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Division septation in the absence of chromosome termination in Bacillus subtilis
(1979) 134, 251-264
J. Mol. BioZ.
Division Septation in the Absence of Chromosome Termination in Bacillus subtilis T. MCGINNESS
Department
AND R. G. WAKE
of Biochemistry, University Australia
(Received 20 March
of Sydney
1979, and in revised form 29 June 1979)
Germinating
spores of the temperature-sensitive DNA initiation mutants of TsB134 and &a-I(Ts), were allowed to undergo a single round of replication by shifting to the restrictive temperature shortly after its initiation. To monitor the progress of the round 5-bromouracil was added at various times and DNA extracted after a further time, sufficient to allow completion of the chromosome. Average replication was measured from the relative amounts of LL and LH material in CszSO, gradients. The replication state of origin (pzlrA), intermediate (ZeuA) and terminus (metB) markers at the times of 5bromouracil addition were obtained from genetic analysis of the density species fractionated in gradients of CsCl. The DNA replication inhibitor, 6-(p-hydroxyphenylazo)~uracil (HPUra), was added at various stages of the single round and the outgrown cells examined at later times for the frequency and type of septation. Under the conditions of the experiment, central division septation was blocked if HPUra (20 pM) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrstions of HPUra, 40 and 100 pM, it was shown that central division septation would occur at about its normal time if replication was blocked after this 70% stage but before termination. In these circumstances there was a distinct tendency for the DNA to remain close to the septum on both sides of it. The B. subtilis spore contains a single chromosome, which means that the central septum that forms in the absence of termination must pass through a partially completed chromosome. Electron microscopic evidence for such a situation has already been described (Van Iterson & Aten, 1976). It is concluded that, at least under the restrictive conditions of the present experiments, termination of chromosome replication is not obligatory for the formation of the division septum with which it is normally coupled.
BaciZZus m&i&,
1. Introduction The bacterial division cycle results in the segregation of two newly replicated daughter chromosomes and the formation of a central division septum between them. Obviously, there must be co-ordination between the DNA replication and septation processes and it has been suggested that completion (or termination) of the round of DNA replication could be a crucial event in septum initiation (see reviews by Pardee et al., 1973; Donachie et al., 1973). It is well known that DNA replication and septum formation can be uncoupled (see reviews by Donachie et al., 1973; Pritchard, 1974), and uncoupling appears to be more readily achieved in Bacillus subtilis than in Escherichia coli. Wild-type strains of B. subtilis, for example, continue to divide and form anucleate cells when DNA synthesis is blocked (Donachie et al., 1971). 251 0022.-2836/79~300261-14
$02.00/0
(%) 1979 Academic
Press Inc. (London)
Ltd.
262
McGINNESS
T.
AND
R. G. WAKE
Recently it was shown that when a single round of DNB replication, following germination and outgrowth of B. subtilis spores. was allowed to proceed to completion, a central division septum formed readily between the segregated daughter chromosomes (Callister & Wake, 1977). But when replication was slowed to such an extent that the first round could not terminate, an asymmetric septum result,ed. ,4 plausible explanation for these observations is that central division septat’ion is dependent upon chromosome termination, and possibly triggered by it,. When termination is blocked, a septum forms independently of replication. However, it is excluded from the central, DNA-containing area and is positioned to one side of t,hC partially replicated chromosome, i.e. at an asymmetric location. Perhaps, in B. subtilix at least, it is primarily the positioning of the septum rather Ohan its formation tOhat is coupled to chromosome replication. In B. subtilis, the progress of a round of replication can be monitored by use of a density shift approach in conjunction with the measurement, of genetic marker activities in fractionated DNA density species by transformation (O’Sullivan &, Sueoka, 1967). The availability of the drug 6-(p-hydroxyphenylazo)-uracil: a specific inhibitor of DNA replication in gram-positive organisms with minimal side elects (see Cozzarelli, 1977), affords the opportunity to study precisely the dependence of central division septation on the extent of replication of t,he round that is coupled to it. Such a study, with particular emphasis on the relationship between chromosome termination and septation, is reported here.
2. Materials and Methods (a) Strains
and
spore
preparations
The B. subtilis strains used and their origins were as follows: 168 trpC, dnaB134(Ts) thyA thyB (Mendelson & Gross, 1967, referred to as TsB134), 168 trpC2 dna-I(Ts) thyA and 168 purA16 ZeuA8 metB5 ihA 1 thyB (White & Sueoka, 1973, referred to as &a-I(Ts)) (Dubnau et al., 1967, referred to as BD72). Sp ores were prepared from the first 2 strains on plates of supplemented potato ext’ract as described by Callister & Wake (1974), thymine being increased to 50 pg/ml. BD72 was used as the recipient in transformation analyses. (b) Spore
germination,
temperahre
shifL
and
density
labelling
procedures
with rotary Spores (TsB134 at 108/m1 or dna-Z(Ts) at 2~ 108/ml) were germinated shaking in the medium described previously (Callist,er & Wake, 1977) with thymine always present, at 20 &ml. The temperature shift was accomplished by transferring all or part of the germination mixture into an equal volume of the same medium at 45.5%. 5-Bromouracil at 455°C was added, when required, at 200 pg/ml t,o give a final BrUrat :T Imperial Chemical Industries weight ratio of 10: 1. HPUra was a gift from Dr B. Langley, Ltd. (c) Sampling
These were fixing samples
PO%,
performed at various
for
septation
measurements
as described previously times in an equal volume
and scoring procedure (Callister & Wake, 1977) except for of formalin of a higher concentratiorl
v/v).
(d) Extraction Details
of DNA,
dewily
of these procedures
t Abbreviations
used: BrUra,
gradient have
sedimentation aiaalysis
been described
5.bromouraoil;
HPUm,
equilibrium
previously
and
transformational
(see Callister
(i-(p-hydroxy~henylazo).uracil.
et al., 1977).
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3. Results (a) Experimental
approach
The B. subtik spore contains a single, completed chromosome (Oishi et al., 1964 ; Callister & Wake, 1974; Wake, 1976). When spores of temperature-sensitive DNA initiation mutants are allowed to start a round of replication at the permissive temperature and then transferred to the restrictive temperature, a single round of replication followed by central division septation ensues (Callister & Wake, 1977). The progress of replication after the t.emperature shift can be followed in thyminerequiring strains by adding 5bromouracil at various times, allowing the round to proceed to completion and examining the extract.ed DNA in analytical gradient,s of C&SO,. DNA replicated in the presence of BrUra will be shifted to the hybrid (LH) position so that the percentage of unlabelled or LL material will give a direct measure of the average extent of replication of the first round at the time of addition of BrUra. The purA marker is close to the origin of replication and metB close to the terminus of the B. subtilis chromosome (see O’Sullivan BESueoka, 1967). Thus, after fractionation in a preparative gradient of CsCl, the purA marker will be found exclusively in the LL species regardless of the time of BrUra addition. But metB will shift to the LH position in proportion to the number of chromosomes that terminate after BrUra is added. In other words, the percentage of chromosomes that had completed the first round of replication at the time of BrUra addition will be given by the relative amount of metB remaining in the LL species. In the experiments to be described here spores of both TsB134 (Mendelson & Gross, 1967) and dna-I(Ts) (White & Sueoka, 1973) were germinated at the permissive temperature (34°C). After a time sufficient to allow most spores to initmte a single round of replication the culture was shifted to the restrictive temperature (455°C). At various times, extending to when the round terminated, samples were generally treated in two ways at the higher temperature: to one BrUra was added and replication allowed to complete before extraction of DNA, which was then examined for both the average extent of replication and the percentage of chromosomes terminated at the time of BrUra addition; to the other, HPUra (sometimes in the presence of BrUra) was added to block replication and portions fixed at later times for measurement of the percentage of cells septated, as well as the septum position and average cell length. In this way a direct estimation of dependence of the first central division septation on the extent of completion of the first round of replication could be made. The toxicity of BrUra incorporation into DNA is well established. It should be noted that, in the approach outlined, it is used primarily to monitor the progress of the round of replication. It is not actually incorporated into DNA in the septation experiments. (b) Initiation
and completion
of a single round of replication
after spore germination
In a previous study it was shown that 45°C is not sufficient to block initiation of the first round of replication after germination of TsB134 spores (Callister et al., 1977). But it will block the second. Higher temperatures, for example 49”C, are effective in blocking initiation at both stages, but appear to have an adverse effect on the first division septation that normally follows completion of the first round of replication (Callister & Wake, 1977). For this reason, 45*5”C was chosen as the restrictive temperature in these experiments. TsB134 spores were germinated at 34°C in the presence
254
T.
McGINNESS
AND
It.
G.
WAKE
of BrUra and shifted to 455°C at 80 minutes. At 200 minutes the outgrown cells were collected and the DNA extracted. Figure 1 (left panel) shows the analysis of the extract in a Cs,SO, gradient. 87% of the DNA is hybrid, LH, or once-replicated material. After fractionation in a preparative gradient of CsCI, the LH species was found to contain 90% and 84% of the purA (origin) and metB (terminus) marker activities, respectively. Thus, shifting the germinating spores to the higher temperature at 80 minutes allows the first round of replication to start in the majority (approx. 80%) of the spores. This round then proceeds to completion. Shifting to the higher temperature later than 80 minutes might allow more spores to initiate a round of replication but there is the increased possibility of second round initiations. I
LL
LH
JN90
min
J-L .A?_ A-L 95 min
100
min
110 min
FIG. 1. Initiation and completion of a single round of replication after germination of TsB134 spores. The left panel relates to an experiment in which spores were germinated at 34°C in BrUra medium (250 ml) and shifted with no dilution to 45.5”C at 80 min. DNA was extracted at 200 min and analysed in a CszSO, gradient. In the right panel, spores were germinated at 34°C and at 80 min added to an equal volume of medium at 46.5’C. At the times indicated, BrUra was added to separate samples (90 ml). DNA was extracted at 200 min and analysed in Cs,SO, gradients. To an additional sample at 96 min, HPUra (20 pM) was added along with BrUra and the DNA extracted and anelysed as before (extreme right).
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265
It should also be pointed out that while 16% of the m&B activity remained in the LL species in the experiment just described, the value was significantly lower in other experiments (see below), presumably because more of the spores initiated the first round in those cases. That there is very little or no HH material in the analytical gradient of Figure 1 (left panel) suggests that initiation of a second round of replication has been effectively blocked in this particular experiment. However, small amounts of second round initiation probably occur in the later experiments with TsB134 which use a different labelling protocol. The progress of the single round of replication, initiated at 34°C and allowed to proceed to completion at 45.5”C in the presence of thymine alone, has been followed by the approach outlined in the previous section. BrUra was added to the germination mixture at various times (90 to 110 min) following the temperature shift at 80 minutes, and DNA was extracted at 200 minutes. Figure 1 (right panel) shows the Cs,SO, density gradient analyses of the extracts. As expected, the proportion of LH material decreases when the BrUra is added at later times. This is because the single round that was initiated draws nearer to completion so there is less replication in the presence of BrUra. It has already been shown that some spores do not initiate replication by 80 minutes (Fig. 1, left panel) so that unreplicated DNA would contribute to the LL species. The relative amounts of LL and LH material in Figure 1 (right panel), after correcting for an approximate amount of unreplicated DNA as found in Figure 1 (left panel), have been used to plot the progress of the first round in TsB134 (Fig. 2, lower line). The data from some other experiments are also included, and it can be seen that approximately 50% of the round, on the average, has been accomplished by 100 minutes. The effectiveness of HPUra in blocking the round of replication has been measured in two ways. When a relatively low level (20 PM) of the drug was added along with BrUra at any time during the 95 to 105 minute period, the formation of LH material
I-----l
L
100
90
1 110
bin)
FIG. 2. Rate of progress of the single round of replication after spore germination. The lower line shows the data for TsB134. The values of the solid circles (a) were calculated from the relative amounts of LL and LH material in the right panel of Fig. 1 after correcting for an amount of unreplicated LL material as found in the left panel. Data from some other experiments are also included. The upper line shows the results of a similar experiment with the dna-I(Ts) strain in which germinating spores were shifted from 34°C to 455°C at 80 min and BrUra added at the times iudicated; DNA was extracted at 230 min.
256
T. McGINNESS
AND
It.
G. WAKE
measured in analytical C&SO, gradients was virtually eliminated (Pig. 1: extreme right, 95 min result only shown). However a small amount of LH was detectable when the ultracentrifuge cell was overloaded. In another experiment, without BrUra, [3H]thymine was added with and without HPUra (20 and 100 pM) at 90 minutes, and the radioactivity in fractionated LL material obtained at 180 minutes compared. By this time, 20 pM and 100 PM-HPUra allowed the equivalent of 5 to 7% and 1 to 2% of a single round of replication, respectively, to proceed. The samples analysed in Figure 1 (right panel) were also fractionated in preparative gradients of CsCl and the density species analysed for pura and ,metB activities. Figure 3 (left panel) shows the results for the 90 minute sample. As expected, most of the origin marker, purA, remains at the LL position: but a minor proportion is present as LH. This shows that some chromosome initiations did indeed occur at 45*5”C after 90 minutes in this experiment. (Nuclear staining in conjunction with further experiments at 47°C showed that these were due to one of the two daughter chromosomes from the first round initiating again in a small percentage of the cases McGinness & Wake, unpublished observations). These second round initiations should not prevent detection of termination of the first rounds. but the sensitivity could be lowered. Also, it is possible that they do not t*erminate by 200 minutes in the presence of BrUra as no significant HH was found.) Figure 3 (left panel) also shows that the terminus marker, metB, is present mainly in the LH position confirming that most chromosomes terminate under the conditions of the experiment,.
I
dno-I
‘I
(Tsl
I9
,r A
pur
A
Frociion Pm. 3. Distribution of origin and terminus markers amongst the DNA density species following completion of the single round of replication in the presence of BrUra. The left panel shows the result for TsB134 in which BrUra was added at 90 min (the temperature shift was at 80 min, see Fig. 1, right panel). After separation in a gradient of CsCl, fractions were analysed for purA (0) and m&B (0) activities. The right panel shows the result for dna-I(Ts) in which BrUra was added at 85 mm, following the temperature shift at SO min (see legend to Fig. 2 ; the purA peak fractions gave > 1000 transformants per plate and the points (6) are positioned at the equivalent of 1009 transformants in the analysis).
REPLICATION
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267
More extensive genetic marker analyses of the 90 to 110 minute samples of Figure 1 (right panel) are considered in Figure 5 along with septation data. The data in Figures 2 and 3 relating to dnu-l(Ts) are discussed later.
(c) Effect of blocking the round of reyplication in TsB134
on subsequent septation
In a preliminary study, in which 12.5 to 50 PM-HPUra was added at 90 or 95 minutes, it was found that the percentage of cells able to form a septum during subsequenb outgrowth was low and about the same for the lowest and highest concentrations of the drug used. But there was a tendency for the outgrowing cells to distort in shape (bulbous forms) upon prolonged incubation following addition of the higher concentrations of HPUra. This was not a problem when up to 100 PM-HPUra was added at 100 minutes or later. Because 20 PM-HPUra is sufficient to inhibit replication almost completely (see previous section) this level was used in the first study of the effect of blocking replication at various stages of the round on subsequent septation. To separate samples of the same mixtures to which BrUra was added at various times (see Fig. 1, right panel) 20 PM-HPUra was added at the same times and portions taken over the 120 to 200 minute period and fixed in formalin. Figure 4 shows the curves describing the percentage of cells at various times containing a septum for each time of addition of HPUra. In the absence of the drug, central septation becomes obvious in a small proportion of the cells soon after 130 minutes; by 160 minutes most have septated. (With longer times of incubation (>180 min) additional septa formed and gave rise to DNA-less cells, but at 160 minutes most cells (87%) that had septated contained just one septum which was central. A more detailed consideration of the positioning of the septum is given below.) When
I
I
I40
1
*
150 160 170 Time (days)
I
180
I
FIQ. 4. Effect of HPUra added at various times during the single round of replication in TsB134 on septation. At the same times as adding BrUra at 456°C in the experiment described in the legend to Fig. 1 (right panel) separate samples (6 ml) were treated with HPUra (20 pM) at 455W. Portions were fixed in formalin at later times for measurement of the percentage of outgrown cells containing a septum. For each point on the graph, 100 cells were scored.
258
T. McGINNESS
AND
R. G. WAKE
chromosome replication is blocked by 20 piv-HPUra during the 90 to 110 minute period it can be seen that reduced levels of septation, appearing at about the same time as the control and levelling off at 160 minutes, are obtained. But the level cycle achieved is higher when the HPUra is added at a later stage in the replication (distortion of shape occurred to an extent of <100/b at 170 min following addition of HPUra at 90 and 95 min). For example, when the drug is added at 90 minutes (the average extent of replication being 30%, see Fig. 2) most cells do not septate by 170 minutes ; but if the round is allowed to progress further (700,; by 110 min) the majority of cells can subsequently form a septum. Again, upon further incubation, additional (asymmetric) septa form and give rise to DNA-less cells (data not shown). The rates of cell length extension for the various times of HPUra addition in Figure 4 were also measured. There was no significant difference in the case of no drug addition and when it was added at 100 and 110 minut’es, the mean cell lengths at. 160 minutes being 7.4*2*0, 7.3k1.8 and 7*3& 2.0 pm, respectively. For HPCra, addition at 90 and 95 minutes the length at 160 minutes was reduced to 5911.6 and 6.6~1.8 pm, respectively (6.812.3 and 6.9i2.1 pm at, 170 min). The relationship between the level of septation obtained following HPUra addition at various times and the extent of chromosome replication achieved at those times was examined in more detail. The samples to which BrUra was added, at the same time as the separate samples of Figure 4 were treated with HPUra for septation measurements, were fractionated into LL and LH species (see Fig. 3) and the pooled fractions analysed for metB as well as the intermediate replicating marker. ZeuA. In Figure 5 the percentage of each of these markers remaining in the LL species (curves) and the amount of septation achieved (solid triangles, from Fig. 4) are plotted against the time of BrUra or HPUra addition. Consider the marker activities first’. As a fork moves past a particular marker in a single round of replication, that ma.rker will remain at the LL position upon subsequent addition of BrUra. There is a low but, constant amount (8*2o/o) of metB in the LL species up until 100 minutes. Obviously, I
90 Time
95
100
105
of BrUm (or HPUm) addiiion
I IO (mln)
FIG. 5. Progress of the single round of replication in TsB134 as measured by ZeuA and metB eplioation and the effect of blocking the round at various stages on subsequent septation. The samples analysed in Fig. 1 (right panel) were separated into LL and LH species as shown in Fig. 3. The pooled LL and LH species were re-analysed for ZeuA ( 0) and m&B (a) activities. The septation data ( A) are taken from the plateau levels in Fig. 4. (-----) Percentage genetic marker in LL.
REPLICATION
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259
this arises from those spores that did not initiate a round of replication by 80 minutes. More importantly, the fact that there is no significant increase shows that no rounds terminate before 100 minutes. But some termination has occurred at 110 minutes. (In interpreting the results of this type of experiment more significance has been attached to changes in the relative amounts of genetic marker activity in the various DNA density species than to absolute levels. It is possible that the specific transforming activity is significantly different for LL and LH DNA and to different extents for various markers. Note, for example, that the amount of ZeuA in LL DNA at 90 minutes is less than that of metB.) As expected, the amount of ZeuA in the LL species starts to increase much earlier, from 90 minutes. These results are consistent with the established replication order of these markers (O’Sullivan & Sueoka, 1967). Considering the septation and marker activities in Figure 5 together, the surprising result is that a significant amount of septation (approx. 40%) is obtained in the absence of any termination. It appears that septation can occur once replication has proceeded as far as the ZeuA marker, or somewhat before it. Approximately 30% of the chromosome replicates after ZeuA (Kejzlarova-Lepesant et al., 1975) so that the small amount of replication (5 to 7%) that might have occurred by 160 minutes following 20 p&r-HPUra addition at 100 minutes would still leave aEZchromosomes significantly uncompleted. An additional and more direct experiment using a higher concentration of HPUra in the presence of BrUra has been performed with TsB134 to confirm the finding of central septation in the absence of termination of the round of replication. After shifting germinating TsB134 spores to 455°C at 80 minutes, separate samples at 90, 100 and 105 minutes were split. The smaller portion in each case was mixed with HPUra (40 pM) and BrUra; to the larger, BrUra alone was added. DNA was extracted from the latter at 200 minutes for measurement of the percentage of rrLetB replicated at the time of BrUra addition, and portions were taken at various times from the mixtures containing HPUra for septation measurements. To test directly the effectiveness of the block in chromosome termination by 40 PM-HPUra, it was added along with BrUra to a separate sample at 101 minutes. Small portions were removed for septation measurements, and from the bulk of it DNA wa,s extracted at 200 minutes for analysis of the distribution of metB between the LL and LH density positions. In this last case, there was no transfer (<0*5%) of metB activity to the LH position (purA and metB were coincident at the LL position, data not shown). Thus, no chromosome terminations occurred after BrUra plus HPUra (40 pM) were added at 101 minutes. The metB replication and septation data for the other samples are shown in Figure 6 (see legend for calculation of metB replication). As before no chromosome terminations have occurred by 100 minutes, and it has already been established that 40 PM-HPUra added at 101 minutes blocks subsequent terminations completely. But, again, there is substantial septation, 30 to 40%, when replication is blocked at this stage. In this experiment, an additional sample was treated with 100 +f-HPUra, in the absence of BrUra, at 100 minutes. The amount of septation at 160 minutes was still significant, but reduced, at 27%. There was no distortion in cell shape. The location of the septum in the cells obtained after 40 PM-HPUra addition at 100, 101 and 105 minutes was very similar to the situation where no drug was present. Figure 7 compares the septum location at 170 minutes in the absence of HPUra and in its presence (from 100 min). At least 80% of the septa are central (0.50 to O-55) in both cases. That the central septum which formed in the presence of
260
T. McGINNESS
AND
It.
G. WAKE
,h
50-
_
I I , ,/Septatmn i
% 4030 -
1
/ If 20-
/
IO -
,6
//
/
met e 5’
5%
95
/I 100
105
Time of BrUra (or &Urn +HPUra)adJ~t~n ImInI FIG. 6. Division septation in TsB134 in the absence of chromosome t,ermination. Germinating TsB134 spores were shifted from 34°C to 456°C at 80 min. St 90, 100 and 105 min separate samples were treated in 2 ways: to a larger one (80 ml) BrUra was added and DNA extracted at 200 min for density gradient analysis and fractionation; to the other (10 ml) HPUra (40 pM) plus BrUra were added and portions fixed in formalin at 160 and 170 min for septation analysis. To another sample (80 ml) at 101 min, HPUra (40 pM) plus BrUra were added; portions were fixed in formalin at 160 and 170 min and the bulk used for DNA extraction at 200 min. (0) and (0) are the septation data (100 cells scored in each case) for portions fixed at 160 and 170 min, respectively. The distribution of metB between the fractionated LL and LH species (after correcting for a base level of 9.9% remaining in the LL species in the 90 and 100 min cases) was used to determine the percentage m&B replicated (mean of 4 measurements + average deviation) at t,he times shown.
HPUra
Septum location (froctmn
+ BrUra
of cell length)
FIG. 7. Location of the septum in TsB134 which forms in the absence of termination, The left panel shows the position of the septum in the absence of HPUra and, in the right panel, HPUra was added along with BrUra at 100 min (see legend to Fig. 6). In both cases, cells fixed at 170 min were examined. Mean cell lengths for the total cell populations in the absence and presence of HPUra were 9.0& 2-4 pm and 8.6 i 2.7 pm, respectively.
REPLICATION
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HPUra (added at 100 min) was a true division septum was confkmed by acridine orange staining of nuclear bodies. DNA was always (>95%) found on both sides of this septum, and in at least one third of the cases it remained very close to it, even at 180 minutes. (d) Effect of blocking the round of replication
in dna-1 (Ts) on subsequent septation
Using the same procedure as for TsB134 it was found that, with dnu-l(Ts), no new initiations occurred after transfer to 45.5”C (see Fig. 3, right panel). The crucial experiments suggesting that central division septation can occur in the absence of chromosome termination were therefore repeated with dna-l(Ts) in order to avoid a(ny possible complications in interpretation due to the small but significant number of new chromosome initiations that occurred with TsB134 after transfer to the restrictive temperature. Upon shifting germinating spores of &a-l(Ts) from 34°C to 455°C at 80 minutes the single round of replication proceeds at a slightly faster rate than in the case of TsB134 (see Fig. 2). A preliminary experiment indicated that the first chromosome terminations were occurring between 95 and 100 minutes. Figure 8 summarizes the results of a more extensive experiment in which the progress of replication over the 85
70 -
A
60-
0 &A
0
50-
n
/
40-
r:-o
95/’
Time of BrUra (or HPUro+BrUra
100 I addition
105 bin)
Fra. 8. Division septation in &a-1(Ts) in the absence of chromosome termination. Germinating dna-l(Ts) spores were shifted from 34°C to 466’C at 80 min. At 86, 90,94, 100 and 106 min separate samples were treated as follows : to a larger one (80 ml) BrUra, was added and DNA extracted at 230 min for C&SO4 density gradient analysis (see Fig. 2, upper line) and fractionation in CsCl; to 2 x 10 ml lots, et 94 and 100 min only, HPUra (40 pM) plus BrUra and HPUra (100 pM) plus BrUra were added and portions fixed in formalin at later times for septation analysis. To a further sample (100 ml) at 96 min, HPUra (40 PM) plus BrUra, were added, portions were fixed in formalin at later times and the bulk used for DNA extraction at 230 min. The septation data (0, 40 q-HPUre; n , 100 PM-HPUre) were obtained from the portions taken at ISO min (100 to 200 cells scored for each point). The distribution of metB activity between pooled LL and LH fractions from the CsCl gradients (after correcting for a constant base level of 16*6+O*6°~ remaining in the LL species in the 86, 90 end 94 min oases) was used to determine the percentage metB T o calculate the percentage ZeuA replicated replicated at the times indicated (-- O-- a--). (-A-A-), an amount of 12.6% remaining in the LL species of the 86 min sample was used 84 a baseline correction for all other times.
262
T. McGINNESS
AND
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to 105 minute period was monitored in the same way as in Figure 6 for TsB134. There was no significant shift of metB from LH to LL over the 85 to 94 minute period. although the constant amount of this marker activity remaining at the LL position was higher than in Figure 6 (16.5% versus 9*9”/“). To a separate sample at 95 minutes, HPUra (40 pM) and BrUra were added. There was no detectable transfer (SO% of the cases. Clearly, these results with &au-l(Ts) confirm the finding with TsB134, that central division septation can occur in the absence of chromosome termination. As with TsB134, a higher concentration of HPUra (100 pM) added before termination reduced the amount of septation eventually achieved (see Fig. 8. solid squares). but it is still substantial. There is no doubt that 40 PM-HPUra blocks all terminations when added at 94 minutes in the case of dna-l(Ts) and 100 minutes with TsB134. The lowered amount of septation achieved with 100 PaI-HPUra could be the result, of a more rapid cessation of replication fork movement.
4. Discussion It has been shown here that germinating spores of temperature-sensitive DNA initiation mutants of B. subtilis, shifted from the permissive to restrictive temperature after initiation of the first round of replication, afford just a single round which goes on to terminate. About 30 minutes after termination, the first central division septum can be seen under the light microscope. It would be expected that if termination of replication is obligatory for division septation under these conditions, interference with the progress of the first round of replication after germination would block the formation of the central septum. The results presented here, in which HPUra has been used as a specific inhibitor of DNA replication, suggest that this is so only if the round of replication is blocked before a certain stage is reached. At about the time when the chromosome is 70% replicated, on the basis of the behaviour of ZeuB, blocking termination by HPUra no longer prevents central division septation occurring at about its normal time. Presumably, in the previous experiments (Callister Sr Wake, 1977) central septation did not occur at the low level of thymine, which reduced DNA replication to < 10% of its normal rate, because the necessary minimum amount of replication could not be achieved within the time available.
REPLICATION
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263
Clearly, if the B. subtilis spore contains a single chromosome, the central septum that forms when termination is blocked must pass through a partially replicated chromosome. This would be consistent with the observations of Van Iterson & Aten (1976). With germinating spores of B. subtilis to which a low level of mitomycin C was added at a certain time, these workers obtained electron micrographs showing a septum passing through an area of nucleoplasm. They suggested that the division process could proceed in the presence of the replication inhibitor so that the septum could close although the nuclear division clock was arrested. While no electron microscopy has been performed with samples obtained under the conditions of inhibition used here, the observation that in a significant percentage of the cases the DNA remains close to and on both sides of the septum could be due to such a situation. It is not surprising that septation can occur in the absence of chromosome termination, as this is now well established (Mendelson, 1968; Donachie et al., 1971; Sargent, 1975). That the septum can be centrally located, requiring that it “divide” the DNA of a partially replicated chromosome, was unexpected, although Van Iterson & Aten (1976) had already provided evidence for such a situation. Certainly, in the normal situation, there must be a mechanism to ensure the formation of a septum preferentially between two completed chromosomes, but there is no definitive information on how this is accomplished. Perhaps changes in the folding or membrane associations of the nucleoid are involved, so that what is normally achieved at 100% in the situation of replication can occur after a minimum of about 700/ replication continued cell extension. It could well be that segregation of membrane-bound origins, each pulling with it the bulk of the replicated portion of each daughter, could provide the situation that would enable central septation in the absence of termination. It should be kept in mind that under the conditions used in the present experiments, where division septation occurs in the absence of termination, the dnaB gene product is inactive (it is possible that ha-1 is also a B group mutation, but it is not known how many genes comprise this group (White & Sueoka, 1973; Karamata & Gross, 1970)). Sargent (1975) has presented evidence suggesting that the dnaB gene product, under the permissive conditions, functions as a repressor of septation. The present results would be entirely consistent with such a role for this gene product. In the normal situation, termination of replication could act to derepress the dnaB gene product and so trigger the formation of the septum. However, at the restrictive temperature where the repressor activity is destroyed, septum formation at the normal division site (perhaps a site of chromosome attachment to the membrane) might proceed as soon as other prerequisites, not dependent on chromosome termination, are accomplished. This possibility is being investigated. This work has been supported by grants from the University Fund and the Australian Research Grants Committee.
of Sydney Cancer Research
REFERENCES Callister, H. & Wake, R. G. (1974). J. Bacterial. 120, 579-582. Callister, H. & Wake, R. G. (1977). J. Mol. Biol. 117, 71-84. Callister,
Cozzarelli, Donachie, 10
H., Le Mesurier, S. & Wake, R. G. (1977). J. Bacterial. N. R. (1977). Annu. Rev. Biochem. 46, 641-668. W. D., Martin, D. T. M. & Begg, K. J. (1971). Natwe
130, 1030-1037. New Biol.
231, 274-276.
264
T. McGINNESS
AND
H. 0.
WAKE
Donachie, W. D., Jones, N. C. & Teather, R. (1973). flynap. Sot. @en. Microbial. 23, 9-44. Dubnau, D., Goldthwaite, C., Smith, I. & Marmur, J. (1967). J. Mol. Biol. 27, 163-185. Karamata, D. & Gross, J. D. (1970). Mol. Gem. Genet. 108, 177-187. Kejzlarova-Lepesant, J., Harford, N., Lepesant, J.-A. & Dedonder, R. (1975). InSpores 1’1 (Gerhardt, P., Costilow, R. N. & Sadoff, H. L., eds), pp. 592--595, American Society fol Microbiology, Washington, DC. Mendelson, N. H. (1968). CoZd Spring Harbor Xymp. Quant. Biol. 33, 313-316. Mendelson, N. H. & Gross, J. D. (1967). J. Bacterial. 94, 1603-1608. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature (London), 204, 1069-1073. O’Sullivan, A. & Sueoka, N. (1967). J. Mol. BioZ. 27, 349%368. Pardee, A. B., Wu, P. C. bt Zusman, D. R. (1973). In Racterial Membran.es and Walls (Lieve, L., ed.), pp. 357-412, Marcel Dekker, Inc.. New York. Pritchard, R. H. (1974). Phil. Trans. Roy. Sot. (London). 267, 303.-336. Sargent, M. G. (1975). J. Bacterial. 123, 1218-1234. Van Iterson, W. & Aten, J. A. (1976). In Microbiology 1976’ (Schlessinger, D. rd.), pp. 128140, American Societ,y for Microbiology, Washington, D.C. Wake, R. G. (1976). .J. Bacterial. 127, 433- 439. White, K. & Sueoka, N. (1973). GenIetics, 73, 185-214.
J. Mol. BioZ.
Division Septation in the Absence of Chromosome Termination in Bacillus subtilis T. MCGINNESS
Department
AND R. G. WAKE
of Biochemistry, University Australia
(Received 20 March
of Sydney
1979, and in revised form 29 June 1979)
Germinating
spores of the temperature-sensitive DNA initiation mutants of TsB134 and &a-I(Ts), were allowed to undergo a single round of replication by shifting to the restrictive temperature shortly after its initiation. To monitor the progress of the round 5-bromouracil was added at various times and DNA extracted after a further time, sufficient to allow completion of the chromosome. Average replication was measured from the relative amounts of LL and LH material in CszSO, gradients. The replication state of origin (pzlrA), intermediate (ZeuA) and terminus (metB) markers at the times of 5bromouracil addition were obtained from genetic analysis of the density species fractionated in gradients of CsCl. The DNA replication inhibitor, 6-(p-hydroxyphenylazo)~uracil (HPUra), was added at various stages of the single round and the outgrown cells examined at later times for the frequency and type of septation. Under the conditions of the experiment, central division septation was blocked if HPUra (20 pM) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrstions of HPUra, 40 and 100 pM, it was shown that central division septation would occur at about its normal time if replication was blocked after this 70% stage but before termination. In these circumstances there was a distinct tendency for the DNA to remain close to the septum on both sides of it. The B. subtilis spore contains a single chromosome, which means that the central septum that forms in the absence of termination must pass through a partially completed chromosome. Electron microscopic evidence for such a situation has already been described (Van Iterson & Aten, 1976). It is concluded that, at least under the restrictive conditions of the present experiments, termination of chromosome replication is not obligatory for the formation of the division septum with which it is normally coupled.
BaciZZus m&i&,
1. Introduction The bacterial division cycle results in the segregation of two newly replicated daughter chromosomes and the formation of a central division septum between them. Obviously, there must be co-ordination between the DNA replication and septation processes and it has been suggested that completion (or termination) of the round of DNA replication could be a crucial event in septum initiation (see reviews by Pardee et al., 1973; Donachie et al., 1973). It is well known that DNA replication and septum formation can be uncoupled (see reviews by Donachie et al., 1973; Pritchard, 1974), and uncoupling appears to be more readily achieved in Bacillus subtilis than in Escherichia coli. Wild-type strains of B. subtilis, for example, continue to divide and form anucleate cells when DNA synthesis is blocked (Donachie et al., 1971). 251 0022.-2836/79~300261-14
$02.00/0
(%) 1979 Academic
Press Inc. (London)
Ltd.
262
McGINNESS
T.
AND
R. G. WAKE
Recently it was shown that when a single round of DNB replication, following germination and outgrowth of B. subtilis spores. was allowed to proceed to completion, a central division septum formed readily between the segregated daughter chromosomes (Callister & Wake, 1977). But when replication was slowed to such an extent that the first round could not terminate, an asymmetric septum result,ed. ,4 plausible explanation for these observations is that central division septat’ion is dependent upon chromosome termination, and possibly triggered by it,. When termination is blocked, a septum forms independently of replication. However, it is excluded from the central, DNA-containing area and is positioned to one side of t,hC partially replicated chromosome, i.e. at an asymmetric location. Perhaps, in B. subtilix at least, it is primarily the positioning of the septum rather Ohan its formation tOhat is coupled to chromosome replication. In B. subtilis, the progress of a round of replication can be monitored by use of a density shift approach in conjunction with the measurement, of genetic marker activities in fractionated DNA density species by transformation (O’Sullivan &, Sueoka, 1967). The availability of the drug 6-(p-hydroxyphenylazo)-uracil: a specific inhibitor of DNA replication in gram-positive organisms with minimal side elects (see Cozzarelli, 1977), affords the opportunity to study precisely the dependence of central division septation on the extent of replication of t,he round that is coupled to it. Such a study, with particular emphasis on the relationship between chromosome termination and septation, is reported here.
2. Materials and Methods (a) Strains
and
spore
preparations
The B. subtilis strains used and their origins were as follows: 168 trpC, dnaB134(Ts) thyA thyB (Mendelson & Gross, 1967, referred to as TsB134), 168 trpC2 dna-I(Ts) thyA and 168 purA16 ZeuA8 metB5 ihA 1 thyB (White & Sueoka, 1973, referred to as &a-I(Ts)) (Dubnau et al., 1967, referred to as BD72). Sp ores were prepared from the first 2 strains on plates of supplemented potato ext’ract as described by Callister & Wake (1974), thymine being increased to 50 pg/ml. BD72 was used as the recipient in transformation analyses. (b) Spore
germination,
temperahre
shifL
and
density
labelling
procedures
with rotary Spores (TsB134 at 108/m1 or dna-Z(Ts) at 2~ 108/ml) were germinated shaking in the medium described previously (Callist,er & Wake, 1977) with thymine always present, at 20 &ml. The temperature shift was accomplished by transferring all or part of the germination mixture into an equal volume of the same medium at 45.5%. 5-Bromouracil at 455°C was added, when required, at 200 pg/ml t,o give a final BrUrat :T Imperial Chemical Industries weight ratio of 10: 1. HPUra was a gift from Dr B. Langley, Ltd. (c) Sampling
These were fixing samples
PO%,
performed at various
for
septation
measurements
as described previously times in an equal volume
and scoring procedure (Callister & Wake, 1977) except for of formalin of a higher concentratiorl
v/v).
(d) Extraction Details
of DNA,
dewily
of these procedures
t Abbreviations
used: BrUra,
gradient have
sedimentation aiaalysis
been described
5.bromouraoil;
HPUm,
equilibrium
previously
and
transformational
(see Callister
(i-(p-hydroxy~henylazo).uracil.
et al., 1977).
REPLICATION
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CYCLE
253
3. Results (a) Experimental
approach
The B. subtik spore contains a single, completed chromosome (Oishi et al., 1964 ; Callister & Wake, 1974; Wake, 1976). When spores of temperature-sensitive DNA initiation mutants are allowed to start a round of replication at the permissive temperature and then transferred to the restrictive temperature, a single round of replication followed by central division septation ensues (Callister & Wake, 1977). The progress of replication after the t.emperature shift can be followed in thyminerequiring strains by adding 5bromouracil at various times, allowing the round to proceed to completion and examining the extract.ed DNA in analytical gradient,s of C&SO,. DNA replicated in the presence of BrUra will be shifted to the hybrid (LH) position so that the percentage of unlabelled or LL material will give a direct measure of the average extent of replication of the first round at the time of addition of BrUra. The purA marker is close to the origin of replication and metB close to the terminus of the B. subtilis chromosome (see O’Sullivan BESueoka, 1967). Thus, after fractionation in a preparative gradient of CsCl, the purA marker will be found exclusively in the LL species regardless of the time of BrUra addition. But metB will shift to the LH position in proportion to the number of chromosomes that terminate after BrUra is added. In other words, the percentage of chromosomes that had completed the first round of replication at the time of BrUra addition will be given by the relative amount of metB remaining in the LL species. In the experiments to be described here spores of both TsB134 (Mendelson & Gross, 1967) and dna-I(Ts) (White & Sueoka, 1973) were germinated at the permissive temperature (34°C). After a time sufficient to allow most spores to initmte a single round of replication the culture was shifted to the restrictive temperature (455°C). At various times, extending to when the round terminated, samples were generally treated in two ways at the higher temperature: to one BrUra was added and replication allowed to complete before extraction of DNA, which was then examined for both the average extent of replication and the percentage of chromosomes terminated at the time of BrUra addition; to the other, HPUra (sometimes in the presence of BrUra) was added to block replication and portions fixed at later times for measurement of the percentage of cells septated, as well as the septum position and average cell length. In this way a direct estimation of dependence of the first central division septation on the extent of completion of the first round of replication could be made. The toxicity of BrUra incorporation into DNA is well established. It should be noted that, in the approach outlined, it is used primarily to monitor the progress of the round of replication. It is not actually incorporated into DNA in the septation experiments. (b) Initiation
and completion
of a single round of replication
after spore germination
In a previous study it was shown that 45°C is not sufficient to block initiation of the first round of replication after germination of TsB134 spores (Callister et al., 1977). But it will block the second. Higher temperatures, for example 49”C, are effective in blocking initiation at both stages, but appear to have an adverse effect on the first division septation that normally follows completion of the first round of replication (Callister & Wake, 1977). For this reason, 45*5”C was chosen as the restrictive temperature in these experiments. TsB134 spores were germinated at 34°C in the presence
254
T.
McGINNESS
AND
It.
G.
WAKE
of BrUra and shifted to 455°C at 80 minutes. At 200 minutes the outgrown cells were collected and the DNA extracted. Figure 1 (left panel) shows the analysis of the extract in a Cs,SO, gradient. 87% of the DNA is hybrid, LH, or once-replicated material. After fractionation in a preparative gradient of CsCI, the LH species was found to contain 90% and 84% of the purA (origin) and metB (terminus) marker activities, respectively. Thus, shifting the germinating spores to the higher temperature at 80 minutes allows the first round of replication to start in the majority (approx. 80%) of the spores. This round then proceeds to completion. Shifting to the higher temperature later than 80 minutes might allow more spores to initiate a round of replication but there is the increased possibility of second round initiations. I
LL
LH
JN90
min
J-L .A?_ A-L 95 min
100
min
110 min
FIG. 1. Initiation and completion of a single round of replication after germination of TsB134 spores. The left panel relates to an experiment in which spores were germinated at 34°C in BrUra medium (250 ml) and shifted with no dilution to 45.5”C at 80 min. DNA was extracted at 200 min and analysed in a CszSO, gradient. In the right panel, spores were germinated at 34°C and at 80 min added to an equal volume of medium at 46.5’C. At the times indicated, BrUra was added to separate samples (90 ml). DNA was extracted at 200 min and analysed in Cs,SO, gradients. To an additional sample at 96 min, HPUra (20 pM) was added along with BrUra and the DNA extracted and anelysed as before (extreme right).
REPLICATION
AND
DIVISION
CYCLE
265
It should also be pointed out that while 16% of the m&B activity remained in the LL species in the experiment just described, the value was significantly lower in other experiments (see below), presumably because more of the spores initiated the first round in those cases. That there is very little or no HH material in the analytical gradient of Figure 1 (left panel) suggests that initiation of a second round of replication has been effectively blocked in this particular experiment. However, small amounts of second round initiation probably occur in the later experiments with TsB134 which use a different labelling protocol. The progress of the single round of replication, initiated at 34°C and allowed to proceed to completion at 45.5”C in the presence of thymine alone, has been followed by the approach outlined in the previous section. BrUra was added to the germination mixture at various times (90 to 110 min) following the temperature shift at 80 minutes, and DNA was extracted at 200 minutes. Figure 1 (right panel) shows the Cs,SO, density gradient analyses of the extracts. As expected, the proportion of LH material decreases when the BrUra is added at later times. This is because the single round that was initiated draws nearer to completion so there is less replication in the presence of BrUra. It has already been shown that some spores do not initiate replication by 80 minutes (Fig. 1, left panel) so that unreplicated DNA would contribute to the LL species. The relative amounts of LL and LH material in Figure 1 (right panel), after correcting for an approximate amount of unreplicated DNA as found in Figure 1 (left panel), have been used to plot the progress of the first round in TsB134 (Fig. 2, lower line). The data from some other experiments are also included, and it can be seen that approximately 50% of the round, on the average, has been accomplished by 100 minutes. The effectiveness of HPUra in blocking the round of replication has been measured in two ways. When a relatively low level (20 PM) of the drug was added along with BrUra at any time during the 95 to 105 minute period, the formation of LH material
I-----l
L
100
90
1 110
bin)
FIG. 2. Rate of progress of the single round of replication after spore germination. The lower line shows the data for TsB134. The values of the solid circles (a) were calculated from the relative amounts of LL and LH material in the right panel of Fig. 1 after correcting for an amount of unreplicated LL material as found in the left panel. Data from some other experiments are also included. The upper line shows the results of a similar experiment with the dna-I(Ts) strain in which germinating spores were shifted from 34°C to 455°C at 80 min and BrUra added at the times iudicated; DNA was extracted at 230 min.
256
T. McGINNESS
AND
It.
G. WAKE
measured in analytical C&SO, gradients was virtually eliminated (Pig. 1: extreme right, 95 min result only shown). However a small amount of LH was detectable when the ultracentrifuge cell was overloaded. In another experiment, without BrUra, [3H]thymine was added with and without HPUra (20 and 100 pM) at 90 minutes, and the radioactivity in fractionated LL material obtained at 180 minutes compared. By this time, 20 pM and 100 PM-HPUra allowed the equivalent of 5 to 7% and 1 to 2% of a single round of replication, respectively, to proceed. The samples analysed in Figure 1 (right panel) were also fractionated in preparative gradients of CsCl and the density species analysed for pura and ,metB activities. Figure 3 (left panel) shows the results for the 90 minute sample. As expected, most of the origin marker, purA, remains at the LL position: but a minor proportion is present as LH. This shows that some chromosome initiations did indeed occur at 45*5”C after 90 minutes in this experiment. (Nuclear staining in conjunction with further experiments at 47°C showed that these were due to one of the two daughter chromosomes from the first round initiating again in a small percentage of the cases McGinness & Wake, unpublished observations). These second round initiations should not prevent detection of termination of the first rounds. but the sensitivity could be lowered. Also, it is possible that they do not t*erminate by 200 minutes in the presence of BrUra as no significant HH was found.) Figure 3 (left panel) also shows that the terminus marker, metB, is present mainly in the LH position confirming that most chromosomes terminate under the conditions of the experiment,.
I
dno-I
‘I
(Tsl
I9
,r A
pur
A
Frociion Pm. 3. Distribution of origin and terminus markers amongst the DNA density species following completion of the single round of replication in the presence of BrUra. The left panel shows the result for TsB134 in which BrUra was added at 90 min (the temperature shift was at 80 min, see Fig. 1, right panel). After separation in a gradient of CsCl, fractions were analysed for purA (0) and m&B (0) activities. The right panel shows the result for dna-I(Ts) in which BrUra was added at 85 mm, following the temperature shift at SO min (see legend to Fig. 2 ; the purA peak fractions gave > 1000 transformants per plate and the points (6) are positioned at the equivalent of 1009 transformants in the analysis).
REPLICATION
AND
DWISION
CYCLE
267
More extensive genetic marker analyses of the 90 to 110 minute samples of Figure 1 (right panel) are considered in Figure 5 along with septation data. The data in Figures 2 and 3 relating to dnu-l(Ts) are discussed later.
(c) Effect of blocking the round of reyplication in TsB134
on subsequent septation
In a preliminary study, in which 12.5 to 50 PM-HPUra was added at 90 or 95 minutes, it was found that the percentage of cells able to form a septum during subsequenb outgrowth was low and about the same for the lowest and highest concentrations of the drug used. But there was a tendency for the outgrowing cells to distort in shape (bulbous forms) upon prolonged incubation following addition of the higher concentrations of HPUra. This was not a problem when up to 100 PM-HPUra was added at 100 minutes or later. Because 20 PM-HPUra is sufficient to inhibit replication almost completely (see previous section) this level was used in the first study of the effect of blocking replication at various stages of the round on subsequent septation. To separate samples of the same mixtures to which BrUra was added at various times (see Fig. 1, right panel) 20 PM-HPUra was added at the same times and portions taken over the 120 to 200 minute period and fixed in formalin. Figure 4 shows the curves describing the percentage of cells at various times containing a septum for each time of addition of HPUra. In the absence of the drug, central septation becomes obvious in a small proportion of the cells soon after 130 minutes; by 160 minutes most have septated. (With longer times of incubation (>180 min) additional septa formed and gave rise to DNA-less cells, but at 160 minutes most cells (87%) that had septated contained just one septum which was central. A more detailed consideration of the positioning of the septum is given below.) When
I
I
I40
1
*
150 160 170 Time (days)
I
180
I
FIQ. 4. Effect of HPUra added at various times during the single round of replication in TsB134 on septation. At the same times as adding BrUra at 456°C in the experiment described in the legend to Fig. 1 (right panel) separate samples (6 ml) were treated with HPUra (20 pM) at 455W. Portions were fixed in formalin at later times for measurement of the percentage of outgrown cells containing a septum. For each point on the graph, 100 cells were scored.
258
T. McGINNESS
AND
R. G. WAKE
chromosome replication is blocked by 20 piv-HPUra during the 90 to 110 minute period it can be seen that reduced levels of septation, appearing at about the same time as the control and levelling off at 160 minutes, are obtained. But the level cycle achieved is higher when the HPUra is added at a later stage in the replication (distortion of shape occurred to an extent of <100/b at 170 min following addition of HPUra at 90 and 95 min). For example, when the drug is added at 90 minutes (the average extent of replication being 30%, see Fig. 2) most cells do not septate by 170 minutes ; but if the round is allowed to progress further (700,; by 110 min) the majority of cells can subsequently form a septum. Again, upon further incubation, additional (asymmetric) septa form and give rise to DNA-less cells (data not shown). The rates of cell length extension for the various times of HPUra addition in Figure 4 were also measured. There was no significant difference in the case of no drug addition and when it was added at 100 and 110 minut’es, the mean cell lengths at. 160 minutes being 7.4*2*0, 7.3k1.8 and 7*3& 2.0 pm, respectively. For HPCra, addition at 90 and 95 minutes the length at 160 minutes was reduced to 5911.6 and 6.6~1.8 pm, respectively (6.812.3 and 6.9i2.1 pm at, 170 min). The relationship between the level of septation obtained following HPUra addition at various times and the extent of chromosome replication achieved at those times was examined in more detail. The samples to which BrUra was added, at the same time as the separate samples of Figure 4 were treated with HPUra for septation measurements, were fractionated into LL and LH species (see Fig. 3) and the pooled fractions analysed for metB as well as the intermediate replicating marker. ZeuA. In Figure 5 the percentage of each of these markers remaining in the LL species (curves) and the amount of septation achieved (solid triangles, from Fig. 4) are plotted against the time of BrUra or HPUra addition. Consider the marker activities first’. As a fork moves past a particular marker in a single round of replication, that ma.rker will remain at the LL position upon subsequent addition of BrUra. There is a low but, constant amount (8*2o/o) of metB in the LL species up until 100 minutes. Obviously, I
90 Time
95
100
105
of BrUm (or HPUm) addiiion
I IO (mln)
FIG. 5. Progress of the single round of replication in TsB134 as measured by ZeuA and metB eplioation and the effect of blocking the round at various stages on subsequent septation. The samples analysed in Fig. 1 (right panel) were separated into LL and LH species as shown in Fig. 3. The pooled LL and LH species were re-analysed for ZeuA ( 0) and m&B (a) activities. The septation data ( A) are taken from the plateau levels in Fig. 4. (-----) Percentage genetic marker in LL.
REPLICATION
AND
DIVISION
CYCLE
259
this arises from those spores that did not initiate a round of replication by 80 minutes. More importantly, the fact that there is no significant increase shows that no rounds terminate before 100 minutes. But some termination has occurred at 110 minutes. (In interpreting the results of this type of experiment more significance has been attached to changes in the relative amounts of genetic marker activity in the various DNA density species than to absolute levels. It is possible that the specific transforming activity is significantly different for LL and LH DNA and to different extents for various markers. Note, for example, that the amount of ZeuA in LL DNA at 90 minutes is less than that of metB.) As expected, the amount of ZeuA in the LL species starts to increase much earlier, from 90 minutes. These results are consistent with the established replication order of these markers (O’Sullivan & Sueoka, 1967). Considering the septation and marker activities in Figure 5 together, the surprising result is that a significant amount of septation (approx. 40%) is obtained in the absence of any termination. It appears that septation can occur once replication has proceeded as far as the ZeuA marker, or somewhat before it. Approximately 30% of the chromosome replicates after ZeuA (Kejzlarova-Lepesant et al., 1975) so that the small amount of replication (5 to 7%) that might have occurred by 160 minutes following 20 p&r-HPUra addition at 100 minutes would still leave aEZchromosomes significantly uncompleted. An additional and more direct experiment using a higher concentration of HPUra in the presence of BrUra has been performed with TsB134 to confirm the finding of central septation in the absence of termination of the round of replication. After shifting germinating TsB134 spores to 455°C at 80 minutes, separate samples at 90, 100 and 105 minutes were split. The smaller portion in each case was mixed with HPUra (40 pM) and BrUra; to the larger, BrUra alone was added. DNA was extracted from the latter at 200 minutes for measurement of the percentage of rrLetB replicated at the time of BrUra addition, and portions were taken at various times from the mixtures containing HPUra for septation measurements. To test directly the effectiveness of the block in chromosome termination by 40 PM-HPUra, it was added along with BrUra to a separate sample at 101 minutes. Small portions were removed for septation measurements, and from the bulk of it DNA wa,s extracted at 200 minutes for analysis of the distribution of metB between the LL and LH density positions. In this last case, there was no transfer (<0*5%) of metB activity to the LH position (purA and metB were coincident at the LL position, data not shown). Thus, no chromosome terminations occurred after BrUra plus HPUra (40 pM) were added at 101 minutes. The metB replication and septation data for the other samples are shown in Figure 6 (see legend for calculation of metB replication). As before no chromosome terminations have occurred by 100 minutes, and it has already been established that 40 PM-HPUra added at 101 minutes blocks subsequent terminations completely. But, again, there is substantial septation, 30 to 40%, when replication is blocked at this stage. In this experiment, an additional sample was treated with 100 +f-HPUra, in the absence of BrUra, at 100 minutes. The amount of septation at 160 minutes was still significant, but reduced, at 27%. There was no distortion in cell shape. The location of the septum in the cells obtained after 40 PM-HPUra addition at 100, 101 and 105 minutes was very similar to the situation where no drug was present. Figure 7 compares the septum location at 170 minutes in the absence of HPUra and in its presence (from 100 min). At least 80% of the septa are central (0.50 to O-55) in both cases. That the central septum which formed in the presence of
260
T. McGINNESS
AND
It.
G. WAKE
,h
50-
_
I I , ,/Septatmn i
% 4030 -
1
/ If 20-
/
IO -
,6
//
/
met e 5’
5%
95
/I 100
105
Time of BrUra (or &Urn +HPUra)adJ~t~n ImInI FIG. 6. Division septation in TsB134 in the absence of chromosome t,ermination. Germinating TsB134 spores were shifted from 34°C to 456°C at 80 min. St 90, 100 and 105 min separate samples were treated in 2 ways: to a larger one (80 ml) BrUra was added and DNA extracted at 200 min for density gradient analysis and fractionation; to the other (10 ml) HPUra (40 pM) plus BrUra were added and portions fixed in formalin at 160 and 170 min for septation analysis. To another sample (80 ml) at 101 min, HPUra (40 pM) plus BrUra were added; portions were fixed in formalin at 160 and 170 min and the bulk used for DNA extraction at 200 min. (0) and (0) are the septation data (100 cells scored in each case) for portions fixed at 160 and 170 min, respectively. The distribution of metB between the fractionated LL and LH species (after correcting for a base level of 9.9% remaining in the LL species in the 90 and 100 min cases) was used to determine the percentage m&B replicated (mean of 4 measurements + average deviation) at t,he times shown.
HPUra
Septum location (froctmn
+ BrUra
of cell length)
FIG. 7. Location of the septum in TsB134 which forms in the absence of termination, The left panel shows the position of the septum in the absence of HPUra and, in the right panel, HPUra was added along with BrUra at 100 min (see legend to Fig. 6). In both cases, cells fixed at 170 min were examined. Mean cell lengths for the total cell populations in the absence and presence of HPUra were 9.0& 2-4 pm and 8.6 i 2.7 pm, respectively.
REPLICATION
AND
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HPUra (added at 100 min) was a true division septum was confkmed by acridine orange staining of nuclear bodies. DNA was always (>95%) found on both sides of this septum, and in at least one third of the cases it remained very close to it, even at 180 minutes. (d) Effect of blocking the round of replication
in dna-1 (Ts) on subsequent septation
Using the same procedure as for TsB134 it was found that, with dnu-l(Ts), no new initiations occurred after transfer to 45.5”C (see Fig. 3, right panel). The crucial experiments suggesting that central division septation can occur in the absence of chromosome termination were therefore repeated with dna-l(Ts) in order to avoid a(ny possible complications in interpretation due to the small but significant number of new chromosome initiations that occurred with TsB134 after transfer to the restrictive temperature. Upon shifting germinating spores of &a-l(Ts) from 34°C to 455°C at 80 minutes the single round of replication proceeds at a slightly faster rate than in the case of TsB134 (see Fig. 2). A preliminary experiment indicated that the first chromosome terminations were occurring between 95 and 100 minutes. Figure 8 summarizes the results of a more extensive experiment in which the progress of replication over the 85
70 -
A
60-
0 &A
0
50-
n
/
40-
r:-o
95/’
Time of BrUra (or HPUro+BrUra
100 I addition
105 bin)
Fra. 8. Division septation in &a-1(Ts) in the absence of chromosome termination. Germinating dna-l(Ts) spores were shifted from 34°C to 466’C at 80 min. At 86, 90,94, 100 and 106 min separate samples were treated as follows : to a larger one (80 ml) BrUra, was added and DNA extracted at 230 min for C&SO4 density gradient analysis (see Fig. 2, upper line) and fractionation in CsCl; to 2 x 10 ml lots, et 94 and 100 min only, HPUra (40 pM) plus BrUra and HPUra (100 pM) plus BrUra were added and portions fixed in formalin at later times for septation analysis. To a further sample (100 ml) at 96 min, HPUra (40 PM) plus BrUra, were added, portions were fixed in formalin at later times and the bulk used for DNA extraction at 230 min. The septation data (0, 40 q-HPUre; n , 100 PM-HPUre) were obtained from the portions taken at ISO min (100 to 200 cells scored for each point). The distribution of metB activity between pooled LL and LH fractions from the CsCl gradients (after correcting for a constant base level of 16*6+O*6°~ remaining in the LL species in the 86, 90 end 94 min oases) was used to determine the percentage metB T o calculate the percentage ZeuA replicated replicated at the times indicated (-- O-- a--). (-A-A-), an amount of 12.6% remaining in the LL species of the 86 min sample was used 84 a baseline correction for all other times.
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to 105 minute period was monitored in the same way as in Figure 6 for TsB134. There was no significant shift of metB from LH to LL over the 85 to 94 minute period. although the constant amount of this marker activity remaining at the LL position was higher than in Figure 6 (16.5% versus 9*9”/“). To a separate sample at 95 minutes, HPUra (40 pM) and BrUra were added. There was no detectable transfer (
4. Discussion It has been shown here that germinating spores of temperature-sensitive DNA initiation mutants of B. subtilis, shifted from the permissive to restrictive temperature after initiation of the first round of replication, afford just a single round which goes on to terminate. About 30 minutes after termination, the first central division septum can be seen under the light microscope. It would be expected that if termination of replication is obligatory for division septation under these conditions, interference with the progress of the first round of replication after germination would block the formation of the central septum. The results presented here, in which HPUra has been used as a specific inhibitor of DNA replication, suggest that this is so only if the round of replication is blocked before a certain stage is reached. At about the time when the chromosome is 70% replicated, on the basis of the behaviour of ZeuB, blocking termination by HPUra no longer prevents central division septation occurring at about its normal time. Presumably, in the previous experiments (Callister Sr Wake, 1977) central septation did not occur at the low level of thymine, which reduced DNA replication to < 10% of its normal rate, because the necessary minimum amount of replication could not be achieved within the time available.
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Clearly, if the B. subtilis spore contains a single chromosome, the central septum that forms when termination is blocked must pass through a partially replicated chromosome. This would be consistent with the observations of Van Iterson & Aten (1976). With germinating spores of B. subtilis to which a low level of mitomycin C was added at a certain time, these workers obtained electron micrographs showing a septum passing through an area of nucleoplasm. They suggested that the division process could proceed in the presence of the replication inhibitor so that the septum could close although the nuclear division clock was arrested. While no electron microscopy has been performed with samples obtained under the conditions of inhibition used here, the observation that in a significant percentage of the cases the DNA remains close to and on both sides of the septum could be due to such a situation. It is not surprising that septation can occur in the absence of chromosome termination, as this is now well established (Mendelson, 1968; Donachie et al., 1971; Sargent, 1975). That the septum can be centrally located, requiring that it “divide” the DNA of a partially replicated chromosome, was unexpected, although Van Iterson & Aten (1976) had already provided evidence for such a situation. Certainly, in the normal situation, there must be a mechanism to ensure the formation of a septum preferentially between two completed chromosomes, but there is no definitive information on how this is accomplished. Perhaps changes in the folding or membrane associations of the nucleoid are involved, so that what is normally achieved at 100% in the situation of replication can occur after a minimum of about 700/ replication continued cell extension. It could well be that segregation of membrane-bound origins, each pulling with it the bulk of the replicated portion of each daughter, could provide the situation that would enable central septation in the absence of termination. It should be kept in mind that under the conditions used in the present experiments, where division septation occurs in the absence of termination, the dnaB gene product is inactive (it is possible that ha-1 is also a B group mutation, but it is not known how many genes comprise this group (White & Sueoka, 1973; Karamata & Gross, 1970)). Sargent (1975) has presented evidence suggesting that the dnaB gene product, under the permissive conditions, functions as a repressor of septation. The present results would be entirely consistent with such a role for this gene product. In the normal situation, termination of replication could act to derepress the dnaB gene product and so trigger the formation of the septum. However, at the restrictive temperature where the repressor activity is destroyed, septum formation at the normal division site (perhaps a site of chromosome attachment to the membrane) might proceed as soon as other prerequisites, not dependent on chromosome termination, are accomplished. This possibility is being investigated. This work has been supported by grants from the University Fund and the Australian Research Grants Committee.
of Sydney Cancer Research
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H., Le Mesurier, S. & Wake, R. G. (1977). J. Bacterial. N. R. (1977). Annu. Rev. Biochem. 46, 641-668. W. D., Martin, D. T. M. & Begg, K. J. (1971). Natwe
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Donachie, W. D., Jones, N. C. & Teather, R. (1973). flynap. Sot. @en. Microbial. 23, 9-44. Dubnau, D., Goldthwaite, C., Smith, I. & Marmur, J. (1967). J. Mol. Biol. 27, 163-185. Karamata, D. & Gross, J. D. (1970). Mol. Gem. Genet. 108, 177-187. Kejzlarova-Lepesant, J., Harford, N., Lepesant, J.-A. & Dedonder, R. (1975). InSpores 1’1 (Gerhardt, P., Costilow, R. N. & Sadoff, H. L., eds), pp. 592--595, American Society fol Microbiology, Washington, DC. Mendelson, N. H. (1968). CoZd Spring Harbor Xymp. Quant. Biol. 33, 313-316. Mendelson, N. H. & Gross, J. D. (1967). J. Bacterial. 94, 1603-1608. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature (London), 204, 1069-1073. O’Sullivan, A. & Sueoka, N. (1967). J. Mol. BioZ. 27, 349%368. Pardee, A. B., Wu, P. C. bt Zusman, D. R. (1973). In Racterial Membran.es and Walls (Lieve, L., ed.), pp. 357-412, Marcel Dekker, Inc.. New York. Pritchard, R. H. (1974). Phil. Trans. Roy. Sot. (London). 267, 303.-336. Sargent, M. G. (1975). J. Bacterial. 123, 1218-1234. Van Iterson, W. & Aten, J. A. (1976). In Microbiology 1976’ (Schlessinger, D. rd.), pp. 128140, American Societ,y for Microbiology, Washington, D.C. Wake, R. G. (1976). .J. Bacterial. 127, 433- 439. White, K. & Sueoka, N. (1973). GenIetics, 73, 185-214.