJ foci (Table 3;

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Co-localization of oriC and Spo0J 947

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Fig. 1N–Q). The number of BrdU foci per cell showed aslight decrease (Table 3), mainly as a result of cell division.When the first cell division septum forms, it should occurbetween the labelled oriC regions arising during the firstround of DNA replication. Thus, in the chain of cellsshown in Fig. 1N–Q the two BrdU foci had segregatedeither side of a division septum into separate cells. Thepresence of two septa in this chain, rather than theexpected three or more, was as a result of the partial inhi-bition of division resulting from the delay in DNA replication(Donachie et al., 1971; Sharpe and Errington, 1995).Nevertheless, both of the BrdU foci in the chain clearlyco-localized with a Spo0J focus (Fig. 1Q) and in the popu-lation of cells as a whole, as at earlier time points, the BrdUfoci almost invariably co-localized with SpoOJ foci (Table3). Therefore, there appears to be a close association ofSpoOJ foci with the oriC region of the chromosomethroughout cell growth.

SpoOJ does not co-localize with oriC-distal regionsof the chromosome

To show that SpoOJ is specifically associated with theoriC region of the chromosome, we attempted to specifi-cally label the terC region of the chromosome with BrdUby adopting methods developed by Adams and Wake(1980) and Sargent (1980). Cells were grown for inductionof sporulation by standard methods (Partridge and Erring-ton, 1993), resuspended in starvation medium containingthymine and allowed to sporulate for 1 h. The cells werethen resuspended in medium containing BrdU and grownfor a further 10 min before HPUra was added to inhibitany further DNA replication. The only spores formedwould be from cells that had completed DNA replication

and not reinitiated any new rounds, so the only BrdU labelshould be in the terC region (see Adams and Wake,1980; Sargent, 1980). Unfortunately, we were unable toidentify a single germinating spore with detectable BrdU-substituted DNA from two separate spore preparations.We are unable to explain this observation at present, butnote that previous authors have also reported problemsin labelling spore DNA around terC (Adams and Wake,1980; Binnie and Coote, 1986).

As an alternative means of testing the specificity of theco-localization of Spo0J and the oriC region of the chromo-some, we repeated the spore outgrowth experimentsdescribed above but with a BrdU pulse 30 min after DNAreplication was initiated with thymine. Therefore, onlysequences far from the origin should be labelled. In princi-ple, four DNA segments should be labelled in each cellbecause two daughter duplex DNAs arise at each replica-tion fork. (This might or might not give rise to an increasednumber of visible BrdU foci, depending on the proximity ofthe clockwise- and anticlockwise-replicated arms of thechromosome.) Fig. 1R–U shows a set of images of a typi-cal chain of cells taken at t120 in such an experiment. Incontrast to the oriC-labelled chain in Fig. 1N–Q, the BrdUfoci lay between Spo0J foci. Quantification of cells at thisand an earlier time point (T50) confirmed that the SpoOJand BrdU foci tended not to co-localize after this labellingprocedure (Table 3). It should be noted that a low degreeof co-localization would be expected if some cells werecapable of initiating a second round of DNA replication dur-ing the labelling period.

Taken together, these results confirmed that Spo0J fociare specifically associated with the oriC region of the chro-mosome. They also show that the oriC and mid-chromo-some regions labelled in these experiments are spatiallywell separated during vegetative growth, supporting thenotion that the overall orientation of the chromosome iscontrolled by the cell.

oriC and SpoOJ remain closely associated afterinhibition of DNA replication

As an alternative means of confirming the close assoc-iation between the oriC region of the chromosome andSpoOJ, we examined whether the association could resistperturbation of DNA replication and segregation. Germi-nation experiments were performed as before to label

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Fig. 1. Use of immunofluorescence microscopy to examine the extent of co-localization of SpoOJ protein with chromosomal DNA labelled withBrdU in cells derived from germinated spores. Cells labelled with BrdU in their oriC region harvested at T10 are shown in A–D; at T50 in E–M;at T120 in N–Q. Origin-distal labelled cells harvested at T120 are shown in R–U. Phase contrast images are shown in A, E, J, N and R. BrdUsignals detected in the FITC (green) channel are shown in B, F, K, O and S. SpoOJ signals detected in the Cy-3 (red) channel are shown inC, G, L, P and T. Overlays of the FITC and Cy3 images are shown in D, H, M, Q and U. Cartoons showing the proposed arrangement ofSpoOJ and BrdU-substituted DNA in cells harvested at T50 are shown adjacent to H and M. The black lines represent chromosomal DNA,green if labelled with BrdU. The red spots represent SpoOJ foci assumed to be associated with sequences in the oriC region. Division septaare marked on the cell chain in N. SpoOJ foci in cells harvested at T120 are marked in P and T.

Table 2. Cellular distribution of oriC-labelled BrdU foci in outgrowingcells.

Number of BrdU foci per cell(% of total cells counted)

Time of Number ofsample cells counted 0 1 2 3 4

T10 110 47 41 11 – 1T50 138 38 23 36 3 –T120 174 55 39a 5 – 0.6

a. These single foci mainly lay in cell pairs or chains containing a totalof two foci.

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the oriC region. HPUra, a specific inhibitor of DNA poly-merase III in Gram-positive organisms (Brown et al.,1972; Mackenzie et al., 1973), was used to stop DNA repli-cation at various time points, as detailed in Experimentalprocedures. Table 1 shows that the cells continued togrow after addition of HPUra, and that DNA replicationwas, as expected, immediately stopped. Nevertheless, asshown previously (Sharpe and Errington, 1995), thenucleoids were still able to expand in parallel with cellgrowth. Images of BrdU foci from cells after treatmentwith HPUra from T10 or T30 are shown in Fig. 2 (D andE). It is clear that BrdU foci in these cells were significantlyfurther separated than when the inhibitor was added(compare with Fig. 2A and B; see also Table 1). Thus,the oriC-labelled sequences undergo some furtherseparation in the absence of DNA replication.

Figure 2F–M shows co-detection of BrdU and Spo0Jfoci in cells treated with HPUra from T10 or T30. In bothof the typical cells shown, there are only two well-sepa-rated Spo0J foci, and in each case they co-localize withthe BrdU foci. Thus, despite the fact that DNA replicationwas stopped and that nucleoid expansion and oriC move-ment were perturbed, there was still almost complete co-localization of BrdU and Spo0J foci (Table 1).

Discussion

We have developed methods to label relatively specificregions of the B. subtilis chromosome with the thymineanalogue BrdU and to determine the localization of thelabelled DNA in situ using immunofluorescence. Germina-tion and outgrowth of spores have previously been shownto provide a useful means of studying cell cycle control(Siccardi et al., 1975; McGinness and Wake, 1979; Par-tridge and Wake, 1995). By use of a thymine-requiringmutant, DNA replication can be further synchronized inthe outgrowing spores by withholding thymine until aftera large proportion of the cells have grown sufficiently to

be ready to initiate. A short exposure to the thymine ana-logue BrdU should result in incorporation only into a rela-tively small region of the chromosome either side of oriC,and only one strand of each daughter chromosome shouldbe labelled. Thus, two BrdU foci should segregate whensuch cells are allowed to complete their round of replica-tion and continue growing. The results we obtained follow-ing the behaviour of the foci using immunofluorescencemicroscopy were consistent with this prediction. Thus, twoBrdU foci were observed in almost all of the labelled cellsor cell chains. Moreover, the two labelled DNA moleculesappeared to be replicated and segregated as relativelystable units for three or more generations. BrdU labellingand detection using immunofluorescence microscopythus seems to be a useful means of following the segrega-tion and inheritance of chromosomes.

An interesting feature of the behaviour of the oriC-labelled foci was that they had separated by a relativelylarge distance well before the first DNA replication cycleshould have finished (Fig. 2B and Table 1). This wouldbe consistent with the emerging concept of an active par-titioning apparatus (see Introduction ), and with the pre-viously observed bipolar pattern of oriC regions detectedin growing cells by an indirect labelling procedure (Webbet al., 1997). Another noteworthy observation concernsthe apparent concentration of the BrdU label into relativelydiscrete foci. This would be compatible with the idea thatthe chromosome has a well organized structure compris-ing a number of ordered, well defined and highly con-densed domains (Higgins, 1994).

Various lines of research have recently implicated thespo0J gene of B. subtilis in the putative active partitioningapparatus (see Introduction ). There were several reasonsfor expecting that Spo0J function might involve a directinteraction with oriC. First, proteins functionally relatedto Spo0J, being required for the stable partitioning oflow-copy number plasmids, and which exhibit significantsequence similarity to Spo0J are known to be site-specific

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Fig. 2. SpoOJ remains associated with oriC after inhibition of DNA replication with HPUra. BrdU signals of untreated cells labelled at oriC andharvested at T10, T30 and T60 are shown in A, B and C respectively. BrdU signals of cells treated with HPUra at T10 and T30 are shown in Dand E respectively. Cells treated with HPUra at T10 and T30, respectively, are shown in F and J (phase contrast), G and K (oriC label), H andL (SpoOJ foci) and I and M (oriC/SpoOJ overlays).

Table 3. Co-localization of Spo0J foci andBrdU-labelled DNA. Location Time of Nucleoids Spo0J foci BrdU foci BrdU/Spo0J

of label sample per cella per cell per cell co-localization (%)

oriC T10 1.0 1.1 1.1 90T50 1.3 2.0 1.6 75T120 2.9 5.6 1.0 80

Mid-chromosome T50 1.6 3.6 2.3 21T120 3.0 6.8 1.4 22

a. Determined by counting nucleoids in a parallel sample of DAPI-stained cells.

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DNA-binding proteins (e.g. ParB and Sop; Hiraga, 1992).In some cases, these proteins have been shown to bindto specific cis-acting sites that are needed for partitioning(Hiraga, 1992). Furthermore, Mohl and Gober (1997)recently found that a SpoOJ homologue in Caulobactercrescentus can bind directly to DNA. Second, we haveshown previously that Spo0J is required for correct posi-tioning of the oriC region of the chromosome close to thepole of the cell at the onset of sporulation (Sharpe andErrington, 1996). Unfortunately, it has not proved possibleto detect a specific binding of SpoOJ protein to DNA.Nevertheless, to test the possibility of a close associationbetween Spo0J and the oriC region in vivo, we developeda protocol for co-detection of Spo0J and the BrdU-labelledchromosomal DNA. When the label was incorporated intothe oriC region there was almost complete co-localizationwith the Spo0J signal (Tables 1 and 3). In contrast, whenthe label was incorporated into a distal region of the chro-mosome, well away from oriC, the Spo0J and BrdU sig-nals were mainly not co-localized. When DNA replicationwas blocked by use of the specific inhibitor HPUra, col-localization with oriC was maintained (Fig. 2). These resultsstrongly support the notion of a tight association betweenthe Spo0J assemblies previously observed (Glaser et al.,1997; Lin et al., 1997) and the oriC region of the chromo-some. They also support the notion that the chromosomehas a relatively fixed orientation both during vegetativegrowth (Webb et al., 1997) and during sporulation (Wuand Errington, 1994; Sharpe and Errington, 1996). Giventhe properties of the family of proteins similar to SpoOJdiscussed above, we think it likely that SpoOJ will interactdirectly with DNA but its binding sites may be relativelynon-specific: for example the bias for binding to the oriCregion could be achieved by co-operative binding to mul-tiple partially specific sites. Mohl and Gober (1997) haveshown that despite showing binding of ParB (a SpoOJhomologue) to a specific DNA fragment, there was alsosome binding to other AT-rich DNA fragments.

In conclusion, a number of new problems arise from thiswork. The most important of these will be to identify thenature of the interaction between Spo0J and the oriCregion and to find and characterize the factors responsiblefor active movement of these putative complexes withinthe cell.

Experimental procedures

Bacterial strains

Bacillus subtilis thy-A (trpC2 thyA thyB ; laboratory stock) wasused in all experiments.

Spore preparation

Spores were prepared as follows. Exponentially growingB. subtilis thy-A were spread onto 15-cm-diameter Oxoid

nutrient agar plates, and sporulation allowed to proceed for6 days at 308C. The spores were harvested by washingeach plate with 5 ml of sterile water. The suspension was cen-trifuged and resuspended in TE buffer. Lysozyme was addedto 3 mg ml¹1 and the mixture incubated at 378C for 1 h. SDSwas added to a final concentration of 6.25% (v/v) and the mix-ture incubated for a further 30 min at 378C. The lysate wasthen centrifuged and the purified spores washed repeatedlywith sterile water at 48C over a period of 5 days. The finalspore preparation was stored at an A600 of 80.

Spore germination

Spore germination was carried out in the defined medium, asdescribed previously (McGinness and Wake, 1979). Germi-nation medium was supplemented with thymine or BrdU(Sigma) at 20 mg ml¹1, as required. The medium was inocu-lated with spores at an A600 of 1.0 and germination and out-growth were allowed to proceed for approximately 2.5 h at378C with shaking. Samples of cultures were checked bymicroscopy to ensure outgrowth had begun before DNA repli-cation was initiated by the addition of thymine or BrdU. A10 min pulse labelling with BrdU was followed by quenchingwith 200 mg ml¹1 thymine and immediate centrifugation andresuspension in fresh pre-warmed medium supplementedwith 20 mg ml¹1 thymine. In experiments in which DNA repli-cation was stopped, an aliquot of culture was transferred toa fresh flask and the specific DNA polymerase III inhibitor6-(para-hydroxyphenylazo)-uracil (HPUra) was added to afinal concentration of 50 mg ml¹1.

Immunofluorescence procedures

Samples of cells (from 0.5 ml of culture) were fixed as des-cribed by Lewis et al. (1996). Permeabilization of cells wasessentially the same as described by Lewis et al. (1996),except that samples were incubated with 2 mg ml¹1 lysozyme,0.01% (v/v) Triton X-100 for 5 min. Affinity-purified rabbitpolyclonal anti-SpoOJ antibodies (Glaser et al., 1997) wereused at a 1:500 dilution and incubation was carried out over-night at 48C. Cy3-conjugated sheep anti-rabbit antibodies wereused at a dilution of 3:1000 and incubation carried out for 1 h atroom temperature in the dark. All subsequent steps were per-formed with minimal exposure of the samples to light. Afterwashing, the samples were fixed again with 2% (w/v) parafor-maldehyde 0.01% (v/v) glutaraldehyde in PBS for 15 min atroom temperature. After washing, samples in which BrdU sig-nals were to be detected were treated with 4 M HCl for 1 h atambient temperature. Samples were then washed and a sec-ond immunostaining procedure was performed with mousemonoclonal anti-BrdU antibodies (Sigma) at a 1:100 dilutionand goat anti-mouse FITC-conjugated antibodies at a 3:1000dilution. All samples were mounted for epifluorescence micro-scopy in Vectashield antifade (Vector Laboratories, Burlin-game, CA, USA) supplemented with 0.2 mg ml¹1 48,6-diamidino-2-phenylindole (DAPI). (Note that a DAPI signalwas not detectable in the HCl-treated samples).

Microscopy, image acquisition and image analysis

Epifluorescence microscopy was performed as described byLewis et al. (1996) and images were acquired using a cooled

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CCD camera (Digital Pixel) with a 1536 × 1024 pixel, 9 mmpitch chip. FITC exposures were for 5 s, Cy3 and DAPI expo-sures were for 2 s. Processing was carried out on the 12 bitimages using IPLab Spectrum V3.1.1 (Signal Analytics,Vienna, VA, USA). Final images were assembled in Adobephotoshop V. 3.0.5 for printing. With the narrow bandpass fil-ters used in these experiments, no spectral cross-over of sig-nals was detected in single-staining control experiments (datanot shown).

Acknowledgements

This work was supported by grants from the Biotechnologyand Biological Sciences Research Council. We thankMichaela Sharpe and Ling-Juan Wu for helpful commentson the manuscript, and Dean Jackson for advice on immunos-taining of BrdU-labelled DNA.

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