Molecular Microbiology (1997) 25(5), 945–954
Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the Spo0J partitioning protein Peter J. Lewis* and Jeffery Errington Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
is collapsed by inhibition of protein synthesis, there are indications for an underlying discontinuous movement (Hiraga et al ., 1990; Begg and Donachie, 1991). In support of the existence of a more active chromosome partitioning apparatus, a gene required for accurate partitioning in E . coli , mukB , was found to encode a myosin-like protein (Niki et al ., 1991; 1992). Moreover, during sporulation in B . subtilis , aspects of the phenotype of mutants affected in the spoIIIE gene suggested that the chromosome has a specific orientation, with the oriC regions located close to the cell poles (Wu and Errington, 1994; L. J. Wu and J. Errington, unpublished). This orientation of the B . subtilis chromosome was recently confirmed and extended to vegetative cells by use of a green fluorescent protein (GFP) derivative that would bind to specific sites in either the oriC or terC regions of the chromosome (Webb et al ., 1997). Mutations in the spo0J gene of B . subtilis affect the polar orientation of the chromosome at the onset of sporulation (Sharpe and Errington, 1996) and produce a mild partitioning defect in vegetative cells (Ireton et al ., 1994). These phenotypes and the similarity of Spo0J to the ParB family of proteins needed for partitioning of stable low copy number plasmids (Hoch, 1993), suggested that spo0J might be involved in an active chromosome partitioning mechanism. Support for this idea has recently been obtained by examination of the subcellular localization of the Spo0J protein during growth and sporulation (Glaser et al ., 1997; Lin et al ., 1997). Thus, the Spo0J protein forms discrete foci closely associated with the nucleoid. The number of foci closely parallels the number of copies of oriC per cell at a range of different growth rates (Glaser et al ., 1997). Examination of the behaviour of the foci in living cells indicated that the foci duplicate at about the same time as oriC replicates and that the foci then move apart towards opposite poles of the cell (Glaser et al ., 1997). These results strongly point to the existence of a mitotic-like mechanism that actively segregates the products of a round of chromosome replication to daughter cells at division. We have now devised a means of directly labelling the B . subtilis chromosome either at oriC or at some distance from the origin. We show that the labelled oriC sequences move apart relatively early in the DNA replication cycle, and that the Spo0J partitioning protein is closely associated with these sequences, even when DNA replication
Summary We have developed methods for labelling regions of the Bacillus subtilis chromosome with the nucleotide analogue 5-bromodeoxyuridine (BrdU) and for subcellular visualization of the labelled DNA. Examination of oriC -labelled chromosomes in outgrowing spores has provided direct evidence for active segregation of sister chromosomes. Co-immunodetection of Spo0J and BrdU-labelled DNA has directly confirmed the expected close association between this chromosome partitioning protein and the oriC region of the chromosome. The results provide further support for the notion that bacterial cells use an active mitotic-like mechanism to segregate their chromosomes. Introduction During the cell cycle the bacterial chromosome must be replicated and the resultant daughter chromosomes accurately segregated (partitioned) into the progeny cells. Surprisingly little is known about the mechanism of partitioning, despite many decades of research (reviewed by Wake and Errington, 1995). There are no clear homologues of proteins involved in eukaryotic mitosis, and no obvious spindle-like apparatus has been detected. For many years, it was supposed that chromosome segregation was effected by association of the replicating chromosomes with cell envelope attachment sites, which draw the chromosomes apart during cell growth. In accordance with such models, careful measurements on growing cells of Escherichia coli (van Helvoort and Woldringh, 1994) and Bacillus subtilis (M. E. Sharpe, P. M. Hauser, R. Sharpe and J. Errington, in preparation) have revealed that the nucleoid expands more or less continuously during growth. However, in experiments in which the nucleoid Received 21 May, 1997; revised 15 July, 1997; accepted 15 July, 1997. *For correspondence. E-mail:
[email protected]; Tel. (1865) 275582; Fax (1865) 275556. Q 1997 Blackwell Science Ltd
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946 P. J. Lewis and J. Errington Table 1. Cell cycle parameters, BrdU and Spo0J foci, in outgrowing cells, and the effects of HPUra treatment.
Time of sample
HPUra from time
Cell length (mm)
DNA content per nucleoid (× 106)a
Nucleoid length (mm)
BrdU foci per cell b
Separation of BrdU foci (mm)b
Spo0J foci per cell b
BrdU co-localization with Spo0J (%)b
T10 T30 T60 T60 T60
– – – T10 T30
3.0 6 1.0 4.3 6 1.4 5.6 6 2.5 5.2 6 1.4 5.8 6 1.8
0.67 6 0.24 0.90 6 0.26 1.5 6 0.76 0.74 6 0.28 1.0 6 0.46
1.1 6 0.44 1.8 6 0.46 2.7 6 0.83 1.9 6 1.3 2.7 6 1.2
1.2 1.4 1.2 1.5 1.7
0.77 6 0.29 1.2 6 0.46 2.3 6 1.1 1.0 6 0.70 1.6 6 1.0
1.2 1.6 4.0 1.5 1.7
90 80 100 100 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).
is inhibited. The results reinforce the likelihood of there being an active, mitotic-like partitioning mechanism in bacteria based on movement apart of newly replicated sequences in or near oriC . Results
Specific labelling of the oriC region of the B. subtilis chromosome and co-detection of Spo0J protein To localize specific regions of the B . subtilis chromosome in situ , we attempted to adapt methods used to label eukaryotic chromosomes with the thymine analogue 5bromo-28-deoxyuridine (BrdU) (Dolbeare et al ., 1990; Dolbeare, 1995). Incorporation of BrdU into the DNA was facilitated by use of a thymine-requiring mutant of B . subtilis . To detect the BrdU-substituted DNA, it was necessary to partially hydrolyse the DNA by treatment with HCl. In preliminary experiments, samples were treated with 0.5, 1.5, 2.5 and 4 M HCl for 5, 15, 30 and 60 min before incubation with anti-BrdU antibodies and secondary fluorescent antibody. Treatment with 4 M HCl for 60 min was found to give the most reproducible and bright signals (data not shown). To co-detect the Spo0J chromosome partitioning protein, we initially attempted to use FITC-conjugated secondary antibodies, as described by Glaser et al . (1997), but no signal was detected. We were able to overcome this problem by using Cy3-conjugated secondary antibodies. Presumably, FITC is sensitive to HCl treatment, whereas Cy3 is not. Thus, to detect both antigens the cells were first stained for Spo0J by use of Cy3-conjugated secondary antibodies, and then gently fixed. The preparations were then treated with HCl and stained for BrdU with FITC-conjugated secondary antibodies (see Experimental procedures).
Labelling and subcellular localization of the oriC region 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 approximately 2.5 h. After this time, almost all of the spores had germinated and begun to outgrow as judged by phase contrast microscopy. BrdU was then added to allow DNA replication to initiate and to label the nascent DNA (BrdU is incorporated in place of thymine). Labelling was terminated after 10 min by addition of a large excess of thymine. The cells were then allowed to grow on in the presence of thymine only. About half of the cells showed BrdU staining under these conditions. Presumably, the remaining cells had failed either to initiate DNA replication during the period of labelling or to incorporate sufficient BrdU to be detected. Table 1 shows that from T10 (the end of the labelling period) through to T60 , the cells increased in length (i.e. they grew), and that growth was accompanied, as expected, by increases in both relative DNA content and nucleoid length (the latter measurements were performed on a parallel sample of cells because the HCl treatment needed to visualize that the BrdU resulted in degradation of the DNA). In accordance with a DNA replication (‘C’) time of about 55 min (Ephrati-Elizur and Borenstein, 1971; Dunn et al ., 1978; Hauser and Errington, 1995; M. E. Sharpe et al ., preparation), and the likelihood of some cells having initiated second rounds of DNA replication by T60 (see also below), the overall increase in DNA content was just over twofold. These data were thus consistent with the population of cells undergoing a fairly synchronous round of DNA replication. A 10 min exposure to BrdU should result in a segment corresponding to at most one-fifth of the chromosome being labelled, centred approximately on oriC . Owing to semiconservative DNA replication, the BrdU would be incorporated into one strand of each daughter DNA duplex. As chromosome replication was initiated by the addition of BrdU, two oriC -labelled foci would be expected, and at cell division these signals should segregate into the daughter cells. During the next round of DNA replication, the labelled and unlabelled strands of each duplex should segregate to give two labelled and two unlabelled copies of oriC . Images of the BrdU staining patterns of representative cells from Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 945–954
Co-localization of oriC and Spo0J 947 samples taken at various times are shown in Fig. 1 (green channels; B, F, K and O). Numbers of BrdU foci per cell at different time points are summarized in Table 2. In general, the results were in accordance with expectation. Immediately after the BrdU labelling ( T10) about half of the cells showed a BrdU signal (Table 2), mostly in the form of a single well-defined focus near mid-cell (Fig. 1B). In a few cells the signal was elongated (Fig. 2A) or there were two discrete but closely juxtaposed foci (not shown). We interpret these cells as having a pair of labelled segments that were beginning to separate. Later in the DNA replication cycle ( T50), a similar proportion of the cells showed label (Table 2), but now the majority of cells showed two discrete foci, as expected for two segregating oriC regions moving away from each other (Fig. 1F and K). When the population of cells had undergone several mass doublings ( T120), the elongated cell chains typically still contained a total of two BrdU foci. These had generally segregated into sister cells (Fig. 1O), so that most labelled cells now only contained one focus (Table 2). The detection of discrete pairs of BrdU foci after only 10 min suggested that the newly replicated sister copies of oriC begin to separate very early in the replication cycle. To confirm this, we measured the distance between pairs 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 rare pairs 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 interestingly, however, at an intermediate time, well before the first round of DNA replication should have been completed ( T30), the foci were significantly more separated than at T10 (1.2 mm; Fig. 2B). This strongly suggests that sister copies of oriC begin to segregate early in the replication cycle.
Co-localization of SpoOJ and oriC region labels The above results were consistent with the BrdU foci representing oriC regions of the chromosome, labelled during the first round of DNA replication and then stably segregated and inherited. The staining procedure described above allowed us to test directly whether the Spo0J chromosome-partitioning protein was indeed associated with the oriC region of the chromosome, as previous results had suggested (Glaser et al ., 1997). In Fig. 1, the red channels show immunodetection of the Spo0J protein stained with Cy3. In these doubly stained HCl-treated preparations, the SpoOJ foci were not as clear and precisely outlined as in our previous single immunostained images (Glaser et al ., 1997), but the general distribution of the foci was still visible and consistent with the previous results. Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 945–954
An additional complication arose from autofluorescence of the residual spore coats that were often attached to one end of the outgrowing cell (see phase-contrast images in Figs 1 and 2). However, control experiments showed that this autofluorescence was restricted to the spore residues and did not occur in the outgrowing cells (data not shown). In accordance with our previous observations (Glaser et al ., 1997), the Spo0J protein formed discrete foci that appeared to increase in number in parallel with rounds of DNA replication, and later to become equipartitioned either side of future division sites. Arrows are included in Fig. 1 (P and T) to indicate the positions of Spo0J foci. As a result of the acid treatment, the foci were slightly less clear, and our interpretation of their positions was partly based on our previous experience observing Spo0J behaviour (Glaser et al ., 1997) and on the appearance of parallel samples of cells that were immunostained without HCl treatment. Note that some of the foci are relatively elongated; presumably these are in the process of duplicating (see Glaser et al ., 1997). Overlays of the red and green images in Fig. 1 allowed the possibility of co-localization of BrdU-substituted DNA and SpoOJ foci to be assessed. At the earliest time point, 10 min after the initiation of chromosome replication (t10), cells such as the one shown in Fig. 1 (A-D) usually contained a single focus of SpoOJ and of BrdU-substituted DNA that co-localized. Table 3 shows that 90% of the BrdU foci co-localized with a SpoOJ focus at this time, confirming 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 shown in Fig. 1 (E-M) (taken at T50) each contained four Spo0J foci, as was expected if a second round of DNA replication had been initiated. As noted previously, the SpoOJ foci tended to be grouped in pairs, with the newly separated foci lying closer together (Glaser et al ., 1997). The images clearly show that the BrdU foci in these cells still co-localized with Spo0J foci. Moreover, the BrdU foci were always associated with well separated Spo0J foci, rather than with closely paired (and thus newly separated) Spo0J foci. Cartoon representations of the probable topology of the chromosomes and the positioning of Spo0J foci in these cells are shown alongside the images. Interestingly, in one of the cells (Fig. 1E–H) the BrdU foci co-localized with the first and third SpoOJ foci (left to right), whereas in the other cell shown (Fig. 1J–M) the co-localization was with the first and fourth SpoOJ foci. We did not detect enough cells of this class to be able to quantify these two states accurately, but the fact that both classes were readily detectable is important because it suggests that segregation of sister oriC regions does not follow a rigid linear pattern. At T120 the cells contained multiple SpoOJ foci (Table 3;
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Co-localization of oriC and Spo0J 949 Table 2. Cellular distribution of oriC -labelled BrdU foci in outgrowing cells. Number of BrdU foci per cell (% of total cells counted) Time of sample
Number of cells counted
0
1
2
3
4
T10 T50 T120
110 138 174
47 38 55
41 23 39a
11 36 5
– 3 –
1 – 0.6
a. These single foci mainly lay in cell pairs or chains containing a total of two foci.
Fig. 1N–Q). The number of BrdU foci per cell showed a slight decrease (Table 3), mainly as a result of cell division. When the first cell division septum forms, it should occur between the labelled oriC regions arising during the first round of DNA replication. Thus, in the chain of cells shown in Fig. 1N–Q the two BrdU foci had segregated either side of a division septum into separate cells. The presence of two septa in this chain, rather than the expected three or more, was as a result of the partial inhibition 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 clearly co-localized with a Spo0J focus (Fig. 1Q) and in the population of cells as a whole, as at earlier time points, the BrdU foci almost invariably co-localized with SpoOJ foci (Table 3). Therefore, there appears to be a close association of SpoOJ foci with the oriC region of the chromosome throughout cell growth.
SpoOJ does not co-localize with oriC-distal regions of the chromosome To show that SpoOJ is specifically associated with the oriC region of the chromosome, we attempted to specifically label the terC region of the chromosome with BrdU by adopting methods developed by Adams and Wake (1980) and Sargent (1980). Cells were grown for induction of sporulation by standard methods (Partridge and Errington, 1993), resuspended in starvation medium containing thymine and allowed to sporulate for 1 h. The cells were then resuspended in medium containing BrdU and grown for a further 10 min before HPUra was added to inhibit any further DNA replication. The only spores formed would be from cells that had completed DNA replication
and not reinitiated any new rounds, so the only BrdU label should be in the terC region (see Adams and Wake, 1980; Sargent, 1980). Unfortunately, we were unable to identify a single germinating spore with detectable BrdUsubstituted DNA from two separate spore preparations. We are unable to explain this observation at present, but note that previous authors have also reported problems in labelling spore DNA around terC (Adams and Wake, 1980; Binnie and Coote, 1986). As an alternative means of testing the specificity of the co-localization of Spo0J and the oriC region of the chromosome, we repeated the spore outgrowth experiments described above but with a BrdU pulse 30 min after DNA replication was initiated with thymine. Therefore, only sequences far from the origin should be labelled. In principle, four DNA segments should be labelled in each cell because two daughter duplex DNAs arise at each replication fork. (This might or might not give rise to an increased number of visible BrdU foci, depending on the proximity of the clockwise- and anticlockwise-replicated arms of the chromosome.) Fig. 1R–U shows a set of images of a typical chain of cells taken at t120 in such an experiment. In contrast to the oriC -labelled chain in Fig. 1N–Q, the BrdU foci lay between Spo0J foci. Quantification of cells at this and an earlier time point ( T50) confirmed that the SpoOJ and BrdU foci tended not to co-localize after this labelling procedure (Table 3). It should be noted that a low degree of co-localization would be expected if some cells were capable of initiating a second round of DNA replication during the labelling period. Taken together, these results confirmed that Spo0J foci are specifically associated with the oriC region of the chromosome. They also show that the oriC and mid-chromosome regions labelled in these experiments are spatially well separated during vegetative growth, supporting the notion that the overall orientation of the chromosome is controlled by the cell.
oriC and SpoOJ remain closely associated after inhibition of DNA replication As an alternative means of confirming the close association between the oriC region of the chromosome and SpoOJ, we examined whether the association could resist perturbation of DNA replication and segregation. Germination experiments were performed as before to label
Fig. 1. Use of immunofluorescence microscopy to examine the extent of co-localization of SpoOJ protein with chromosomal DNA labelled with BrdU 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. BrdU signals 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 in C, 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 of SpoOJ 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 septa are marked on the cell chain in N. SpoOJ foci in cells harvested at T120 are marked in P and T. Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 945–954
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Co-localization of oriC and Spo0J 951 Table 3. Co-localization of Spo0J foci and BrdU-labelled DNA.
Location of label
Time of sample
Nucleoids per cella
Spo0J foci per cell
BrdU foci per cell
BrdU/Spo0J co-localization (%)
oriC
T10 T50 T120
1.0 1.3 2.9
1.1 2.0 5.6
1.1 1.6 1.0
90 75 80
Mid-chromosome
T50 T120
1.6 3.0
3.6 6.8
2.3 1.4
21 22
a. Determined by counting nucleoids in a parallel sample of DAPI-stained cells.
the oriC region. HPUra, a specific inhibitor of DNA polymerase III in Gram-positive organisms (Brown et al ., 1972; Mackenzie et al ., 1973), was used to stop DNA replication at various time points, as detailed in Experimental procedures. Table 1 shows that the cells continued to grow after addition of HPUra, and that DNA replication was, as expected, immediately stopped. Nevertheless, as shown previously (Sharpe and Errington, 1995), the nucleoids were still able to expand in parallel with cell growth. Images of BrdU foci from cells after treatment with HPUra from T10 or T30 are shown in Fig. 2 (D and E). It is clear that BrdU foci in these cells were significantly further separated than when the inhibitor was added (compare with Fig. 2A and B; see also Table 1). Thus, the oriC -labelled sequences undergo some further separation in the absence of DNA replication. Figure 2F–M shows co-detection of BrdU and Spo0J foci in cells treated with HPUra from T10 or T30 . In both of the typical cells shown, there are only two well-separated Spo0J foci, and in each case they co-localize with the BrdU foci. Thus, despite the fact that DNA replication was stopped and that nucleoid expansion and oriC movement were perturbed, there was still almost complete colocalization of BrdU and Spo0J foci (Table 1). Discussion We have developed methods to label relatively specific regions of the B . subtilis chromosome with the thymine analogue BrdU and to determine the localization of the labelled DNA in situ using immunofluorescence. Germination and outgrowth of spores have previously been shown to provide a useful means of studying cell cycle control (Siccardi et al ., 1975; McGinness and Wake, 1979; Partridge and Wake, 1995). By use of a thymine-requiring mutant, DNA replication can be further synchronized in the outgrowing spores by withholding thymine until after a large proportion of the cells have grown sufficiently to
be ready to initiate. A short exposure to the thymine analogue BrdU should result in incorporation only into a relatively small region of the chromosome either side of oriC , and only one strand of each daughter chromosome should be labelled. Thus, two BrdU foci should segregate when such cells are allowed to complete their round of replication and continue growing. The results we obtained following the behaviour of the foci using immunofluorescence microscopy were consistent with this prediction. Thus, two BrdU foci were observed in almost all of the labelled cells or cell chains. Moreover, the two labelled DNA molecules appeared to be replicated and segregated as relatively stable units for three or more generations. BrdU labelling and detection using immunofluorescence microscopy thus seems to be a useful means of following the segregation and inheritance of chromosomes. An interesting feature of the behaviour of the oriC labelled foci was that they had separated by a relatively large distance well before the first DNA replication cycle should have finished (Fig. 2B and Table 1). This would be consistent with the emerging concept of an active partitioning apparatus (see Introduction ), and with the previously observed bipolar pattern of oriC regions detected in growing cells by an indirect labelling procedure (Webb et al ., 1997). Another noteworthy observation concerns the apparent concentration of the BrdU label into relatively discrete foci. This would be compatible with the idea that the chromosome has a well organized structure comprising a number of ordered, well defined and highly condensed domains (Higgins, 1994). Various lines of research have recently implicated the spo0J gene of B . subtilis in the putative active partitioning apparatus (see Introduction ). There were several reasons for expecting that Spo0J function might involve a direct interaction with oriC . First, proteins functionally related to Spo0J, being required for the stable partitioning of low-copy number plasmids, and which exhibit significant sequence similarity to Spo0J are known to be site-specific
Fig. 2. SpoOJ remains associated with oriC after inhibition of DNA replication with HPUra. BrdU signals of untreated cells labelled at oriC and harvested 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 D and 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 and L (SpoOJ foci) and I and M (oriC /SpoOJ overlays). Q 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 945–954
952 P. J. Lewis and J. Errington DNA-binding proteins (e.g. ParB and Sop; Hiraga, 1992). In some cases, these proteins have been shown to bind to specific cis -acting sites that are needed for partitioning (Hiraga, 1992). Furthermore, Mohl and Gober (1997) recently found that a SpoOJ homologue in Caulobacter crescentus can bind directly to DNA. Second, we have shown previously that Spo0J is required for correct positioning of the oriC region of the chromosome close to the pole of the cell at the onset of sporulation (Sharpe and Errington, 1996). Unfortunately, it has not proved possible to detect a specific binding of SpoOJ protein to DNA. Nevertheless, to test the possibility of a close association between Spo0J and the oriC region in vivo , we developed a protocol for co-detection of Spo0J and the BrdU-labelled chromosomal DNA. When the label was incorporated into the oriC region there was almost complete co-localization with the Spo0J signal (Tables 1 and 3). In contrast, when the label was incorporated into a distal region of the chromosome, well away from oriC , the Spo0J and BrdU signals were mainly not co-localized. When DNA replication was blocked by use of the specific inhibitor HPUra, collocalization with oriC was maintained (Fig. 2). These results strongly support the notion of a tight association between the Spo0J assemblies previously observed (Glaser et al ., 1997; Lin et al ., 1997) and the oriC region of the chromosome. They also support the notion that the chromosome has a relatively fixed orientation both during vegetative growth (Webb et al ., 1997) and during sporulation (Wu and Errington, 1994; Sharpe and Errington, 1996). Given the properties of the family of proteins similar to SpoOJ discussed above, we think it likely that SpoOJ will interact directly with DNA but its binding sites may be relatively non-specific: for example the bias for binding to the oriC region could be achieved by co-operative binding to multiple partially specific sites. Mohl and Gober (1997) have shown that despite showing binding of ParB (a SpoOJ homologue) to a specific DNA fragment, there was also some binding to other AT-rich DNA fragments. In conclusion, a number of new problems arise from this work. The most important of these will be to identify the nature of the interaction between Spo0J and the oriC region and to find and characterize the factors responsible for active movement of these putative complexes within the cell.
nutrient agar plates, and sporulation allowed to proceed for 6 days at 308C. The spores were harvested by washing each plate with 5 ml of sterile water. The suspension was centrifuged and resuspended in TE buffer. Lysozyme was added to 3 mg ml¹1 and the mixture incubated at 378C for 1 h. SDS was added to a final concentration of 6.25% (v/v) and the mixture incubated for a further 30 min at 378C. The lysate was then centrifuged and the purified spores washed repeatedly with sterile water at 48C over a period of 5 days. The final spore preparation was stored at an A 600 of 80.
Spore germination Spore germination was carried out in the defined medium, as described previously (McGinness and Wake, 1979). Germination medium was supplemented with thymine or BrdU (Sigma) at 20 mg ml¹1, as required. The medium was inoculated with spores at an A 600 of 1.0 and germination and outgrowth were allowed to proceed for approximately 2.5 h at 378C with shaking. Samples of cultures were checked by microscopy to ensure outgrowth had begun before DNA replication was initiated by the addition of thymine or BrdU. A 10 min pulse labelling with BrdU was followed by quenching with 200 mg ml¹1 thymine and immediate centrifugation and resuspension in fresh pre-warmed medium supplemented with 20 mg ml¹1 thymine. In experiments in which DNA replication was stopped, an aliquot of culture was transferred to a fresh flask and the specific DNA polymerase III inhibitor 6-(para-hydroxyphenylazo)-uracil (HPUra) was added to a final concentration of 50 mg ml¹1.
Immunofluorescence procedures
Bacillus subtilis thy-A (trpC2 thyA thyB ; laboratory stock) was used in all experiments.
Samples of cells (from 0.5 ml of culture) were fixed as described by Lewis et al . (1996). Permeabilization of cells was essentially 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 rabbit polyclonal anti-SpoOJ antibodies (Glaser et al ., 1997) were used at a 1:500 dilution and incubation was carried out overnight at 48C. Cy3-conjugated sheep anti-rabbit antibodies were used at a dilution of 3:1000 and incubation carried out for 1 h at room temperature in the dark. All subsequent steps were performed with minimal exposure of the samples to light. After washing, the samples were fixed again with 2% (w/v) paraformaldehyde 0.01% (v/v) glutaraldehyde in PBS for 15 min at room temperature. After washing, samples in which BrdU signals were to be detected were treated with 4 M HCl for 1 h at ambient temperature. Samples were then washed and a second immunostaining procedure was performed with mouse monoclonal anti-BrdU antibodies (Sigma) at a 1:100 dilution and goat anti-mouse FITC-conjugated antibodies at a 3:1000 dilution. All samples were mounted for epifluorescence microscopy in Vectashield antifade (Vector Laboratories, Burlingame, CA, USA) supplemented with 0.2 mg ml¹1 48,6diamidino-2-phenylindole (DAPI). (Note that a DAPI signal was not detectable in the HCl-treated samples).
Spore preparation
Microscopy, image acquisition and image analysis
Spores were prepared as follows. Exponentially growing B . subtilis thy-A were spread onto 15-cm-diameter Oxoid
Epifluorescence microscopy was performed as described by Lewis et al . (1996) and images were acquired using a cooled
Experimental procedures
Bacterial strains
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Co-localization of oriC and Spo0J 953 CCD camera (Digital Pixel) with a 1536 × 1024 pixel, 9 mm pitch chip. FITC exposures were for 5 s, Cy3 and DAPI exposures were for 2 s. Processing was carried out on the 12 bit images using IPLab Spectrum V3.1.1 (Signal Analytics, Vienna, VA, USA). Final images were assembled in Adobe photoshop V. 3.0.5 for printing. With the narrow bandpass filters used in these experiments, no spectral cross-over of signals was detected in single-staining control experiments (data not shown).
Acknowledgements This work was supported by grants from the Biotechnology and Biological Sciences Research Council. We thank Michaela Sharpe and Ling-Juan Wu for helpful comments on the manuscript, and Dean Jackson for advice on immunostaining of BrdU-labelled DNA.
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