- Email: [email protected]
On the process of cellular division in Escherichia coli
J. Mol. Biol. (1970) 63, 369-387
On the Process of Cellular Division in Escherichia coli III?. Thermosensitive Mutants of Escherichiu coli Altered in the Process of DNA Initiation YUEINORI
HIROTA, Josh MORDOH AND FR~QOIS
JACOB
Laboratoire de GknBique celluluire de l’lnstitut Pasteur et du Colk?gede France, Paris, France (Received 10 Februuy
1970, and in revised form 10 June 1970)
Genetic and physiological properties of a temperature conditional mutant of Eecherichia coli K12 are described. After a shift from 30°C to 41”C, the mutant exhibits a residual synthesis of DNA, which stops after an hour. All the characteristics of the mutant indicate that at high temperature, it can complete a DNA cycle already started, but not initiate a new one. After shift back from 41°C to 3O”C, protein synthesis is required in order for DNA synthesis to resume. Reinitiation of DNA synthesis appears always to occur in the same area of the bacterial chromosome. In correlation with this alteration in DNA initiation, several lines of evidence indicate an alteration in the structure of the cell membrane.
1. Introduction The cell division cycle of Es&e&h&z coli involves the regulation of many cellular activities, including DNA initiation and replication, formation of the bacterial membrane and equipartition of DNA copies. Little is known about the co-ordination of these operations, and in particular about the way in which the rate of DNA synthesis might be co-ordinated with the growth rate. There is some direct evidence that changes in the rate of DNA synthesis are determined primarily by changes in the frequency of initiation (Oishi, Yoshikawa & Sueoka, 1964; Maalee & Kjeldgaard, 1966; Helmstetter & Cooper, 1968; Bird & Lark, 1968). The replicon model (Jacob, Brenner & Cuzin, 1963) predicted that a replication cycle of the E. coli chromosome is initiated at a specific point on the chromosome, and that replication follows in the replication machinery, which is integrated as a part of the membrane structure. Thus, the bacterial membrane could possibly regulate in some way the functioning of these processes. A class of thermosensitive mutants is known in which initiation of DNA synthesis appears to be blocked at high temperature. Isolation and some preliminary characterization have already been described (Kohiyama, Lamfrom, Brenner $ Jacob, 1963; Kohiyama, 1968; Hirota, Jacob, Ryter, Buttin & Nakai, 1968; Hirota, Ryter & Jacob, 1968). Evidence for alteration in membrane structure of such mutants is presented in paper II of this series (Shapiro, Siccardi, Hirota t Jacob, 1970). In the t Paper II in this series is Shapiro, Siccardi, Hirota & Jacob, 1970. 369
370
Y.
HIROTA,
present paper, the evidence synthesis is reported.
J. MORDOH
supporting
AND
F. JACOB
a defect in the process of initiation
of DNA
2. Materials and Methods (a) Bacterial
strains
The following strains of E. co&i K12 have been used: CRT46 : F- Thr- Leu- ThyB,- Ilv- lac,- Mal- T46s Xr : F- Thy- lac - T46S hS derived from CRT46 after cross with Hfr P13 (Jacob Rc CRT464 Wollman, 19y61). HfrT46 : IlvThyT46S Hfr, injects its chromosome in the order: O-ProA-LeuThr- - - lac-F AT1243 : PyrEMet- Hfr, injects its chromosome in the order: 0-Thr-Leu-lac - - -MetF (Ramakrishnan & Adelberg, 1965). CRTS3 : F- Thr- Leu- B,- Ilv- Mtl- T838. (b) Media Nutrient broth contained 10 g of polypeptone, 10 g of meat extract, and 5 g of NaCl in 1 litre of water adjusted to pH 7.0 with KOH. For growth of thymineless bacteria, 60 pg of thymine/ml. were added. Minimal eosine methyleno blue (EMB) medium was prepared as described by Lederberg (1950). Minimal synthetic medium 63 (Monod, Cohen-Bazire & Cohn, 1951) supplemented with 0.3% glucose, 60 pg thymine/ml., 0.1 pg thiamine/ml. and 60 pg each of L-threonine, L-leucine, L-isoleucine and L-valine/ml. was also used. Casamino acids medium was prepared, supplementing the same medium 63 with 0.3% glucose, 0.3% Casamino acids, 0.1 rg thiamine/ml. and 60 rg thymine/ml. (c) Isotope Two
methods
(i) Measure
incorporation
and radioactivity
assay
were used:
of total
amount
of DNA
synthesis
Cultures were grown overnight in the media previously described, to which [l%]thymine (C.E.A. France, spec.act. 43.3 me/m-mole) was added to a final concentration of 1 @/ml. The next morning cultures were diluted 1 to 20 in the same medium containing the same concentration of [14C]thymine. The experiments were carried out when the cultures were in the exponential phase. l-ml. portions were added to 5 ml. of ice-cold 5% trichloroacetic acid and stood on ice for 1 hr. They were then filtered through HA Millipore filters and washed with 30 ml. of 5% trichloroacetic acid. Filters were dried and counted invials containing 10 ml. of toluene-PPO-POPOP mixture (Packard Instrument Co.) (11. : 3g : 0.3 g) in a Packard Tricarb scintillator. (ii) Measure
of the rate
of DNA
synthesis
To 2 ml. of the bacterial culture, a pulse was given of [3H]thymidine (C.E.A. France, spec.act. 18 c/m-mole) with a final concentration of 4 PC/ml., adding 1 rg of cold thymidine/ml. Further manipulations were carried out as previously described. (d) Cesium
chloride
density-gradient
centrifugation
of DNA
labelled
with
[3H]thymidine
and
heavy isotopes Procedures for radioactive pulse labelling of DNA followed by density labelling, extraction of DNA and CsCl centrifugation were a modification of those described by Lark, Repko & Hoffman (1963). An overnight culture of CRT46 in synthetic medium 63 supplemented as described above was diluted in fresh medium to adjust the cell concentration to 4 x lo7 cells/ml. Two lo-ml. samples of this suspension were incubated at 30°C for about three generations (generation time, 100 min). (i) Thymidine
pulse labelling
40 PC of [3H]thymidine (C.E.A. France, spec.act. 18 c/m-mole) were added to the cultures for a given time, one before (control) and the other after a shift to 41°C (experimental). After this pulse, the cells were quickly washed by filtration through a Millipore filter with
THERMOSENSITIVE
DNA
371
1NITIATION
100 ml. of the same medium containing 50 pg of cold thymidine/ml. Cells were collected and resuspended in 20 ml. of non-radioactive medium. They were kept at 21°C for overnight growth without shaking, in order to randomize growth. The next morning, the cultures had reached a density of 4.3 x lo* cells/ml. 6-ml. samples from each culture were added to 14 ml. of fresh medium and incubated at 30°C for about one more generation. Final density of the culture was 3.2 x 10s cells/ml. (ii) Density
label&g
Both cultures were shifted to 41°C for 1 hr. After this treatment, cells were harvested on Millipore filters, and washed with 100 ml. of medium 63 without NH,Cl. Cells ware resuspended in 20 ml. of medium 63 supplemented as-described above, with the exception that NH,Cl and glucose had been replaced by 2 mg each of 15NH,Cl/ml. (97.2 atom y. as r5N, Isomet Corporation) and [2H]glucose/ml. (98.2 atom o/o as 2H, Volk Radiochemical Co.). Cultures were shaken at 30°C and after various times (20, 40 and 60 min), g-ml. portions were taken. Cells were harvested by centrifugation at low temperature and washed once with 0.01 M-Tris buffer, pH 7.5, containing 0.001 M-EDTA. (iii) D,VA extraction and CsCl eentrifugation The pellet was resuspended in 0.45 ml. of the same buffer, and 0.05 ml. of 25% Sarkosyl was added. After 5 min at room temperature, this mixture was diluted tenfold with buffer, and 0.05 ml. of a 10 mg/ml. protease solution (from Streptomyces g&sew, type VI, Sigma Chem. Corp.) were added. After 30 min at 37’C, the same amount of protease was added again and incubation was continued for another 3 hr at 37°C. Cells debris were removed by low speed centrifugation. The supernatant fraction was removed, and 2.24 ml. were mixed with 2.861 g of cesium chloride (The Harshaw Chem. Co.). Centrifugation was carried out at 25,000 rev./m& 20°C; 3 days, in the SWBOLl swinging bucket rotor of the Spinco model L preparative ultracentrifuge. l-drop fractions were collected from the bottom of the tubes directly into an aqueous scintillation fluid (Pritchard BE Lark, 1964) and radioactivity was estimated in a Packard Tricarb scintillator after overnight cooling in the dark.
(e) Estimation
of cell number
A Coulter counter model F (Coultronics France, S.A.) was used. The counter was used to estimate the total cell number, a fraction of which may have no colony-forming capacity. It was operated with a 20-p diameter orifice, maximum amplification and a current setting of 5.8 and 11. Coulter counter readings were maintained in the 2 to 20 x lo3 range after dilution in normal saline which had been filtered through a type HA Millipore filter. (f) Measurement of fluorescence intensity
and polarization sulphonate
of I-aniline-8-naphthalene
Fluorescence intensity and polarization of 1-anilino-8-naphthalene sulphonate measured according to the method of Monnerie & Neel (1969). Portions of the cultures taken at different times of incubation, centrifuged at low speed and washed twice chilled medium 63. The cells were resuspended in the same medium, and intensity depolarization were followed after 1-anilino-8-naphthalene sulphonate was added concentration: 0.05 M). The wavelength of excitation was 365 rnp and fluorescence observed at 483 mp; in both instances, interference filters were used.
were were with and (final was
3. Results (a) Properties
of mutant CRT46
.
As described in a previous paper (Hirota, Jacob, Ryter, Buttin & Nakai, 1968) after a shift from 30 to 40°C total DNA synthesis in a culture decreases progressively for about 60 minutes and then stops. The increase in DNA content at 41°C is about 30 to 40%. After the arrest of DNA synthesis, the optical density continues to increase linearly and proteins continue to be synthesized. During the same time, there is a
372
Y.
HIRCTA,
J. MCRDOH
AND
Y. JACOB
rapid decline in viability. Cell division proceeds at 41°C for a few hours at a decreasing rate after the arrest of DNA synthesis (Fig. 3(b)). This results in a decreased number of nuclei per cell and the production of DNA-less bacteria. Two classes of thermosensitive mutants are known in which DNA synthesis is disturbed: (1) one in which the temperature shift results in an immediate stop in synthesis; (2) the second only after a residual synt’hesis. Mutant CRT46 belongs to the latter class. In order to determine whether the synthesis of any genct,ic element or only of the chromosome was affected by the mutation, bacteria previously incubated at 41°C for 60 minutes were infected with phage h. The results of this experiment are given in Table 1. The burst size at 41 “C was 510, as compared with 56 in the control at 30°C. TABLE
1
Production of yhage lambda by CRT464 celb at 30°C and 41°C
Input phage adsorbed Infectious centres Phage released Burst size
after 10 min (%)
30°C
41°C
98 1.2 x 10’ 6.7 x 10s 66
99 1.3 x 107 6.6 x 108 510
An exponentially growing culture of the mutant CRT464 in broth supplemented with 0.1% maltose at 30°C was used. At 2 x 10s cells/ml., the culture was divided into two parts. One was shaken at 3O”C, the other at 41’C. After 90 min, to O-9 ml. of each culture was added 0.1 ml. of h suspension containing 1.3 x lo* phages/ml. (time 0). The mixtures were maintained at 30 and 41”C, respectively, and at 10 min phage adsorption was stopped by dilution without temperature change. At various times, samples were assayed for plaque formation on E. coli K12 C600 at 37%. Unadsorbed phage was measured after killing the cells with chloroform.
It is concluded therefore that mutation CRT46 stops DNA synthesis only in the chromosome. Mutant CRT83 behaves in the same way (Kohiyama et al., 1963). Other mutations of class 2, i.e. with residual DNA synthesis at high temperature, are known which are unable to reproduce phage h at high temperature (Hirota, Ryter & Jacob, 1968; Fangman & Novick, 1968). By this criterion, a subdivision can therefore be introduced among mutants of class 2. The subclass to which mutants CRT46 and CRT83 belong exhibits the properties which can be expected from a lesion in a specific initiation process of chromosome replication. (b) Effects of cultural condition
on mutant CRT46
(i) Effect of temperature Residual synthesis of DNA was measured after shifting a culture growing at 30°C to various temperatures: 39.0, 41-O and 42.5%. The rationale of the experiment was as follows: if the initiation process is affected in this type of mutant, the residual synthesis of DNA should be somewhat independent of temperature once the denaturing temperature has been attained, the opposite being true if the replication enzyme were the element affected. The results are shown in Figure 1. It can be seen that there is a little escape at 39”C, but that at 41.0 and 425°C the amount of residual synthesis remains unchanged.
THERMOSENSITIVE
DNA
t-----------High
0
373
INITIATION
tempera~“re-------
I
I
-60
60
I
120
Time (min)
FIO. 1. Residual synthesis of DNA at different temperatures. C!u&rea of CRT40 growing exponentially at 30°C in different media were shifted to high temperstares (--O--O-, 39-O%; --A--A--, 41.0°C; --m--n--, 42PC). The amount of DNA was measured by [14C]thymine incorporation 8s described under Materials and Methods.
(ii) Effect of composition
of medium
There are reasons to believe that the rate of DNA synthesis in a culture is controlled, not by the speed at which the replicating enzymes work, but by the number of replicating forks working at constant speed. Bacteria grown in rich medium would thus show more rapid DNA synthesis because of more frequent re-initiation (Yoshikawa, O’Sullivan & Sueoka, 1964; MaaIoe $ Kjeldgaard, 1966; Helmstetter & Cooper, 1968). If CRT46 mutation does result in an alteration of DNA initiation, the amount of DNA synthesized at 41°C should depend on the medium. Since a cell grown in rich medium contains more replicating forks than a cell grown in a poor one, the former should synthesize more DNA at 41°C than the latter. This is the case, as can be seen in the results of the experiment reported in Figure 2. (iii) Resumption of DNA eynt?hesisupm 8hififmn
41°C to 30°C
After one hour at 41”C?,mutant CRT46 is unable to synthesize DNA. The resumption of DNA synthesis when the culture is returned to 30°C was studied. The results of such an experiment are reported in Figure 3(a). In the control at 30% the rate of
374
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
Time (min) FIG. 2. Residual synthesis of DNA in different media. Cultures of CRT46 growing exponentially at 30°C in different media (-O-O-, minimal synthetic medium; --n-o-, Casamino acids medium; --A-A-, nutrient broth) were shifted to 41°C and the residual increase of DNA was measured by [14C]thymine incorporation as described under Materials and Methods. The values are normalized to the values at zero time. The compositon of the media is described under Materials and Methods. The generation times of CRT46 in these media at 30°C are approximately 100 min (minimal synthetic medium), 86 min (Casamino acids medium) and 62 min (nutrient broth).
DNA synthesis, measured by the incorporation of [3H]thymidine into the trichloroacetic acid-insoluble fraction during a series of three-minute pulses, increases exponentially. In the population kept for 60 minutes at 41°C and then shifted to 3O”C, however, after a small lag, this rate increases, reaches a plateau and then decreases. After 100 minutes, i.e. approximately the generation time under these conditions, whereas the rate of DNA synthesis has decreased, the rate of cell division increases sharply (Fig. 3(b)). In order to determine whether protein synthesis is required for resumption of DNA synthesis, chloramphenicol was added to a sample of the culture when shifted from 41 to 30°C: no DNA synthesis was then observed. The resumption of DNA synthesis upon a shift from 41 to 30°C can also be measured in the following way. A population of CRT46 grown at 30°C is shifted to 41°C for 60 minutes. It is then shifted back to 30°C. At various times, samples are returned to 41°C and the amount of residual DNA synthesized is measured. As shown in Figures 4 and 5, the DNA content increased up to 300% of the original amount when the culture is allowed to stay 60 minutes at 30°C before being transferred again to
(b)
Time (mid
120
I80
0
60 CC)
120
I(
FIG. 3. Resumption of DNA synthesis, cell division and cell growth after 1 hour at 41°C. (a) An exponemially growing culture of CRT46 in minimal synthetic medium at 36°C was shifted to 41°C for 1 hr, and then put back to 30°C (time 6) or 200 pg/ml, (-n-a--). Two CultureS Were used either in the absence (-O-O-) or in the presence of chloramphenicol, 100 ~g/ml. ( ~n-n--) ), The rate of DNA synthesis as controls, ono growing at 36°C all the time (-m-m-) and the other kept at 41°C throughout bhe experiment (-0 0 W&S measured by [3H]thymidine incorporation (see Materials and Methods). (b) CRT46 growing exponentially in minimal medium at 30°C was treated as doscribed in (a), and cell number was measured as described under Materials and Methods. Symbols are the same as in (a). (c) A culture of CRT46 growing exponentially in minimal medium at 30°C was divided into three part’s: one culture was kept at 30°C all the time and another shifted to 41°C for 1 hr and then put back to 30°C (-m-m--), another was shifted t,o 41°C and kept at that temperature (-O-O-), (-e-e--). Optical density of the cultures was followed with a Zeiss spcctrophotomctcr (type PM& II) at 550 mp.
(a)
60
I c
I
: 50
I
I20
I
180 Time (min)
240
FIG. 4. Residual synthesis of DNA at 41°C after a previous temperature shift treatment. A culture of CRT46 growing exponentially at 30°C in minimal synthetic medium was shifted to 41°C for 1 hr. After this treatment, it was put back again to 30°C (-O--O-), and at different times, indicated by arrows, portions were replaced at 41% (------). The time at 30°C before being shifted at 41°C were, for the different samples : 6 min (--Q--m--) ; 20 mm (-- 0 -- 0 --) ; 40 min (--A -- A--); 60 min (--V --V --); 36 min (-- l -- a--); 105 min (-- n -- n --) was kept at 41’C all the time. DNA syn120 min (--A--A--). One sample (-*-a-) thesis was measured by [r*C]thymine incorporation as described under Materials and Methods.
I
I
I 1 I Beginning of cell division
1
I
1
I
I
I
20 40 60 60 80 100 I20 0 Time at 30°C before shift bock to 41°C (min)
80
100
120
500
2
AI, 0
I 20
40
(a)
I
I
I
w
(b)
Fxa. 5. DNA synthesis at 41’C after a previous temperature shift treatment. The data used for this Figure are those of the experiment reported in Fig. 4. (a) The results are expressed as percentage of DNA increase during the last 41°C treatment, over the amount found immediately before every last shift to 41°C at various times taken as 100. (b) The results are expressed as percentage of DNA increase at 41”C, over the amount foundat the time (80 min) of the shift down from 41 to 3O”C, taken as 100. The beginning of cell division was determined in an experiment similar to that reported in Fig. 3(b).
THERMOSENSITIVE
DNA
377
INITIATION
41°C. These figures may be easily understood if mutation CRT46 blocks re-initiation of DNA synthesis at high temperature. During the first 30 minutes at 30”, all cells would progressively restart DNA synthesis ; every chromosome initially completed at 41°C would initiate replication twice or more after 80 minutes at 3O”C, i.e. before cellular division has occurred (compare time in Figs 3(b) and 5). (iv) Comparison of the effect of inhibition DNA
synthesis and of CRT46 mutation on
ofprotein
synthesis
In wild-type E. co&, inhibition of protein synthesis, either by amino acid starvation or by chloramphenicol addition, results in an arrest of DNA replication after a residual synthesis of about 40%. This was interpreted as a requirement for protein synthesis for the re-initiation of a new DNA cycle (Maalee t Hanawalt, 1961; Lark et al., 1963). The results previously reported with mutant CRT46 point to a similar interpretation. In order to compare the temperature shift and amino acid starvation effects, the following experiment was performed. Mutant CRT46 (which requires threonine and leucine) was grown at 30°C in minimal medium containing the two amino acids. The culture was then incubated at 30°C without the two amino acids. After a residual synthesis of aproximately 40%, DNA synthesis stopped. The culture was then divided in three parts. Part 1 was incubated at 30°C in the presence of the amino acids ; part 2 at 41°C in the presence of the amino acids; part 3 at 41°C in the absence of the amino acids. If the same process of DNA initiation is altered by amino acid starvation
',5 -
,o -
..5-
Od
I
I
60
I
I
I20
180
240
Time (mid Fm. 6. DNA synthesis at 41°C after amino acid starvation at 30°C. A culture of CRT46 was grown at 30°C in minimal supplemented medium. At a density of 4 x lo* cells/ml., the culture was shifted to a starvation medium (time 0). Synthetic medium 03 supplemented with 0.2% glucose, 6 pg vitamin B1 and 60 pg of each Gsoleucine, L-valine and thymine/ ml. was used for amino acid starvation. After 2 hr at 30°C; the culture was divided into three portions: one was incubated in the same medium at 41% (-n--O--); the other two were supplemented with 50 pg/ml. of both L-leucine and z-threonine, and incubated at 30% (- O-O-) or 41’C (--A--A--). [‘W]Thyrnine was added at time 0 (final concentration, 2 q/ml.) and radioactivity was measured as described under Materials and Methods.
378
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
as well as high temperature, one would expect no DNA synthesis in part 2. As shown in Figure 6, this is exactly what is observed, in opposition to what was found by Fangman & Novick (1968) with another mutant. This result suggests that the two phenomena, found either after amino acid starvation or at high temperature in CRT46, belong to the same process. CRT46 affects the properties of a protein the requirement of which for DNA initiation is evidenced by amino acid starvation. (c) Place of re-initiation
If CRT46 blocks initiation of DNA cycles at 41”C, a shift back to 30°C should initiate synthesis always in the same area of the chromosome. To investigate this possibility, a culture was submitted to two successive re-initiations, with overnight growth between them. Two parallel cultures of CRT46 in synthetic medium were prepared, one of them serving as a control. The only difference between the control and the experimental cultures was the moment at which the [3H]thymidine pulse was given. The cultures grown at 30°C were maintained at 41°C for 60 minutes, and then shifted back to 30°C. The control was given a six-minute pulse of [3H]thymidine (see Materials and Methods) immediately before the temperature shift up, whereas the experimental culture received a 12-minute pulse eight minutes after returning to 30°C (see Fig. 3(a)). The amount of label incorporated in both cultures was essentially the same and corresponded to about 5% of the chromosome length. After appropriate washing, the cultures were grown overnight in minimal medium for four generations, and were incubated again at 41°C for 1 hour. After this treatment, they were returned to 30°C in the presence of heavy isotopes. At various times (20,40 and 60 min) samples of both cultures were taken, lysates prepared and DNA analysed in a caesium chloride density-gradient (see Materials and Methods). The results of this experiment are shown in Figure 7. In the control culture, where the chromosomes were labelled at random, the radioactivity migrates progressively from the light to the hybrid band. After 60 minutes, which corresponds to half a generation time, there is an equal amount in both peaks. In contrast, in the population maintained at 41°C before the pulse, the radioactive material enters the hybrid peak much earlier ; after 20 minutes, 65% is found in the hybrid peak. This result indicates that after two successive reinitiations, DNA synthesis occurs in the same area of the chromosome. This is the result which is expected if CRT46 mutation inhibits initiation at high temperature. (d) Alteration of cell surface According to the replicon model (Jacob et al., 1963) the cell membrane would be the site where DNA initiation and replication take place. Two approaches were used to investigate the properties of the cell surface of CRT46. First, it is known that some mutants, suspected to have altered membrane properties, such as some clones tolerant to colicin E2 (Nomura, 1964), are sensitive to concentrations of sodium deoxycholate which do not affect the wild type (Nagel de Zwaig $ Luria, 1967). Mutant CRT46 and its parent CR34 were therefore compared as to their relative susceptibility to various concentrations of sodium deoxycholate at 30°C and 41°C. The presence of deoxycholate at a concentration of 0.5% does not affect the growth of the parent CR34, either at 30°C or at 41°C. In contrast, as shown in the results of the experiment reported in Figure 8, the growth of the mutant CRT46, while not altered at 30°C; is affected at 41°C. The cells lyse in the presence of only 0.1% of deoxycholate, but only after one hour at 41”C, indicating that membrane
1
Fractian no.
60
I
70
40 min
T
40
50
60
70
60 min
PIG. 7. Caeaium chloride density-gradient analysis of the re-init.iation place of CE!I’46. The detailed experimental procedure is described under Materials and Met)hods and in the test. Two exponentially growing cultures of CRT46 at 30°C in minimal medium were shifted to 41’C for 1 hour and then replaced at 30°C. The control culture (-O--(j--) received a 6.min. [%]thyrnidine pulse immediately before the temperature shift to 41°C; and the experimental cult,ure (-a--•-.-) received a 1%min [?H]thymidine pulse 8 min after returning to 30°C. After overnight growth, both cultures were shifted again to 41°C fur 1 hr, and replaced at 30°C in synthetic medium containing [2H]glucose and *5NN&I. Saqbs were taken at 20, 40 and 60 min, and the lysatcs were subjected to density-gradient analysis. Density decreases in the Figure from left to right.
r
80
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
Time (mid
Fro. 8. Deoxycholate sensitivity from CRT46 at 30°C and 41% A culture of CRT46 growing exponentially at 30°C in broth supplemented with thymine was transferred to the same medium either in the presence of deoxycholate (final concentrations 0.1 and 0.5%) or in the absence of detergent. The cultures were incubated at 30°C (solid symbols) or 41°C (open symbols) with gentle shaking. Detergent concentrations: 0, 0, 0%; n , 0, O-1 %; A, A, 0.5%. Portions were taken at different times and optical density was measured at 550 rnp with a Zeiss spectrophotometer PMQII.
alteration does not occur immediately but after some time of metabolism. Although the mode of action is not very clear, this result suggests some alteration in the surface properties of the cell. The second method was suggested by Dr B. Shapiro, It takes advantage of the fact that l-anilino-8-naphthalene-sulphonate gives rise to a weak fluorescence in water solution and to a strong one in protein solution by fixation to hydrophobic bonds of the latter (Weber & Young, 1964; Stryer, 1965). When mixed with normal CR34 bacteria, at either temperature, or with the mutant CRT46 at low temperature, the probe produces a weak fluorescence. With the mutant at 41”C, however, a significant
THERMOSENSITIVE
DNA
INITIATION
381
Time (mid
FIG. 9. Fluorescence of CR34 and CRT46 bacteria measured in the presence of l-anilino-8, naphthalene sulphonate. E. coli CRT46 and its parental strain CR34, growing exponentially at 30°C in broth supplemented with thymine, were each divided into two parts (time 0): one remained at 30°C and the other was placed at 41°C. Samples were taken at different times, centrifuged in the cold and washed twice with cold M63 medium. Cell mass of bacteria was adjusted to an optical density of 0.5 at 650 rnp (Zeiss spectrophotometer, PM& 11) in all samples, and fluorescence was measured at 25°C after adding the sulphonate to the bacterial suspensions as described under Materials and Methods. In the arbitrary scale shown in this Figure, three units correspond to the fluorescence of 1 pmole of sulphonate bound to bovine serum albumin in the same conditions. -O-O-, CR34, 41°C; --A--A-CR34, 30°C. CRT48, 41°C; -.--.--, CRT46, 30°C; --A-A--,
increase in fluorescence is observed. It can be detected only after a lag of about 60 minutes, and then the fluorescence increases linearly (Fig. 9). In all cultures, more than 90% of the fluorescent material sediments at low-speed centrifugation (3000 rev./min), indicating that it is bound to the cell itself. From the results shown in Figure 9, fluorescence depolarization was calculated (Kasai, Changeux & Monnerie, 1969). It was observed to increase from 0.19 (30°C) to 0.27 (4 hr at 41°C) in the mutant. Results with both deoxycholate and I-anilino-8-naphthalene aulphonate indicate therefore a change in the organization of the cell surface. In both oases, something, which is masked in the parent at both temperatures or in the mutant at low temperature, has become accessible in the mutant kept at high temperature. This change, however, does not affect some of the properties of the cell surface; for example, the capacity to adsorb T6 and h phages and the specific permeability to /3-galactosides. That the change involves an alteration of the membrane is evidenced by the tiding that, when analysed by electrophoresis on acrylamide gels, one of the membrane proteins exhibits modified properties at 41°C (Shapiro et al., 1970). It is concluded therefore that the perturbation of DNA initiation and the alteration in membrane proteins found at high temperature in mutant CRT46 are associated. However, while the arrest of DNA initiation is observed immediately after a shift to 41”C, the change in membrane properties found with the two techniques used is detected only after
382
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
60 minutes at 41°C. No significant change in deoxycholate sensitivity or in fluorescence was observed in other types of DNA mutants making filament,s nor after thymine starvation of the parental type. (e) Genetic analysis of mutants CRT46 and CRT83 In all the properties examined so far (Kohiyama, 1968; Shapiro et al., 1970), mutant CRT83 behaves in exactly the same way as CRT46. The genetic analysis of both mutations was therefore performed in parallel. (i) Conjugation Preliminary experiments have located CRT46 in the isoleucine-valine (11~) region of the chromosome (Hirota, Jacob, Ryter, Buttin & Nakai, 1968; Hirota, Ryter & Jacob, 1968). More precise mapping was carried out by crossing CRT46 (F-) with a thermoresistant Hfr carrying various markers. Recombinants for nutritional markers or thermoresistance were selected for, and their genetic constitution analysed (Table 2(a) and (b)). The results indicate that T46 is located close to PyrE-Ilv and MetE, the probable order being: -PyrE-T46-Ilv-MetE. An Hfr strain is known in which F is integrated between the Mtl and Ilv loci, so t’hat the chromosome is transferred in the order 0-PyrE-Ilv. . . Xyl-Mtl (Jacob & Wollman, 1961). Kinetic transfer experiments indicate that the CRT46 marker is t,ransferred into the F- cell shortly after PyrE and shortly before Ilv. CRT83 is located very close to CRT46. In order to study their linkage, a cross between an Hfr carrying the T46 allele and an F- carrying T83 was performed. The results are given in Table 3. The results indicate a strong linkage between the two mutations, the most likely order being Mtl-T46-T83-Ilv. (ii) Transduction More precise mapping of CRT46 and CRT83 was done by Pl transduction, with the use of markers known to be in the area, such as ChlB (Puig $ Azoulay, 1967), Tna (Gartner & Riley, 1965), PhoS (Echols, Garen, Garen & Torriani, 1961) and several independent Ilv (Ramakrishnan & Adelberg, 1965). R esults are summarized in Table 4. T46 is closely linked to Ilv, PhoS and Tna but not to ChlB. The most probable order is T46-Tna-PhoS-Ilv. By transduction, the T83 mutation exhibits a similar degree of linkage to Ilv as T46. The most likely order of markers in the area is represented in Figure 10. T46 and T83 appear to be very closely linked. Both appear to have the same alteration of some membrane protein (see Shapiro et al., 1970). Although technical difficulties have hitherto prevented complementation analysis, it seems reasonable to associate them in designating the chromosomal segment where both are located as DnaA. It remains to be seen whether only one or several cistrons are involved in this segment. One should note that among recombinants or transductants, a complete correlation was observed between thermosensitivity and susceptibility to deoxycholate with both T46 and T83 mutations. Although the existence of two mutations, one affecting DNA initiation, the other membrane properties, cannot be completely excluded as yet, it appears very unlikely.
THERMOSENSITIVE
DNA TABLE
383
INITIATION
2(a)
Segregation of themosensitive characters in a cross HfrAT1243
(1)
Ilv+
SmP
Thy
196
Sm
(L)
PyrE
Ilv
MetE
115
196 (100) 392 (85)
156 (79) 270 (59)
(59) (2)
T46+ Sm’
CRT46 Sm’
Frequency of male markers found among recombinants (%)
No. of colonies analysed
Selected markers
x
459
(i.7)
388 (85)
Thre-Leu
T46t 158 (81) 459 (100)
t Determined by the capacity to grow on complete medium at 41°C. Bacteria F- CRT46 SmP and HfrAT1243 exponentially growing at 30°C in broth supplemented with thymine were mixed (lHfr/lOF-) in the same medium. After 2 hr at 3O”C, the cultures were diluted and samples plated on appropriate media. The plate for selection 1 was incubated at 30°C. That for selection 2 was first incubated for 2 hr at 30°C and then at 41°C. Recombinant colonies were isolated from selections 1 and 2 (196 and 459, respectively), purified, and tested for their characters by replica plating on suitable media. HfrAT1243: MetE- PyrE-, injects its chromosome in the order : O-Thre-Leu-lac-Try---His-PyrEM&E-B,. CRT46 Sml‘: Thy- Ilv- Thr- Leu-. TABLE
2(b)
Linkage of T46 locus with PyrE, Ilv and MetE in the cross AT1243-f x CRT46 Sm’ Recombinants
PyrE
(1) Ilv+ Smr
1 0 1 1 0 1 0 0
T46
Ilv
42 19 0.5 15.3 17.4 0.5 4.1 1
1961 1 0 1 1 0 1 0 0
Total
No. of recombinants (%) 83 37 1 30 34 1 8 2
Total
(2) T46+ Smr
MetE
1 1 0 1 0 0 I 0
224 32 8 116 6 40 20 13
52 7.4 1.8 27 1.4 9.2 4.6 3.0
4331
t HfrAT1243: Thy+ SmaPyrE-T46+Ilv+MetE-Thr+Leu+ : 11111111 CRT46 Sm’: Thy-SmrPyrE+T46SIlv-MetE +Thr-Leu: 00000000 3 The total number of recombinants examined was 196 in exp. 1 and 433 in exp. 2. Genetic markers of all the recombinants from the crosses summarized in Table 2(a) were scored and classified. Alleles of markers derived from the Hfr parent are represented by 1, and those derived from the F- parent by 0. 26
384
Y. HIROTA,
J. MORDOH
TABLE
Number
AND
F. JACOB
3
of colonies formed in a cross, HfrT46
Selections
x CRT831
No. of recombinant colonies formed per ml. of mating mixture
Rat’io of recombinant formation (%)
3.4 x 10’ 8.0 x lo1 3.8 x lo3
100 0.0002 0.01
(1) Sm’Mtl+ (2) SmrMtl+T46+T83+ (3) SmrT46+T83+
HfrT46: Ilv-T46sT83+Mtl+Sms injects chromosome in the order: 0-ProA-TL-Ilv-Mtl --- lac F. CRT831: Il~-T83~T46+Mtl-Sm~Thr-Leu-B,-Thy-lac,-F-. Bacteria HfrT46 and F- CRT831, exponentially growing at 30°C in broth supplemented with thymine were mixed (1: 10) in the same medium, and incubated at 30°C for 3 hr without shaking. Cells were washed with medium 63 twice and plated on media listed below at 30°C or 41%. Selection 1: EM synthetic agar plus 1% mannitol, L-threonine 50 pg/ml., L-leucine 50 pg/ml., vitamin B, 5 pg/ml., thymine 50 pg/ml., L-isoleucine 50 pg/ml., L-valine 50 pg/ml. and streptomycin 100 rg/ml. at 30°C. Selection 2: The same medium as above but incubated at 41%. Selection 3: EM synthetic agar plus 1% glucose, L-threonine 60 pg/ml., L-leucine 60 ag/ml., vitamin B1 5 ag/ml., thymine 50 pg/ml., L-isoleucine 50 pg/ml., L-valine 50 pg/ml., and streptomycin 100 pg/ml. at 41°C. Plates were examined after 2 to 3 days of incubation.
TABLE
Frequency of joint-transductions Donor
CRT4619 (Tna-PhoS-)
Recipient
HfrT46 (Il~-T46~)
DnaA
4
of markers located near DnaA
Tna
PhoS
Ilv
l.ocus
Selections
11v+
4%)
Ilv+ and T46 +
(l&) AB2151 (Tna-Ilv-)
CRT46 (T46’Ilv-)
CRT4610 (T46’)
AB2151 (Ilv-Tna-)
115 (100%)
11v+
CRT833 (Ilv-T8SB
AB2151 (Ilv-Tna-)
100 (100%)
11v+
T46 +
Suspensions of phage PlKc were prepared by infection of various donor strains grown overnight at 30°C in broth supplemented with thymine. The recipients were infected according to Lennox (1955). EM sugar agar (Lederberg, 1950) supplemented with 0.15% of sodium citrate and required nutrients of recipient bacteria was used for the selection medium. Transductants were selected on a medium deprived of isoleucine and valine (selection of Ilv+) at 30°C or with the full supplements of required nutrients at 41°C (selection of T46+).
THERMOSENSITIVE
XYl I
Mtl II
:o I 71
DNA
PyrE I Hfr PI3
I
72
DnaA j PhoS IIII II
I
73
385
INITIATION
Ilv II
MetE I
1 I
74
75 min
FIG. 10. A tentative map order of DnaA on the E. coli chromosome. Relative map position of DnaA on the E. cold chromosome is schematiortlly given with the other reference markers used. Numbers under the map indicate the relative time position of reference markers according to Taylor & Trotter (1967).
4. Discussion In the replicon model (Jacob et al., 1963) it was assumed that both the initiation of a DNA cycle of replication at a fixed point of the chromosome and the whole process of replication occurred in the cell membrane, which co-ordinates the series of events taking part in cell division. The model predicts therefore the existence of mutants in which a simple genetic alteration disturbs both membrane properties and DNA synthesis, the latter by preventing either replication or initiation. Mutants CRT46 and CRT83 appear to present the properties expected from the latter type. No alteration can be detected among the enzymes now assumed to be involved in DNA synthesis, repair or degradation (personal communications from Dr G. Buttin and from Dr M. Kohiyame). The lesion appears to be specific to the bacterial chromosome, since phage A does multiply at high temperature. Furthermore, chromosome replication can be shown to occur at 41°C in certain classes of HFrT46 where the integrated F factor appears to supply the missing function(s) (Nishimura, Caro, Berg & Hirots, msnuscript in preperation). Although the nature of the T46 lesion cannot yet be precisely described, there is little doubt that in these mutants, altered membrane properties and DNA synthesis are correlated. On one hand, the band pattern of proteins extracted from membranes in cells grown at 41°C appears to be specifically changed; but the nature of the lesion cannot yet be ascribed. On the other hand, DNA synthesis appears to be halted at a specific point at 41°C; where activity is resumed upon a shift back to 30°C; but it cannot be determined as yet whether or not the point of arrest at 41°C and restart at 30°C does correspond to the physiological origin of the chromosome. One cannot therefore decide which one of the observed alterations corresponds to the primary lesion resulting from T46 or T83 mutations, and which one is secondary. The two possible alternatives are as follows. (1) The altered product of the mutant allele is directly involved in the initiation process. At high temperature, it is unstable and csnnot fit properly into the membrane, which appears secondarily to be altered. (2) The product of the mutant allele is a structural part of the membrane, in which the complex of enzymes involved in initiation and/or replication is inserted. Its alteration results in a change of configuration in the complex so that the enzymes which have to be synthesized for initiation to occur either can not fit, or not be active. There are few arguments as yet to support either hypothesis. On one hand, in order to be detectable by electrophoresis, the change in membrane proteins must affect
386
Y. HIROTA,
J. MORDOH
AND
F. JACOB
several per cent of the total membrane protein; in contrast, the factors directly involved in initiation must be present in very small amounts ; it is not clear therefore how a primary change in the latter could result in such a change in the former. On the other hand, while arrest in DNA initiation appears to follow immediat’ely on the shift to 41°C changes in the membranes can be detected only after 60 minutes at 41°C by the various techniques used. The two arguments work therefore in opposite directions. It is not difficult to produce a model to solve this apparent paradox, but at present it would be of little value. The results of the experiments with the mutant CRT46 can be interpreted by saying that the mutation has altered a protein the presence of which is required for DNA synthesis to be initiated. One can consider this statement in the light of negative verse positive regulation. Negative means that the enzymes ensuring initiation are constantly available during the division cycle, the presence of some factor, acting as a repressor, preventing initiation occurring during most of the cycle ; initiation would then result from derepression occurring for example in repressor dilution during growth (Pritchard, Barth & Collins, 1969) or synthesis of an antirepressor during growth (Rosenberg, Cavalieri & Ungers, 1969). On the other hand, positive regulation means that during most of the cycle, some factor required for initiation to occur is missing and that, in addition, this factor can only be used once; initiation would then result from the synthesis of this factor. One should emphasize that this positive model does not exclude a negative control operating on the synthesis of such an initiating protein. If one assumes the negative model, the lesion of CRT46 cannot be interpreted as an alteration of the regulatory mechanism. Since only 10 minutes at low temperature, i.e. one tenth of the generation time, is enough for re-initiation to occur (Fig. 3(a)), one cannot invoke the dilution of a repressor during this time. If an antirepressor is the affected element, this should only be compatible with a model where the antirepressor is synthesized immediately after DNA synthesis stops, either because a cycle is jlnished or because it is interrupted. Alternatively, CRT46 may affect some initiator enzyme made constitutively. If one considers a positive model, then one has only to assume that the protein altered in CRT46 is some component which acts on the chromosome to allow replication to start; furthermore, this component would act only once, so that its synthesis would be required for initiating every DNA cycle. Finally, a last point should be mentioned. It has been observed for a long time that any arrest in DNA synthesis results in a cessation of septum formation. As a consequence, filaments are formed. This indicates some kind of co-ordinated regulation in the various processes involved in cellular division. CRT46 does not escape this rule: since it divides probably once at 41”C, the DNA cycle already initiated is completed and then forms long filaments. But it underlines another aspect of this co-ordination. After a stay at 41”C, and then a shift back to 3O”C, cellular division occurs only after the new cycle of DNA replication has been completed. We thank Misses C. Barnoux and J. Perreau for their during the course of this work. We also thank Dr M. Kasai and his calculations on fluorescence depolarization.
excellent technical assistance for fluorescence measurements
This work was supported by grants from the Centre National de la Recherche Scientifique, the Commissariat it 1’Energie Atomique, the Delegation G&r&ale a la Recherche Scientifique et Technique and the National Institutes of Health, U.S.A. One of us (J.M.) is a Fellow of the John Simon Guggenheim Memorial Foundation.
THERhlOSENSITIVE
DNA
INITIATION
387
REFERENCES Bird, R. & Lark, K. G. (1968). Cold Spr. Hurb. Symp. Quant. Biol. 33, 799. Echols, H., Garen, A., Garen, S. & Torriani, A. (1961). J. Mol. BioE. 3, 425. Fangman, W. & Novick, A. (1968). Gknetic-s, 60, 1. Gartner, T. K. & Riley, M. (1965). J. Bact. 89, 319. Helmstetter, C. E. L Cooper, S. (1968). J. Mol. Biol. 31, 507. Hirota, Y., Jacob, F., Ryter, A., Buttin, G. & Nakai, T. (1968). J. Mol. Biol. 35, 175. Hirota, Y., Ryter, A. & Jacob, F. (1968). Cold Spr. Harb. Symp. Quant. BioZ. 33, 677. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spr. Harb. Symp. Quunt. BioZ. 26, 329. Jacob, F. & Wollman. E. (1961). Sexuality und Genetics of Bacteria. New York: Academic Press Inc. Kasai, M., Changeux, J. P. & Monnerie, L. (1969). Biochem. Biophys. Res. Comm. 36, 420. Kohiyama, M. (1968). Cold Spr. Harb. Symp. Quant. BioZ. 33, 317. Kohiyama, M., Lamfrom, H., Brenner, S. & Jacob, F. (1963). C. R. &ad. Sci. 265, 1820. Lark, K. G., Repko, T. & Hoffman, E. 5. (1963). Biochim. biophye. Acta, 76, 9. Lederberg, J. (1950). Methods in Medical Research, vol. 3,5. Year Book Publishers, Chicago. Lennox, E. S. (1955). Virology, 1, 190. MaaIm, 0. & Hanawalt, P. C. (1961). J. Mol. BioZ. 3, 144. Maalee, 0. & Kjeldgaard, N. 0. (1966). Control of Macromolecular Synthesis. New York: W. A. Benjamin Inc. Monnerie, L. & Neel, J. (1969). J. Chim. phys. 61, 504. Monod, J., Cohen-Bazire, G. 8; Cohn, M. (1951). Biochim. biophys. Acta, 7, 585. Nagel de Zwaig, R. & Luria, S. E. (1967). J. Butt. 94, 1112. Nomura, M. (1964). Proc. Nat. Acud. Sci., Wash. 52, 1514. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature, 204, 1069. Pritchard, R. H., Barth, P. T. & Collins, J. (1969). Symp. Sot. Gen. Microbial. 19, 263. Pritchard, R. H. & Lark, K. G. (1964). J. Mol. BioZ. 9, 288. Puig, J. & Azoulay, E. (1967). C. R. Acad. Sci. 264, 1916. Ramakrishnan, T. & Adelberg, E. A. (1965). J. Bact. 89, 661. Rosenberg, B. H., Cavalieri, L. F. & Ungers, G. (1969). Proc. iNut. Acad. Sci., W’mh. 63, 1410. Shapiro, B., Siccardi, A., Hirota, Y. & Jacob, F. (1970). J. Mol. BioZ. 52, 75. Stryer, L. (1965). J. Mol. BioZ. 13, 482. Taylor, A. L. & Trotter, 0. D. (1967). Bact. Rev. 31, 332. Weber, G. & Young, L. E. (1964). J. BioZ. Chem. 239, 1415. Yoshikuwa, H., O’Sullivan, A. & Sueoka, N. (1964). Proc. Nat. Acad. Sci., Wash. 52, 973.
On the Process of Cellular Division in Escherichia coli III?. Thermosensitive Mutants of Escherichiu coli Altered in the Process of DNA Initiation YUEINORI
HIROTA, Josh MORDOH AND FR~QOIS
JACOB
Laboratoire de GknBique celluluire de l’lnstitut Pasteur et du Colk?gede France, Paris, France (Received 10 Februuy
1970, and in revised form 10 June 1970)
Genetic and physiological properties of a temperature conditional mutant of Eecherichia coli K12 are described. After a shift from 30°C to 41”C, the mutant exhibits a residual synthesis of DNA, which stops after an hour. All the characteristics of the mutant indicate that at high temperature, it can complete a DNA cycle already started, but not initiate a new one. After shift back from 41°C to 3O”C, protein synthesis is required in order for DNA synthesis to resume. Reinitiation of DNA synthesis appears always to occur in the same area of the bacterial chromosome. In correlation with this alteration in DNA initiation, several lines of evidence indicate an alteration in the structure of the cell membrane.
1. Introduction The cell division cycle of Es&e&h&z coli involves the regulation of many cellular activities, including DNA initiation and replication, formation of the bacterial membrane and equipartition of DNA copies. Little is known about the co-ordination of these operations, and in particular about the way in which the rate of DNA synthesis might be co-ordinated with the growth rate. There is some direct evidence that changes in the rate of DNA synthesis are determined primarily by changes in the frequency of initiation (Oishi, Yoshikawa & Sueoka, 1964; Maalee & Kjeldgaard, 1966; Helmstetter & Cooper, 1968; Bird & Lark, 1968). The replicon model (Jacob, Brenner & Cuzin, 1963) predicted that a replication cycle of the E. coli chromosome is initiated at a specific point on the chromosome, and that replication follows in the replication machinery, which is integrated as a part of the membrane structure. Thus, the bacterial membrane could possibly regulate in some way the functioning of these processes. A class of thermosensitive mutants is known in which initiation of DNA synthesis appears to be blocked at high temperature. Isolation and some preliminary characterization have already been described (Kohiyama, Lamfrom, Brenner $ Jacob, 1963; Kohiyama, 1968; Hirota, Jacob, Ryter, Buttin & Nakai, 1968; Hirota, Ryter & Jacob, 1968). Evidence for alteration in membrane structure of such mutants is presented in paper II of this series (Shapiro, Siccardi, Hirota t Jacob, 1970). In the t Paper II in this series is Shapiro, Siccardi, Hirota & Jacob, 1970. 369
370
Y.
HIROTA,
present paper, the evidence synthesis is reported.
J. MORDOH
supporting
AND
F. JACOB
a defect in the process of initiation
of DNA
2. Materials and Methods (a) Bacterial
strains
The following strains of E. co&i K12 have been used: CRT46 : F- Thr- Leu- ThyB,- Ilv- lac,- Mal- T46s Xr : F- Thy- lac - T46S hS derived from CRT46 after cross with Hfr P13 (Jacob Rc CRT464 Wollman, 19y61). HfrT46 : IlvThyT46S Hfr, injects its chromosome in the order: O-ProA-LeuThr- - - lac-F AT1243 : PyrEMet- Hfr, injects its chromosome in the order: 0-Thr-Leu-lac - - -MetF (Ramakrishnan & Adelberg, 1965). CRTS3 : F- Thr- Leu- B,- Ilv- Mtl- T838. (b) Media Nutrient broth contained 10 g of polypeptone, 10 g of meat extract, and 5 g of NaCl in 1 litre of water adjusted to pH 7.0 with KOH. For growth of thymineless bacteria, 60 pg of thymine/ml. were added. Minimal eosine methyleno blue (EMB) medium was prepared as described by Lederberg (1950). Minimal synthetic medium 63 (Monod, Cohen-Bazire & Cohn, 1951) supplemented with 0.3% glucose, 60 pg thymine/ml., 0.1 pg thiamine/ml. and 60 pg each of L-threonine, L-leucine, L-isoleucine and L-valine/ml. was also used. Casamino acids medium was prepared, supplementing the same medium 63 with 0.3% glucose, 0.3% Casamino acids, 0.1 rg thiamine/ml. and 60 rg thymine/ml. (c) Isotope Two
methods
(i) Measure
incorporation
and radioactivity
assay
were used:
of total
amount
of DNA
synthesis
Cultures were grown overnight in the media previously described, to which [l%]thymine (C.E.A. France, spec.act. 43.3 me/m-mole) was added to a final concentration of 1 @/ml. The next morning cultures were diluted 1 to 20 in the same medium containing the same concentration of [14C]thymine. The experiments were carried out when the cultures were in the exponential phase. l-ml. portions were added to 5 ml. of ice-cold 5% trichloroacetic acid and stood on ice for 1 hr. They were then filtered through HA Millipore filters and washed with 30 ml. of 5% trichloroacetic acid. Filters were dried and counted invials containing 10 ml. of toluene-PPO-POPOP mixture (Packard Instrument Co.) (11. : 3g : 0.3 g) in a Packard Tricarb scintillator. (ii) Measure
of the rate
of DNA
synthesis
To 2 ml. of the bacterial culture, a pulse was given of [3H]thymidine (C.E.A. France, spec.act. 18 c/m-mole) with a final concentration of 4 PC/ml., adding 1 rg of cold thymidine/ml. Further manipulations were carried out as previously described. (d) Cesium
chloride
density-gradient
centrifugation
of DNA
labelled
with
[3H]thymidine
and
heavy isotopes Procedures for radioactive pulse labelling of DNA followed by density labelling, extraction of DNA and CsCl centrifugation were a modification of those described by Lark, Repko & Hoffman (1963). An overnight culture of CRT46 in synthetic medium 63 supplemented as described above was diluted in fresh medium to adjust the cell concentration to 4 x lo7 cells/ml. Two lo-ml. samples of this suspension were incubated at 30°C for about three generations (generation time, 100 min). (i) Thymidine
pulse labelling
40 PC of [3H]thymidine (C.E.A. France, spec.act. 18 c/m-mole) were added to the cultures for a given time, one before (control) and the other after a shift to 41°C (experimental). After this pulse, the cells were quickly washed by filtration through a Millipore filter with
THERMOSENSITIVE
DNA
371
1NITIATION
100 ml. of the same medium containing 50 pg of cold thymidine/ml. Cells were collected and resuspended in 20 ml. of non-radioactive medium. They were kept at 21°C for overnight growth without shaking, in order to randomize growth. The next morning, the cultures had reached a density of 4.3 x lo* cells/ml. 6-ml. samples from each culture were added to 14 ml. of fresh medium and incubated at 30°C for about one more generation. Final density of the culture was 3.2 x 10s cells/ml. (ii) Density
label&g
Both cultures were shifted to 41°C for 1 hr. After this treatment, cells were harvested on Millipore filters, and washed with 100 ml. of medium 63 without NH,Cl. Cells ware resuspended in 20 ml. of medium 63 supplemented as-described above, with the exception that NH,Cl and glucose had been replaced by 2 mg each of 15NH,Cl/ml. (97.2 atom y. as r5N, Isomet Corporation) and [2H]glucose/ml. (98.2 atom o/o as 2H, Volk Radiochemical Co.). Cultures were shaken at 30°C and after various times (20, 40 and 60 min), g-ml. portions were taken. Cells were harvested by centrifugation at low temperature and washed once with 0.01 M-Tris buffer, pH 7.5, containing 0.001 M-EDTA. (iii) D,VA extraction and CsCl eentrifugation The pellet was resuspended in 0.45 ml. of the same buffer, and 0.05 ml. of 25% Sarkosyl was added. After 5 min at room temperature, this mixture was diluted tenfold with buffer, and 0.05 ml. of a 10 mg/ml. protease solution (from Streptomyces g&sew, type VI, Sigma Chem. Corp.) were added. After 30 min at 37’C, the same amount of protease was added again and incubation was continued for another 3 hr at 37°C. Cells debris were removed by low speed centrifugation. The supernatant fraction was removed, and 2.24 ml. were mixed with 2.861 g of cesium chloride (The Harshaw Chem. Co.). Centrifugation was carried out at 25,000 rev./m& 20°C; 3 days, in the SWBOLl swinging bucket rotor of the Spinco model L preparative ultracentrifuge. l-drop fractions were collected from the bottom of the tubes directly into an aqueous scintillation fluid (Pritchard BE Lark, 1964) and radioactivity was estimated in a Packard Tricarb scintillator after overnight cooling in the dark.
(e) Estimation
of cell number
A Coulter counter model F (Coultronics France, S.A.) was used. The counter was used to estimate the total cell number, a fraction of which may have no colony-forming capacity. It was operated with a 20-p diameter orifice, maximum amplification and a current setting of 5.8 and 11. Coulter counter readings were maintained in the 2 to 20 x lo3 range after dilution in normal saline which had been filtered through a type HA Millipore filter. (f) Measurement of fluorescence intensity
and polarization sulphonate
of I-aniline-8-naphthalene
Fluorescence intensity and polarization of 1-anilino-8-naphthalene sulphonate measured according to the method of Monnerie & Neel (1969). Portions of the cultures taken at different times of incubation, centrifuged at low speed and washed twice chilled medium 63. The cells were resuspended in the same medium, and intensity depolarization were followed after 1-anilino-8-naphthalene sulphonate was added concentration: 0.05 M). The wavelength of excitation was 365 rnp and fluorescence observed at 483 mp; in both instances, interference filters were used.
were were with and (final was
3. Results (a) Properties
of mutant CRT46
.
As described in a previous paper (Hirota, Jacob, Ryter, Buttin & Nakai, 1968) after a shift from 30 to 40°C total DNA synthesis in a culture decreases progressively for about 60 minutes and then stops. The increase in DNA content at 41°C is about 30 to 40%. After the arrest of DNA synthesis, the optical density continues to increase linearly and proteins continue to be synthesized. During the same time, there is a
372
Y.
HIRCTA,
J. MCRDOH
AND
Y. JACOB
rapid decline in viability. Cell division proceeds at 41°C for a few hours at a decreasing rate after the arrest of DNA synthesis (Fig. 3(b)). This results in a decreased number of nuclei per cell and the production of DNA-less bacteria. Two classes of thermosensitive mutants are known in which DNA synthesis is disturbed: (1) one in which the temperature shift results in an immediate stop in synthesis; (2) the second only after a residual synt’hesis. Mutant CRT46 belongs to the latter class. In order to determine whether the synthesis of any genct,ic element or only of the chromosome was affected by the mutation, bacteria previously incubated at 41°C for 60 minutes were infected with phage h. The results of this experiment are given in Table 1. The burst size at 41 “C was 510, as compared with 56 in the control at 30°C. TABLE
1
Production of yhage lambda by CRT464 celb at 30°C and 41°C
Input phage adsorbed Infectious centres Phage released Burst size
after 10 min (%)
30°C
41°C
98 1.2 x 10’ 6.7 x 10s 66
99 1.3 x 107 6.6 x 108 510
An exponentially growing culture of the mutant CRT464 in broth supplemented with 0.1% maltose at 30°C was used. At 2 x 10s cells/ml., the culture was divided into two parts. One was shaken at 3O”C, the other at 41’C. After 90 min, to O-9 ml. of each culture was added 0.1 ml. of h suspension containing 1.3 x lo* phages/ml. (time 0). The mixtures were maintained at 30 and 41”C, respectively, and at 10 min phage adsorption was stopped by dilution without temperature change. At various times, samples were assayed for plaque formation on E. coli K12 C600 at 37%. Unadsorbed phage was measured after killing the cells with chloroform.
It is concluded therefore that mutation CRT46 stops DNA synthesis only in the chromosome. Mutant CRT83 behaves in the same way (Kohiyama et al., 1963). Other mutations of class 2, i.e. with residual DNA synthesis at high temperature, are known which are unable to reproduce phage h at high temperature (Hirota, Ryter & Jacob, 1968; Fangman & Novick, 1968). By this criterion, a subdivision can therefore be introduced among mutants of class 2. The subclass to which mutants CRT46 and CRT83 belong exhibits the properties which can be expected from a lesion in a specific initiation process of chromosome replication. (b) Effects of cultural condition
on mutant CRT46
(i) Effect of temperature Residual synthesis of DNA was measured after shifting a culture growing at 30°C to various temperatures: 39.0, 41-O and 42.5%. The rationale of the experiment was as follows: if the initiation process is affected in this type of mutant, the residual synthesis of DNA should be somewhat independent of temperature once the denaturing temperature has been attained, the opposite being true if the replication enzyme were the element affected. The results are shown in Figure 1. It can be seen that there is a little escape at 39”C, but that at 41.0 and 425°C the amount of residual synthesis remains unchanged.
THERMOSENSITIVE
DNA
t-----------High
0
373
INITIATION
tempera~“re-------
I
I
-60
60
I
120
Time (min)
FIO. 1. Residual synthesis of DNA at different temperatures. C!u&rea of CRT40 growing exponentially at 30°C in different media were shifted to high temperstares (--O--O-, 39-O%; --A--A--, 41.0°C; --m--n--, 42PC). The amount of DNA was measured by [14C]thymine incorporation 8s described under Materials and Methods.
(ii) Effect of composition
of medium
There are reasons to believe that the rate of DNA synthesis in a culture is controlled, not by the speed at which the replicating enzymes work, but by the number of replicating forks working at constant speed. Bacteria grown in rich medium would thus show more rapid DNA synthesis because of more frequent re-initiation (Yoshikawa, O’Sullivan & Sueoka, 1964; MaaIoe $ Kjeldgaard, 1966; Helmstetter & Cooper, 1968). If CRT46 mutation does result in an alteration of DNA initiation, the amount of DNA synthesized at 41°C should depend on the medium. Since a cell grown in rich medium contains more replicating forks than a cell grown in a poor one, the former should synthesize more DNA at 41°C than the latter. This is the case, as can be seen in the results of the experiment reported in Figure 2. (iii) Resumption of DNA eynt?hesisupm 8hififmn
41°C to 30°C
After one hour at 41”C?,mutant CRT46 is unable to synthesize DNA. The resumption of DNA synthesis when the culture is returned to 30°C was studied. The results of such an experiment are reported in Figure 3(a). In the control at 30% the rate of
374
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
Time (min) FIG. 2. Residual synthesis of DNA in different media. Cultures of CRT46 growing exponentially at 30°C in different media (-O-O-, minimal synthetic medium; --n-o-, Casamino acids medium; --A-A-, nutrient broth) were shifted to 41°C and the residual increase of DNA was measured by [14C]thymine incorporation as described under Materials and Methods. The values are normalized to the values at zero time. The compositon of the media is described under Materials and Methods. The generation times of CRT46 in these media at 30°C are approximately 100 min (minimal synthetic medium), 86 min (Casamino acids medium) and 62 min (nutrient broth).
DNA synthesis, measured by the incorporation of [3H]thymidine into the trichloroacetic acid-insoluble fraction during a series of three-minute pulses, increases exponentially. In the population kept for 60 minutes at 41°C and then shifted to 3O”C, however, after a small lag, this rate increases, reaches a plateau and then decreases. After 100 minutes, i.e. approximately the generation time under these conditions, whereas the rate of DNA synthesis has decreased, the rate of cell division increases sharply (Fig. 3(b)). In order to determine whether protein synthesis is required for resumption of DNA synthesis, chloramphenicol was added to a sample of the culture when shifted from 41 to 30°C: no DNA synthesis was then observed. The resumption of DNA synthesis upon a shift from 41 to 30°C can also be measured in the following way. A population of CRT46 grown at 30°C is shifted to 41°C for 60 minutes. It is then shifted back to 30°C. At various times, samples are returned to 41°C and the amount of residual DNA synthesized is measured. As shown in Figures 4 and 5, the DNA content increased up to 300% of the original amount when the culture is allowed to stay 60 minutes at 30°C before being transferred again to
(b)
Time (mid
120
I80
0
60 CC)
120
I(
FIG. 3. Resumption of DNA synthesis, cell division and cell growth after 1 hour at 41°C. (a) An exponemially growing culture of CRT46 in minimal synthetic medium at 36°C was shifted to 41°C for 1 hr, and then put back to 30°C (time 6) or 200 pg/ml, (-n-a--). Two CultureS Were used either in the absence (-O-O-) or in the presence of chloramphenicol, 100 ~g/ml. ( ~n-n--) ), The rate of DNA synthesis as controls, ono growing at 36°C all the time (-m-m-) and the other kept at 41°C throughout bhe experiment (-0 0 W&S measured by [3H]thymidine incorporation (see Materials and Methods). (b) CRT46 growing exponentially in minimal medium at 30°C was treated as doscribed in (a), and cell number was measured as described under Materials and Methods. Symbols are the same as in (a). (c) A culture of CRT46 growing exponentially in minimal medium at 30°C was divided into three part’s: one culture was kept at 30°C all the time and another shifted to 41°C for 1 hr and then put back to 30°C (-m-m--), another was shifted t,o 41°C and kept at that temperature (-O-O-), (-e-e--). Optical density of the cultures was followed with a Zeiss spcctrophotomctcr (type PM& II) at 550 mp.
(a)
60
I c
I
: 50
I
I20
I
180 Time (min)
240
FIG. 4. Residual synthesis of DNA at 41°C after a previous temperature shift treatment. A culture of CRT46 growing exponentially at 30°C in minimal synthetic medium was shifted to 41°C for 1 hr. After this treatment, it was put back again to 30°C (-O--O-), and at different times, indicated by arrows, portions were replaced at 41% (------). The time at 30°C before being shifted at 41°C were, for the different samples : 6 min (--Q--m--) ; 20 mm (-- 0 -- 0 --) ; 40 min (--A -- A--); 60 min (--V --V --); 36 min (-- l -- a--); 105 min (-- n -- n --) was kept at 41’C all the time. DNA syn120 min (--A--A--). One sample (-*-a-) thesis was measured by [r*C]thymine incorporation as described under Materials and Methods.
I
I
I 1 I Beginning of cell division
1
I
1
I
I
I
20 40 60 60 80 100 I20 0 Time at 30°C before shift bock to 41°C (min)
80
100
120
500
2
AI, 0
I 20
40
(a)
I
I
I
w
(b)
Fxa. 5. DNA synthesis at 41’C after a previous temperature shift treatment. The data used for this Figure are those of the experiment reported in Fig. 4. (a) The results are expressed as percentage of DNA increase during the last 41°C treatment, over the amount found immediately before every last shift to 41°C at various times taken as 100. (b) The results are expressed as percentage of DNA increase at 41”C, over the amount foundat the time (80 min) of the shift down from 41 to 3O”C, taken as 100. The beginning of cell division was determined in an experiment similar to that reported in Fig. 3(b).
THERMOSENSITIVE
DNA
377
INITIATION
41°C. These figures may be easily understood if mutation CRT46 blocks re-initiation of DNA synthesis at high temperature. During the first 30 minutes at 30”, all cells would progressively restart DNA synthesis ; every chromosome initially completed at 41°C would initiate replication twice or more after 80 minutes at 3O”C, i.e. before cellular division has occurred (compare time in Figs 3(b) and 5). (iv) Comparison of the effect of inhibition DNA
synthesis and of CRT46 mutation on
ofprotein
synthesis
In wild-type E. co&, inhibition of protein synthesis, either by amino acid starvation or by chloramphenicol addition, results in an arrest of DNA replication after a residual synthesis of about 40%. This was interpreted as a requirement for protein synthesis for the re-initiation of a new DNA cycle (Maalee t Hanawalt, 1961; Lark et al., 1963). The results previously reported with mutant CRT46 point to a similar interpretation. In order to compare the temperature shift and amino acid starvation effects, the following experiment was performed. Mutant CRT46 (which requires threonine and leucine) was grown at 30°C in minimal medium containing the two amino acids. The culture was then incubated at 30°C without the two amino acids. After a residual synthesis of aproximately 40%, DNA synthesis stopped. The culture was then divided in three parts. Part 1 was incubated at 30°C in the presence of the amino acids ; part 2 at 41°C in the presence of the amino acids; part 3 at 41°C in the absence of the amino acids. If the same process of DNA initiation is altered by amino acid starvation
',5 -
,o -
..5-
Od
I
I
60
I
I
I20
180
240
Time (mid Fm. 6. DNA synthesis at 41°C after amino acid starvation at 30°C. A culture of CRT46 was grown at 30°C in minimal supplemented medium. At a density of 4 x lo* cells/ml., the culture was shifted to a starvation medium (time 0). Synthetic medium 03 supplemented with 0.2% glucose, 6 pg vitamin B1 and 60 pg of each Gsoleucine, L-valine and thymine/ ml. was used for amino acid starvation. After 2 hr at 30°C; the culture was divided into three portions: one was incubated in the same medium at 41% (-n--O--); the other two were supplemented with 50 pg/ml. of both L-leucine and z-threonine, and incubated at 30% (- O-O-) or 41’C (--A--A--). [‘W]Thyrnine was added at time 0 (final concentration, 2 q/ml.) and radioactivity was measured as described under Materials and Methods.
378
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
as well as high temperature, one would expect no DNA synthesis in part 2. As shown in Figure 6, this is exactly what is observed, in opposition to what was found by Fangman & Novick (1968) with another mutant. This result suggests that the two phenomena, found either after amino acid starvation or at high temperature in CRT46, belong to the same process. CRT46 affects the properties of a protein the requirement of which for DNA initiation is evidenced by amino acid starvation. (c) Place of re-initiation
If CRT46 blocks initiation of DNA cycles at 41”C, a shift back to 30°C should initiate synthesis always in the same area of the chromosome. To investigate this possibility, a culture was submitted to two successive re-initiations, with overnight growth between them. Two parallel cultures of CRT46 in synthetic medium were prepared, one of them serving as a control. The only difference between the control and the experimental cultures was the moment at which the [3H]thymidine pulse was given. The cultures grown at 30°C were maintained at 41°C for 60 minutes, and then shifted back to 30°C. The control was given a six-minute pulse of [3H]thymidine (see Materials and Methods) immediately before the temperature shift up, whereas the experimental culture received a 12-minute pulse eight minutes after returning to 30°C (see Fig. 3(a)). The amount of label incorporated in both cultures was essentially the same and corresponded to about 5% of the chromosome length. After appropriate washing, the cultures were grown overnight in minimal medium for four generations, and were incubated again at 41°C for 1 hour. After this treatment, they were returned to 30°C in the presence of heavy isotopes. At various times (20,40 and 60 min) samples of both cultures were taken, lysates prepared and DNA analysed in a caesium chloride density-gradient (see Materials and Methods). The results of this experiment are shown in Figure 7. In the control culture, where the chromosomes were labelled at random, the radioactivity migrates progressively from the light to the hybrid band. After 60 minutes, which corresponds to half a generation time, there is an equal amount in both peaks. In contrast, in the population maintained at 41°C before the pulse, the radioactive material enters the hybrid peak much earlier ; after 20 minutes, 65% is found in the hybrid peak. This result indicates that after two successive reinitiations, DNA synthesis occurs in the same area of the chromosome. This is the result which is expected if CRT46 mutation inhibits initiation at high temperature. (d) Alteration of cell surface According to the replicon model (Jacob et al., 1963) the cell membrane would be the site where DNA initiation and replication take place. Two approaches were used to investigate the properties of the cell surface of CRT46. First, it is known that some mutants, suspected to have altered membrane properties, such as some clones tolerant to colicin E2 (Nomura, 1964), are sensitive to concentrations of sodium deoxycholate which do not affect the wild type (Nagel de Zwaig $ Luria, 1967). Mutant CRT46 and its parent CR34 were therefore compared as to their relative susceptibility to various concentrations of sodium deoxycholate at 30°C and 41°C. The presence of deoxycholate at a concentration of 0.5% does not affect the growth of the parent CR34, either at 30°C or at 41°C. In contrast, as shown in the results of the experiment reported in Figure 8, the growth of the mutant CRT46, while not altered at 30°C; is affected at 41°C. The cells lyse in the presence of only 0.1% of deoxycholate, but only after one hour at 41”C, indicating that membrane
1
Fractian no.
60
I
70
40 min
T
40
50
60
70
60 min
PIG. 7. Caeaium chloride density-gradient analysis of the re-init.iation place of CE!I’46. The detailed experimental procedure is described under Materials and Met)hods and in the test. Two exponentially growing cultures of CRT46 at 30°C in minimal medium were shifted to 41’C for 1 hour and then replaced at 30°C. The control culture (-O--(j--) received a 6.min. [%]thyrnidine pulse immediately before the temperature shift to 41°C; and the experimental cult,ure (-a--•-.-) received a 1%min [?H]thymidine pulse 8 min after returning to 30°C. After overnight growth, both cultures were shifted again to 41°C fur 1 hr, and replaced at 30°C in synthetic medium containing [2H]glucose and *5NN&I. Saqbs were taken at 20, 40 and 60 min, and the lysatcs were subjected to density-gradient analysis. Density decreases in the Figure from left to right.
r
80
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
Time (mid
Fro. 8. Deoxycholate sensitivity from CRT46 at 30°C and 41% A culture of CRT46 growing exponentially at 30°C in broth supplemented with thymine was transferred to the same medium either in the presence of deoxycholate (final concentrations 0.1 and 0.5%) or in the absence of detergent. The cultures were incubated at 30°C (solid symbols) or 41°C (open symbols) with gentle shaking. Detergent concentrations: 0, 0, 0%; n , 0, O-1 %; A, A, 0.5%. Portions were taken at different times and optical density was measured at 550 rnp with a Zeiss spectrophotometer PMQII.
alteration does not occur immediately but after some time of metabolism. Although the mode of action is not very clear, this result suggests some alteration in the surface properties of the cell. The second method was suggested by Dr B. Shapiro, It takes advantage of the fact that l-anilino-8-naphthalene-sulphonate gives rise to a weak fluorescence in water solution and to a strong one in protein solution by fixation to hydrophobic bonds of the latter (Weber & Young, 1964; Stryer, 1965). When mixed with normal CR34 bacteria, at either temperature, or with the mutant CRT46 at low temperature, the probe produces a weak fluorescence. With the mutant at 41”C, however, a significant
THERMOSENSITIVE
DNA
INITIATION
381
Time (mid
FIG. 9. Fluorescence of CR34 and CRT46 bacteria measured in the presence of l-anilino-8, naphthalene sulphonate. E. coli CRT46 and its parental strain CR34, growing exponentially at 30°C in broth supplemented with thymine, were each divided into two parts (time 0): one remained at 30°C and the other was placed at 41°C. Samples were taken at different times, centrifuged in the cold and washed twice with cold M63 medium. Cell mass of bacteria was adjusted to an optical density of 0.5 at 650 rnp (Zeiss spectrophotometer, PM& 11) in all samples, and fluorescence was measured at 25°C after adding the sulphonate to the bacterial suspensions as described under Materials and Methods. In the arbitrary scale shown in this Figure, three units correspond to the fluorescence of 1 pmole of sulphonate bound to bovine serum albumin in the same conditions. -O-O-, CR34, 41°C; --A--A-CR34, 30°C. CRT48, 41°C; -.--.--, CRT46, 30°C; --A-A--,
increase in fluorescence is observed. It can be detected only after a lag of about 60 minutes, and then the fluorescence increases linearly (Fig. 9). In all cultures, more than 90% of the fluorescent material sediments at low-speed centrifugation (3000 rev./min), indicating that it is bound to the cell itself. From the results shown in Figure 9, fluorescence depolarization was calculated (Kasai, Changeux & Monnerie, 1969). It was observed to increase from 0.19 (30°C) to 0.27 (4 hr at 41°C) in the mutant. Results with both deoxycholate and I-anilino-8-naphthalene aulphonate indicate therefore a change in the organization of the cell surface. In both oases, something, which is masked in the parent at both temperatures or in the mutant at low temperature, has become accessible in the mutant kept at high temperature. This change, however, does not affect some of the properties of the cell surface; for example, the capacity to adsorb T6 and h phages and the specific permeability to /3-galactosides. That the change involves an alteration of the membrane is evidenced by the tiding that, when analysed by electrophoresis on acrylamide gels, one of the membrane proteins exhibits modified properties at 41°C (Shapiro et al., 1970). It is concluded therefore that the perturbation of DNA initiation and the alteration in membrane proteins found at high temperature in mutant CRT46 are associated. However, while the arrest of DNA initiation is observed immediately after a shift to 41”C, the change in membrane properties found with the two techniques used is detected only after
382
Y.
HIROTA,
J. MORDOH
AND
F. JACOB
60 minutes at 41°C. No significant change in deoxycholate sensitivity or in fluorescence was observed in other types of DNA mutants making filament,s nor after thymine starvation of the parental type. (e) Genetic analysis of mutants CRT46 and CRT83 In all the properties examined so far (Kohiyama, 1968; Shapiro et al., 1970), mutant CRT83 behaves in exactly the same way as CRT46. The genetic analysis of both mutations was therefore performed in parallel. (i) Conjugation Preliminary experiments have located CRT46 in the isoleucine-valine (11~) region of the chromosome (Hirota, Jacob, Ryter, Buttin & Nakai, 1968; Hirota, Ryter & Jacob, 1968). More precise mapping was carried out by crossing CRT46 (F-) with a thermoresistant Hfr carrying various markers. Recombinants for nutritional markers or thermoresistance were selected for, and their genetic constitution analysed (Table 2(a) and (b)). The results indicate that T46 is located close to PyrE-Ilv and MetE, the probable order being: -PyrE-T46-Ilv-MetE. An Hfr strain is known in which F is integrated between the Mtl and Ilv loci, so t’hat the chromosome is transferred in the order 0-PyrE-Ilv. . . Xyl-Mtl (Jacob & Wollman, 1961). Kinetic transfer experiments indicate that the CRT46 marker is t,ransferred into the F- cell shortly after PyrE and shortly before Ilv. CRT83 is located very close to CRT46. In order to study their linkage, a cross between an Hfr carrying the T46 allele and an F- carrying T83 was performed. The results are given in Table 3. The results indicate a strong linkage between the two mutations, the most likely order being Mtl-T46-T83-Ilv. (ii) Transduction More precise mapping of CRT46 and CRT83 was done by Pl transduction, with the use of markers known to be in the area, such as ChlB (Puig $ Azoulay, 1967), Tna (Gartner & Riley, 1965), PhoS (Echols, Garen, Garen & Torriani, 1961) and several independent Ilv (Ramakrishnan & Adelberg, 1965). R esults are summarized in Table 4. T46 is closely linked to Ilv, PhoS and Tna but not to ChlB. The most probable order is T46-Tna-PhoS-Ilv. By transduction, the T83 mutation exhibits a similar degree of linkage to Ilv as T46. The most likely order of markers in the area is represented in Figure 10. T46 and T83 appear to be very closely linked. Both appear to have the same alteration of some membrane protein (see Shapiro et al., 1970). Although technical difficulties have hitherto prevented complementation analysis, it seems reasonable to associate them in designating the chromosomal segment where both are located as DnaA. It remains to be seen whether only one or several cistrons are involved in this segment. One should note that among recombinants or transductants, a complete correlation was observed between thermosensitivity and susceptibility to deoxycholate with both T46 and T83 mutations. Although the existence of two mutations, one affecting DNA initiation, the other membrane properties, cannot be completely excluded as yet, it appears very unlikely.
THERMOSENSITIVE
DNA TABLE
383
INITIATION
2(a)
Segregation of themosensitive characters in a cross HfrAT1243
(1)
Ilv+
SmP
Thy
196
Sm
(L)
PyrE
Ilv
MetE
115
196 (100) 392 (85)
156 (79) 270 (59)
(59) (2)
T46+ Sm’
CRT46 Sm’
Frequency of male markers found among recombinants (%)
No. of colonies analysed
Selected markers
x
459
(i.7)
388 (85)
Thre-Leu
T46t 158 (81) 459 (100)
t Determined by the capacity to grow on complete medium at 41°C. Bacteria F- CRT46 SmP and HfrAT1243 exponentially growing at 30°C in broth supplemented with thymine were mixed (lHfr/lOF-) in the same medium. After 2 hr at 3O”C, the cultures were diluted and samples plated on appropriate media. The plate for selection 1 was incubated at 30°C. That for selection 2 was first incubated for 2 hr at 30°C and then at 41°C. Recombinant colonies were isolated from selections 1 and 2 (196 and 459, respectively), purified, and tested for their characters by replica plating on suitable media. HfrAT1243: MetE- PyrE-, injects its chromosome in the order : O-Thre-Leu-lac-Try---His-PyrEM&E-B,. CRT46 Sml‘: Thy- Ilv- Thr- Leu-. TABLE
2(b)
Linkage of T46 locus with PyrE, Ilv and MetE in the cross AT1243-f x CRT46 Sm’ Recombinants
PyrE
(1) Ilv+ Smr
1 0 1 1 0 1 0 0
T46
Ilv
42 19 0.5 15.3 17.4 0.5 4.1 1
1961 1 0 1 1 0 1 0 0
Total
No. of recombinants (%) 83 37 1 30 34 1 8 2
Total
(2) T46+ Smr
MetE
1 1 0 1 0 0 I 0
224 32 8 116 6 40 20 13
52 7.4 1.8 27 1.4 9.2 4.6 3.0
4331
t HfrAT1243: Thy+ SmaPyrE-T46+Ilv+MetE-Thr+Leu+ : 11111111 CRT46 Sm’: Thy-SmrPyrE+T46SIlv-MetE +Thr-Leu: 00000000 3 The total number of recombinants examined was 196 in exp. 1 and 433 in exp. 2. Genetic markers of all the recombinants from the crosses summarized in Table 2(a) were scored and classified. Alleles of markers derived from the Hfr parent are represented by 1, and those derived from the F- parent by 0. 26
384
Y. HIROTA,
J. MORDOH
TABLE
Number
AND
F. JACOB
3
of colonies formed in a cross, HfrT46
Selections
x CRT831
No. of recombinant colonies formed per ml. of mating mixture
Rat’io of recombinant formation (%)
3.4 x 10’ 8.0 x lo1 3.8 x lo3
100 0.0002 0.01
(1) Sm’Mtl+ (2) SmrMtl+T46+T83+ (3) SmrT46+T83+
HfrT46: Ilv-T46sT83+Mtl+Sms injects chromosome in the order: 0-ProA-TL-Ilv-Mtl --- lac F. CRT831: Il~-T83~T46+Mtl-Sm~Thr-Leu-B,-Thy-lac,-F-. Bacteria HfrT46 and F- CRT831, exponentially growing at 30°C in broth supplemented with thymine were mixed (1: 10) in the same medium, and incubated at 30°C for 3 hr without shaking. Cells were washed with medium 63 twice and plated on media listed below at 30°C or 41%. Selection 1: EM synthetic agar plus 1% mannitol, L-threonine 50 pg/ml., L-leucine 50 pg/ml., vitamin B, 5 pg/ml., thymine 50 pg/ml., L-isoleucine 50 pg/ml., L-valine 50 pg/ml. and streptomycin 100 rg/ml. at 30°C. Selection 2: The same medium as above but incubated at 41%. Selection 3: EM synthetic agar plus 1% glucose, L-threonine 60 pg/ml., L-leucine 60 ag/ml., vitamin B1 5 ag/ml., thymine 50 pg/ml., L-isoleucine 50 pg/ml., L-valine 50 pg/ml., and streptomycin 100 pg/ml. at 41°C. Plates were examined after 2 to 3 days of incubation.
TABLE
Frequency of joint-transductions Donor
CRT4619 (Tna-PhoS-)
Recipient
HfrT46 (Il~-T46~)
DnaA
4
of markers located near DnaA
Tna
PhoS
Ilv
l.ocus
Selections
11v+
4%)
Ilv+ and T46 +
(l&) AB2151 (Tna-Ilv-)
CRT46 (T46’Ilv-)
CRT4610 (T46’)
AB2151 (Ilv-Tna-)
115 (100%)
11v+
CRT833 (Ilv-T8SB
AB2151 (Ilv-Tna-)
100 (100%)
11v+
T46 +
Suspensions of phage PlKc were prepared by infection of various donor strains grown overnight at 30°C in broth supplemented with thymine. The recipients were infected according to Lennox (1955). EM sugar agar (Lederberg, 1950) supplemented with 0.15% of sodium citrate and required nutrients of recipient bacteria was used for the selection medium. Transductants were selected on a medium deprived of isoleucine and valine (selection of Ilv+) at 30°C or with the full supplements of required nutrients at 41°C (selection of T46+).
THERMOSENSITIVE
XYl I
Mtl II
:o I 71
DNA
PyrE I Hfr PI3
I
72
DnaA j PhoS IIII II
I
73
385
INITIATION
Ilv II
MetE I
1 I
74
75 min
FIG. 10. A tentative map order of DnaA on the E. coli chromosome. Relative map position of DnaA on the E. cold chromosome is schematiortlly given with the other reference markers used. Numbers under the map indicate the relative time position of reference markers according to Taylor & Trotter (1967).
4. Discussion In the replicon model (Jacob et al., 1963) it was assumed that both the initiation of a DNA cycle of replication at a fixed point of the chromosome and the whole process of replication occurred in the cell membrane, which co-ordinates the series of events taking part in cell division. The model predicts therefore the existence of mutants in which a simple genetic alteration disturbs both membrane properties and DNA synthesis, the latter by preventing either replication or initiation. Mutants CRT46 and CRT83 appear to present the properties expected from the latter type. No alteration can be detected among the enzymes now assumed to be involved in DNA synthesis, repair or degradation (personal communications from Dr G. Buttin and from Dr M. Kohiyame). The lesion appears to be specific to the bacterial chromosome, since phage A does multiply at high temperature. Furthermore, chromosome replication can be shown to occur at 41°C in certain classes of HFrT46 where the integrated F factor appears to supply the missing function(s) (Nishimura, Caro, Berg & Hirots, msnuscript in preperation). Although the nature of the T46 lesion cannot yet be precisely described, there is little doubt that in these mutants, altered membrane properties and DNA synthesis are correlated. On one hand, the band pattern of proteins extracted from membranes in cells grown at 41°C appears to be specifically changed; but the nature of the lesion cannot yet be ascribed. On the other hand, DNA synthesis appears to be halted at a specific point at 41°C; where activity is resumed upon a shift back to 30°C; but it cannot be determined as yet whether or not the point of arrest at 41°C and restart at 30°C does correspond to the physiological origin of the chromosome. One cannot therefore decide which one of the observed alterations corresponds to the primary lesion resulting from T46 or T83 mutations, and which one is secondary. The two possible alternatives are as follows. (1) The altered product of the mutant allele is directly involved in the initiation process. At high temperature, it is unstable and csnnot fit properly into the membrane, which appears secondarily to be altered. (2) The product of the mutant allele is a structural part of the membrane, in which the complex of enzymes involved in initiation and/or replication is inserted. Its alteration results in a change of configuration in the complex so that the enzymes which have to be synthesized for initiation to occur either can not fit, or not be active. There are few arguments as yet to support either hypothesis. On one hand, in order to be detectable by electrophoresis, the change in membrane proteins must affect
386
Y. HIROTA,
J. MORDOH
AND
F. JACOB
several per cent of the total membrane protein; in contrast, the factors directly involved in initiation must be present in very small amounts ; it is not clear therefore how a primary change in the latter could result in such a change in the former. On the other hand, while arrest in DNA initiation appears to follow immediat’ely on the shift to 41°C changes in the membranes can be detected only after 60 minutes at 41°C by the various techniques used. The two arguments work therefore in opposite directions. It is not difficult to produce a model to solve this apparent paradox, but at present it would be of little value. The results of the experiments with the mutant CRT46 can be interpreted by saying that the mutation has altered a protein the presence of which is required for DNA synthesis to be initiated. One can consider this statement in the light of negative verse positive regulation. Negative means that the enzymes ensuring initiation are constantly available during the division cycle, the presence of some factor, acting as a repressor, preventing initiation occurring during most of the cycle ; initiation would then result from derepression occurring for example in repressor dilution during growth (Pritchard, Barth & Collins, 1969) or synthesis of an antirepressor during growth (Rosenberg, Cavalieri & Ungers, 1969). On the other hand, positive regulation means that during most of the cycle, some factor required for initiation to occur is missing and that, in addition, this factor can only be used once; initiation would then result from the synthesis of this factor. One should emphasize that this positive model does not exclude a negative control operating on the synthesis of such an initiating protein. If one assumes the negative model, the lesion of CRT46 cannot be interpreted as an alteration of the regulatory mechanism. Since only 10 minutes at low temperature, i.e. one tenth of the generation time, is enough for re-initiation to occur (Fig. 3(a)), one cannot invoke the dilution of a repressor during this time. If an antirepressor is the affected element, this should only be compatible with a model where the antirepressor is synthesized immediately after DNA synthesis stops, either because a cycle is jlnished or because it is interrupted. Alternatively, CRT46 may affect some initiator enzyme made constitutively. If one considers a positive model, then one has only to assume that the protein altered in CRT46 is some component which acts on the chromosome to allow replication to start; furthermore, this component would act only once, so that its synthesis would be required for initiating every DNA cycle. Finally, a last point should be mentioned. It has been observed for a long time that any arrest in DNA synthesis results in a cessation of septum formation. As a consequence, filaments are formed. This indicates some kind of co-ordinated regulation in the various processes involved in cellular division. CRT46 does not escape this rule: since it divides probably once at 41”C, the DNA cycle already initiated is completed and then forms long filaments. But it underlines another aspect of this co-ordination. After a stay at 41”C, and then a shift back to 3O”C, cellular division occurs only after the new cycle of DNA replication has been completed. We thank Misses C. Barnoux and J. Perreau for their during the course of this work. We also thank Dr M. Kasai and his calculations on fluorescence depolarization.
excellent technical assistance for fluorescence measurements
This work was supported by grants from the Centre National de la Recherche Scientifique, the Commissariat it 1’Energie Atomique, the Delegation G&r&ale a la Recherche Scientifique et Technique and the National Institutes of Health, U.S.A. One of us (J.M.) is a Fellow of the John Simon Guggenheim Memorial Foundation.
THERhlOSENSITIVE
DNA
INITIATION
387
REFERENCES Bird, R. & Lark, K. G. (1968). Cold Spr. Hurb. Symp. Quant. Biol. 33, 799. Echols, H., Garen, A., Garen, S. & Torriani, A. (1961). J. Mol. BioE. 3, 425. Fangman, W. & Novick, A. (1968). Gknetic-s, 60, 1. Gartner, T. K. & Riley, M. (1965). J. Bact. 89, 319. Helmstetter, C. E. L Cooper, S. (1968). J. Mol. Biol. 31, 507. Hirota, Y., Jacob, F., Ryter, A., Buttin, G. & Nakai, T. (1968). J. Mol. Biol. 35, 175. Hirota, Y., Ryter, A. & Jacob, F. (1968). Cold Spr. Harb. Symp. Quant. BioZ. 33, 677. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spr. Harb. Symp. Quunt. BioZ. 26, 329. Jacob, F. & Wollman. E. (1961). Sexuality und Genetics of Bacteria. New York: Academic Press Inc. Kasai, M., Changeux, J. P. & Monnerie, L. (1969). Biochem. Biophys. Res. Comm. 36, 420. Kohiyama, M. (1968). Cold Spr. Harb. Symp. Quant. BioZ. 33, 317. Kohiyama, M., Lamfrom, H., Brenner, S. & Jacob, F. (1963). C. R. &ad. Sci. 265, 1820. Lark, K. G., Repko, T. & Hoffman, E. 5. (1963). Biochim. biophye. Acta, 76, 9. Lederberg, J. (1950). Methods in Medical Research, vol. 3,5. Year Book Publishers, Chicago. Lennox, E. S. (1955). Virology, 1, 190. MaaIm, 0. & Hanawalt, P. C. (1961). J. Mol. BioZ. 3, 144. Maalee, 0. & Kjeldgaard, N. 0. (1966). Control of Macromolecular Synthesis. New York: W. A. Benjamin Inc. Monnerie, L. & Neel, J. (1969). J. Chim. phys. 61, 504. Monod, J., Cohen-Bazire, G. 8; Cohn, M. (1951). Biochim. biophys. Acta, 7, 585. Nagel de Zwaig, R. & Luria, S. E. (1967). J. Butt. 94, 1112. Nomura, M. (1964). Proc. Nat. Acud. Sci., Wash. 52, 1514. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature, 204, 1069. Pritchard, R. H., Barth, P. T. & Collins, J. (1969). Symp. Sot. Gen. Microbial. 19, 263. Pritchard, R. H. & Lark, K. G. (1964). J. Mol. BioZ. 9, 288. Puig, J. & Azoulay, E. (1967). C. R. Acad. Sci. 264, 1916. Ramakrishnan, T. & Adelberg, E. A. (1965). J. Bact. 89, 661. Rosenberg, B. H., Cavalieri, L. F. & Ungers, G. (1969). Proc. iNut. Acad. Sci., W’mh. 63, 1410. Shapiro, B., Siccardi, A., Hirota, Y. & Jacob, F. (1970). J. Mol. BioZ. 52, 75. Stryer, L. (1965). J. Mol. BioZ. 13, 482. Taylor, A. L. & Trotter, 0. D. (1967). Bact. Rev. 31, 332. Weber, G. & Young, L. E. (1964). J. BioZ. Chem. 239, 1415. Yoshikuwa, H., O’Sullivan, A. & Sueoka, N. (1964). Proc. Nat. Acad. Sci., Wash. 52, 973.