J. Mol. Biol.
(1978)
122, 271-286
Studies on the Regulation of Initiation of Chromosome Replication in Escherichia coli JOE
A.
PRALICK
Department of Microbiology Texas Tech University Xchool of Medicine Lubbock, Tex. 79409, U.S.A. (Received 4 August 1977, and in revised form I March 1978) The total initiation frequency of chromosome replication in Escherichia coli is dependent on two factors; the timing or time interval between successive initiations on an individual chromosome (initiation pace) and the number of individual chromosomes which are being replicated per cell. We have examined these parameters in a dnaAtS, conditionally-lethal, “initiation mutant” of an E. co& K12 strain growing at different permissive temperatures. Our results indicate that at temperatures between 30 and 35’C the gene product of the drzaA167 allele becomes limiting with respect to the number of replicating chromosomes per cell, which decreases from two at 30°C to one at 35°C. However, over this same temperature range it is clear that cell growth is balanced and the initiation pace, as determined from the growth rate, increases with temperature and is indistinguishable from that of the dnaA + parent. These results demonstrate that one can alter the total initiation frequency independently of the initiation pa,ce, indicating the involvement of at least two cellular components in the regulation of initiation. They also suggest that while the d%aA product may be involved in determining the total number of initiation events which can occur per cell per doubling time it does not control the timing or pace at which successive initiation events are triggered on each chromosome, i.e. it is not the “pace-maker” for initiation.
1. Introduction The initiation of chromosomal replication has been shown to play a central role in the cell cycle of the bacterium Escherichia coli (Yoshikawa et al., 1964; Maalee & Kjeldgaard, 1966; Cooper & Helmstetter, 1968). The rate of DNA synthesis, the amount of DNA per cell and the timing of cell division have all been shown to be tightly coupled to this event (Helmstetter et al., 1968; Yoshikawa & Haas, 1968; Donachie, 1968; Sompayrac & Maalee, 1973). However, although the need for de novo protein and RNA synthesis in the initiation process has been demonstrated (Maalee & Hanawalt, 1961; Lark & Renger, 1969; Lark, 1972; Messer, 1972), the actual means by which the cell regulates this important activity has remained obscure. Theoretically there are two distinct levels at which the control of initiation could exist. On one hand, regulation could be exerted by determining the rate or frequency at which each chromosome is initiated, a parameter which will be defined as the initiation pace. Under balanced growth conditions this would also determine the rate of termination of chromosomal replication and presumably the rate of cell division (Helmstetter et al., 1968; Donachie, 1968; Pardee & Rosengurt,, 1975). It is clea’r from 271 0022-2836/78/1223-7186
$02.00/O
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Press Inc. (London)
Ltd.
272
S. A. FRALICK
the work of Cooper & Helmstetter (1968) and of Bird& Lark (1968) that the initiation pace can be altered with nutritional shifts. On the other hand, the initiation of chromosomal replication could also be regulated by determining the number of initiation events which occur per cell per unit time, which we wiI1 define as the total initiation frequency and which is equivalent to the product of the initiation pace and the number of replicating chromosomes per cell. The experimenta approach used in this study has been to examine the initiation pace and the total initiation frequency of a dnaAts mutant growing under physiological conditions at. which the temperature labile gene product is limiting (i.e. at semipermissive temperatures). The resuIts demonstrate that the total initiation frequency can be varied independently of the initiation pace in this mutant, indicating that there are at least two separately regulated cellular components which control the initiation of chromosomal replication in E. coli.
2. Materials and Methods (a) Bacterial
strahs
All bacteria used in these experiments were derivatives of E. co&i K12 strains. The pertinent properties of these strains are given in Table 1. The E. coli strain, designated N167, selected for this study has been well-characterized as a temperature sensitive, conditionally lethal mutant unable to initiate new rounds of chromosomal replication at the non-permissive temperature and whose temperature sensitive lesion maps at the dnaA locus (Abe & Tomizawa, 1971; Hirago & Saitoh, 1974, 1975; Saitoh & Hiraga, 1975). The dnaA gene product of this strain is apparently a thermolabile protein which is converted t.o an inactive state at the restrictive temperature but which, upon a shift back to the permissive temperature, can resume an active conformation in the presence of chloramphenicol (i.e. its activity is thermoreversible) (Hirago $ Saitoh, 1974). In our hands, growing in a glucose/Casamino acid minimal salts mediam the non-permissive temperature for this strain lies between 42 and 43°C. Below 42”C, DNA synthesis can continue for more than a single round of replication. (b) Isotopes and chemicals (0.25 mCi/O.GTi mg) were [MethyZ-3H]thymine (5mCi/0.32 mg) and [methyZ-14C]thymine purchased from New England Nuclear Corp. Oleic acid (sodium salt, 99% pure), Brij 58, all amino acids, thymine and rifampicin were purchased from Sigma Chemical Co. Ilford Nuclear Research Emusion L4 was purchased from Polyscience Inc., Warrington, Penn. (c) Growth of bacteria grown in a New Brunswick gyratory water bath Bacterial cultures were routinely (Aquatherm G86) at 200 revs/min. The temperature was monitored with a telethermometer Springs, Ohio). The precision of temperature (Yellow Springs Instrument Co., Yellow measurement and regulation for the instruments is &O-l deg. C. 1972) supplemented with 0.4% Bacteria were grown in minimal A medium (Miller, glucose as a carbon source and containing 0.1 o/0 Casamino acids (Difco, vitamin-free) plus the required supplements which included : thymine (5, 10 or 20 pg/ml), thiamine (2 pg/ml), methionine (50 pg/ml), tryptophan (50 pg/ml), and oleic acid (IO pg/ml) dissolved in,0.02°/0 Brij 58 (polyoxyethylene 20 cetyl ether), depending on the E. coli strain used. (d) Measurement
of cell growth
Extinction of cultures was measured in an Acta CIII Instruments, Inc.) at a wavelength of 550 nm. Uninoculated turbidity control.
spectrophotometer (Beckman medium was used as a zero
strains
thy, met, dnnA167 thy, met
from
J. Wechsler
J. Wechsler
P. Overath
J. Tomizawa J. Tomizawa
Obtained
coli &ailzs
1
revertant
by Overath
Transductant
Described
of JW150
by Wechsler
using E508 as the donor
& Zdzienicka.
of K1059 (1975)
of N167 which can grow at 43°C
(1971)
et (II. (1970)
Thymine-requiring mutant Transductant of K1059-T
Described
a spontaneous
Described by Abe & Tomizawa Parental strain of N167
Remarks
t Genetic abbreviations are as used by Bachmann et al. (1967). The allele number of the dnu mutations is according to Gross (1972). $ A thy mutant of K1059 was selected for with trimethoprim according to the method of Miller (1972). From these thy mutants low t,hymine requirers were selected. §The dnaA167 allele was co-transduced with bgl marker. SZ:Tmutants of N167 were selected according to Miller (1972).Pl transduction was carried out as described by Miller (1972).
thy, lac, rha, str, dnnA508
F-,
E508
JW154
f&B, fadE, thi, th,y
F-,.fabB, f&E, thi, thy, dnaA167 F-, thy, thr, leu, thi, kzc, tonA, &=, dlzaA508
F-,
HfrH, thy, met, R3 F- , fnbl3, fadE, thi
HfrH, HfrH,
Description?
KA22§
K1059 KlOBY-T%
HMT N167-R
N167
Bacterial
Escherjchia
TABLE
274
J. A. FRALICK
Cell number was measured by counting cells in physiological saline (Cutter Laboratories, Inc.) on a Coulter counter model ZF (Coulter Electronics, Inc.) using a 30 pm orifice, l/aperture setting of l/4, l/amplification setting of 1 and a lower threshold of 5 to 10. On occasion, cells were also count,ed microscopically using a Petroff-Hauser counting chamber. Viable cell counts were determined by colony counts on the appropriate media. (e) Labeling
procedure
Exponentially growing cells were labeled at cell densities ranging from 5 x lo7 to 2 x lo* cells/ml. When [14C]thymine was used, cultures were labeled at a spec. act. of 0.5 PCi (5 pg)/ml. In steady-state labeling experiments the cells were labeled for more than 4 doublings before samples were taken. (f) Measurement
of raclioisotope
incorporation
Incorporation of radioactive precursors, [3H]thymine or [14C]thymino, into acidinsoluble material was measured by collecting 0.05 to 0.1 ml samples on Whatman 3 MM filter discs and washing them by passage through trichloroacetic acid and alcohol (Fralick & Lark, 1973). Dried discs were counted in a Beckman scintillation counter, model LS-230 using 0.5% 2,5-bis-2-[5-tert-butylbenzoxazolyll-thiophene (Research Products International Corp., Elk Grove Village, Ill.) dissolved in toluene as a scintillation cocktail. Under our high voltage settings, gain settings and window settings the counting efficiency for 3H was 14% and that for 14C was 61%. (g) Chemical
estimation
of DNA
and
protein
Exponentially growing cells were harvested in a cell density of 1 x lo8 to 2 x IO8 cells/ml by centrifugation. Samples were washed 3 times by centrifugation and resuspended in 5 ml of ice-cold buffered saline containing 0.01% gelatin (usually at about lo9 cells/ml). Cell numbers were determined at this point with a Coulter counter. Concentrated perchloric acid was then added to a final concentration of 1.0 M and then heated to 90°C for 20 min with agitation. The hydrolyzed samples were chilled in an ice-bath and centrifuged to pellet the acid-insoluble cell components. The supernatant was assayed for deoxyribose by the modified diphenylamine method described by Giles & Myers (1965). Calf thymus DNA wa,s extracted similiarly for use as a standard. A DNA standard curve was constructed for each experiment. The acid-insoluble pellet from the extraction described above was resuspended in 5 ml of 1 Jr-NaOH and left in a stoppered tube overnight at room temperature. Protein analysis of this extract was performed by the method of Lowry et al. (1951). Bovine serum albumin in 1 Jr-NaOH was used as a standard, and a standard curve was run for each experiment. (h) Autoradiography Radioactively labeled cells were fixed in 4% formaldehyde (Caro, 1970). Autoradiographs were prepared a.ccording to Lark (1968). Drops of cell suspension were placed on agar from which impression smears were made. The slides were dried, washed twice in 5% cold trichloroacetic acid, rinsed with water and dried. They were then dipped in 1: 1 dilution of Ilford Emulsion L4, dried and exposed at room temperature for a period of time. Slides were developed in Kodak D-19 developer for 3 min, fixed at room temperature and stained in 0.174 gentian violet for 1 min before examination under oil immersion (1200 x ) using a Zeiss phase-contrast microscope. Grain counts were analyzed as described in the text.
3. Results (a) Effect
of growth
temperature
on the initiation
pace of N167 cells
The time interval between successive DNA initiations in cells growing with balanced, steady-state growth has been shown to be equal in time to the interval between successive divisions (Maalme & Kjeldgaard, 1966; Helmstetter et al., 1968;
INITIATION
OF CHROMOSOMAL
REPLICATION
IS
Pa.rdee & Rozengurt, 1975). Thus the frequency of cell division, stances, can be considered equivalent to the initiation pace, (i) Temperature
E. COLI
“75
under there circum-
effect on cell division
The effect of growth temperature on the rate of cell division in the dnaAts strain, N167, and the dnaA+ parent, HMT, is shown in Figure 1. Note that both of these strains had similar, if not identical, temperature coefficients for cell division at temperatures below 35°C. At growth temperatures above 35”C, however, the rate of cell division for the mutant leveled off and began to decline. Therefore, it appears that the dnaA167 gene product became rate limiting for cell division at temperatures greater than 35°C.
l-6
0
I
I
35
30
Temperature
0
(“Cl
FIG. 1. Effect of growth temperature on division rate in N176 and HMT cells. X167 ( 0) and HMT ( 0) cells were grown at the indicated temperature for more than 5 doublings before determining the exponential cell division coefficient (growth rate). The exponential growth rate was determined with a Coulter counter. Greater than 5 different sampling times were used to caldate the growth rate of each oulture.
(ii) Evidence for balanced growth Figure 2 presents the increase in cell mass (A,,,), cell number (particle count) and DNA content (steady-state [14C]thymine corporation) of Nl67 cells which had been growing at the extremes of the temperature range studied (above 30°C and 38°C). Similar results were obtained at the intermediate growth temperature of 34°C (data not shown). From these data it can be seen that N167 cells can grow with balanced,
276
J. A. FRALICK
(b)
I
I
I
1
I
20
40
60
80
100
120
Time (min)
2. Growth characteristics of N167 cells growing at 30°C and 38°C. Cultures of Nl67 cells were grown in glucose/Casamino acid medium (see Materials and Methods) containing [l%]thymine (0.5 @iI5 pg per ml) for 4 doublings before starting the experiment. Samples were removed at the time intervals indicated and the rate of increase in absorbance (-u-C-), particle number (-O-O-) and [‘T!]thymine incorporation (--A-A-) was determined at 30°C (a) and 38°C (b). FIG.
exponential growth at temperatures as high as 38°C. Therefore, it appears that the product of the dnaA167 gene becomes limiting with respect to the initiation pace only at growth temperatures above 35°C. (b) Efj’ect of growth temperature (i) DNA
on chromosome
copy number of N167 cells
content
It has been calculated from the amount of acid-insoluble [14C]thymine incorporation in Figure 2 that the amount of DNA per cell in N167 cells growing at 38°C is approximately one-half that of N167 cells cultured at 30°C (13,000 cts/min per IO* cells and 23,000 cts/min per 10’ cells, respectively). One artifact which could lead to this result would be that the specific activity of the DNA of these cells was temperature dependent. As shown in Table 2, the specific activity of the DNA in N167 cells
1 2 3
1 2 3
30
38
1 2 3
1 2 3
Experiment number
38
30
Growth temperature (“Cl
15.2 15.8 14.5
14.7 13.7 13.9
6.69 7.1 6.8
12.6 14,5 13.7
DNA/cell (pg x 10-y
2146 -.
2288 -
2344 --
-
2292
Specific activity7 of DNA (cts/min per pg)
3.6 3.7 3.4
3.5 3.2 3.3
1.6 1.7 1.6
2.9 3.4 3.2
Chromosome$ equivalents/cell
2.5 2.6 2.4
2.4 2.2 2.6
I.09 1.16 1.09
2.0 2.3 2.2
Heplicating § chromosome equivalents/cell
on chromosome composition
2
355 396
325 330
406 355
327 326
Protein/cell (g x 10-y
0.044 0.036
.0.042 0.042
0.018 0.019
0.044 0.042
DNA/protein ratio
t N167 and HMT ~011sgrowing at 30°C and 38OC were harvested by oentrifugation at a population density of approx. 2 x lo* cells/ml and analyzed for DNA and protein as described in Materials and Methods. In this experiment the cells were grown for more than 4 doublings in media containing [‘*C]thymiuo (0.1 @/ 5 pg per ml). Cell protein was not determined. $ Chromosome equivalents were calculated by assuming the mass of a unit chromosome to be 4.22 x lo-l5 g which corresponds to a molecular weight of 2.5 x 109 daltons for the E. csli chromosome. 8 For comparative purposes the value of 1.44 unit chromosomes per replicating chromosomes was used in this calculation. This value was obtained from the equation of chromosomal replication (Sueoka & Yoshikawa, 1965) assuming the average number of replicative forks (pairs of replicative forks) per chromosome and the replicative position distance (HP-distance) to be one (Table 3) in cells growing at both temperatures.
HMT
N167
Strain
Effect of growth tenxpernture
TABLE
J. A. FRALICK
278
and the dnaA+ parent, HMT, was not affected by growth temperature. However, the actual amount of DNA per cell, and the DNA to protein ratio in the dnaAts mutant growing at 38”C, was decreased by a factor of two compared to the same cells growing at 30°C. This temperature dependent difference was not observed with the dnaA+ strain. That this temperature effect on DNA content in N167 cells was representative of the majority of the population of the culture can be seen from the autoradiographic analysis of Figure 3. The effects of intermediate growth temperatures on the cellular DNA content in N167 and HMT cells is illustrated in Figure 4. Note that the relative amount of DNA per cell for HMT remained relatively constant over the temperature range examined. The dnaA167 mutant, however, demonstrated a decrease in DNA content with increasing growth temperatures between 31°C and 35°C. These results suggest that the dnaA167 gene product becomes limiting for the amount of DNA on a per cell basis at temperatures above 31°C.
3(
Ln = 9 z
2(
B E z’ IC
0123456705
,
Groins /ccl I
FIG. 3. Autoradiographio N167 cells were grown for more per ml) at 38°C (a) and 30°C (b). Materials and Methods (exposure
histograms of N167 cells growing at 30°C and 38°C. than 4 doublings in the presence of [3H]thymine (100 @i/5 p*g Autoradiographs of these cells were prepared as described in time 17 days) and grains were counted.
(ii) Chromosome composition To determine if the temperature dependent difference seen in the DNA content of forks per chromoN167 cells had been due to a difference in the number of replicative some or to a difference in the number of replicating chromosomes per cell, the increment in DNA synthesized at the restrictive temperature by N167 cells which had been growing at 30, 34 and 38°C was measured. The underlying assumption to this experiment was that new rounds of chromosomal replication could not commence after transfer to the restrictive temperature and that existing replication forks would progress to the chromosome terminus (Abe & Tomizawa, 1971). The amount of DNA synthesized at the non-permissive temperature, therefore, would be a measure of the relative number and position of replication forks on the chromosome (Sueoka &
INITIATION
OF CHROMOSOMAL
REPLICATION
Temperature
IN
E. COLI
279
PC)
FIG. 4. Effect of growth temperature on the DNA content of N167 and HMT cells. and HMT (- X-X-) cells were grown at the inCultures of N167 (0, q , A, XjJ, -----) dicated temperatures for more than 4 doubling3 in the presence of [l%]thymine (0.5 $3/B pg per ml) before being assayed for acid-insoluble radioactivity. Cell number was determined with a Coulter counter. Eash symbol type represents a series of independent experiments. Each data point is the average of 6 or more different determinations.
Yoshikawa, 1965). Table 3 presents the results. The calculated increase in DNA, as measured by [14C]thymine steady-state labeling, was essentially the same for all three cultures and is consistent with only one pair of replicating forks per chromosome (39:/, residual increase) (Sueoka & Yoshikawa, 1965). These results are significently different from the 84 and 137% residual increase expected from chromosomes concontaining two and three pairs of replication forks, respectively. Similar results were obtained by blocking new rounds of initiation with the addition of rifampicin (data not shown). Therefore, it appears that the temperature sensitivity of the c&A167 gene product does not affect the number of replication forks per chromosome or the rate of polynucleotide chain elongation per se but more likely affects the number of chromosomes per cell. This explanation is also in agreement with the data of Table 4, which indicate that the amount of DNA per cell in N167 cells growing at 38°C was compatible with a single replicating chromosome that had only one pair of replication forks (Sueoka & Yoshikawa, 1965), while the same cells growing at 30°C contained
J. A. FRALICK
280
TABLE 3 Residzcal increase in the amount of DNA
synthesized at 43°C
Growth temperature before shift to 43°C
Residual increase in DNA synthesized at 43OC
30 34 38
43.4&&S 38.316.1 38.2*&S
Cultures of N167 were grown at the indicated temperatures in media containing [l%]thymine (0.5 @i/B pg per ml). At a titer of 5 x 10’ to 10s cells/ml, they were placed at 43°C and the incorporation of thymine measured. The residual increase in DNA was measured as the difference between the initial radioactivity. These values represent mean and standard deviation from a minimum of 4 independent experiments.
TABLE 4 Effect of growth temperature on the DNA/protein ratio of dnaA initiation mutants and their derivatives
Strain?
Relevant genotype
N167
dn,aAtS
N167-R$
R3
HMT
dnaA +
KA22
dnaAts
K1059-T
dnaA +
E508
dnaAtS
JW154
dnaAt”
Growth
temperature (“C) 38 30 38 30 38 30 38 30 38 30 38 30 38 30
DNA/protein ratio
DNA/protein DNA/protein
0.017 0.041 0.045 0.043 0.048 0.042 0.013 0.036 0.040 0.038 0.02 0.044 0.0125 0,0325
Cultures of the above strains were grown at 30°C or 38°C to a cell density 2 x lo* to 3 x lOa and then harvested by centrifngation. The DNA and proteins and assayed as described in Materials and Methods. t For description of these strains, see Table 1. f Ts+ reversion of the dnaA167 mutation.
at 38°C at 30cc
0.4 1.05 1.1 0.33 1.04 0.45 0.38
of approximately were precipitated
DNA for two such replicating chromosomes. These results therefore suggest that the dnaA167 gene product becomes limiting for the number of replicating chromosomes per cell (chromosome copy number) at temperatures above 30°C.
enough
(c) Effect of growth temperature on total initiation frequency in N16Y cells The total initiation frequency (TIP) is equivalent to the product of the initiation (IP) a.nd the number of replicating chromosomes per cell (N) (i.e. TIF = IP x N). If one assumed that the growth rate (under balanced growth conditions)
pace
INITIATION
OF CHROMOSOMAL
REPLICATION
IN
E. COLI
281
was equivalent to the initiation pace and that the DNA content (as determined from [14C]thymine steady-state incorporation) was directly proportional to the number of replicating chromosomes per cell, then one could estimate the total initiation frequency for N167 and HMT cells growing at different growth temperatures from the data of Figures 1 and 4. Such calculations have been plotted in Figure 5. Note that while the t’otal initiation frequency for HMT cells increased in a linear fashion with increasing temperatures between 31 and 38”C, it decreased for N167 cells growing over this same temperature range. In fact, unlike the effect of growth temperature on initiation pace or DNA content per cell, this decrease in the calculated total initiation frequency for N167 cells is linear over the entire temperature range examined. lt appears, therefore, that the dnaA167 product becomes limiting for the tot,al initiation frequency at growth temperatures great’er than 31°C. I
I
30
I
I
/
I
I
I
,
I
35 Temperature
PC)
5. Effect of growth temperature on the total initiation
frequency of N167 and HMT cells. The values for this Figure were derived from the data of Figs 1 and 4 using the equation: Total initiation frequency = initiation pace x chromosome copy number. In this calculation we have assumed the cell division frequency to be equivalent to initiation pace and that the radioactivity of N167 cells growing at 38°C to be equivalent to one replicating chromosome per cell on the average (see Table 2). FIG.
(d) EJect of growth tempembure on another dnaAtS mutant In an attempt to determine whether the temperature dependent effect seen in A-167 cells was characteristic of the dnaAtS phenotype in general or to the N167 strain in particular, the DNA to protein ratio in an independently derived E. coli K12 dnaAtS mutant and its derivatives were examined. The results are summarized in Table 4. It can be seen that there was little or no temperature dependence of this ratio in the parental HMT and K1059-T cells or in a spontaneous revertant of the dnaAtS phenotype (Nl67-R). However, all of the dnaAts mutants tested show a
282
J. A. FRALICK
decreased DNA to protein ratio at 38°C. From these results it may be concluded that the temperature dependent effects observed with N167 cells were not unique to their specific dnaAts allele nor to the genetic background with which it was associated.
4. Discussion It is an accepted view that’ the bacterial cell regulates DNA synthesis by controlling the initiation frequency of chromosomal replication (Maaloe & Kjeldgaard, 1966; Helmstetter Q Cooper, 1968; Lark, 1968; Bleecken, 1971; Sompayrac & Maalee, 1973 ; Pardee & Rozengurt, 1975). However, there are at least two aspects of initiation frequency which may or may not be subject to the same control mechanism(s). The first aspect deals with the individual chromosome. How often will each chromosome be initiated? Under balanced growth conditions the bacterial cell must be able to monitor its growth rate and accurately adjust the timing between successive initiation events so that new rounds of chromosomal duplication occur once, and only once, each time the cell mass and/or cell number doubles. This aspect of initiation frequency has been termed the initiation pace, which is equiva’lent to the pace at which cells divide under steady-state growth conditions. The second aspect deals with the total number of initiation events that’ take place per cell division. This factor also controls the average number of replicating chromosomes that are present in a cell during balanced growth (chromosome copy number). We have termed this aspect of initiation frequency the total initiation frequency, which is equal to the product of the initiation pace and the number of replicating chromosomes per cell. We have examined the effects of growth temperature on these aspects of initiation in a dnaAts mutant and its dnaA+ parent. The results of this study are summarized in Figure 6 and can be described as follows. (1) The dnaA167 gene product becomes limiting for t’he total initiation frequency at growth temperature greater than 31°C. This is illustrated in Figure 6(a), in which the effect of growth temperature on the derived values for the total initiation frequency is given. Note that the overall initiation frequency for this mutant diminishes linearly over this temperature range, indicating that the dnaA167 gene product, or some fraction of its total concentration, becomes temperature labile at growth temperatures above 31°C and that the magnitude of t’his effect increases with increasing growth temperature. (2) The dnaA167 gene product becomes limiting for the cells’ chromosome composition at growth temperatures above 31°C. This can be seen in Figure 6(b), in which the amount of DNA per cell has been equated to the number of replicating chromosomes per cell (Fig. 4 and Table 2). From this graph it can be seen that the average number of replicating chromosomes per cell in the dnaAts mutant decreases with increasing temperatures over the temperature range of region I (31 to 35”C), after which it levels off to equal approximately one-half that of the parental dnaA+ strain growing at the same temperature. Quantitatively, N167 cells growing at 30°C contain enough DNA for two replicating chromosomes, while those growing at 38°C contain only enough DNA for one (Table 2). At both of these growth temperatures, growth is balanced and each chromosome contains only one pair of replication forks (Table 3). (3) The initiation pace, as determined by the frequency of cell division under balanced growth conditions (Figs 1 and 2), remains normal for the dnaAts mutant compared to the dnaA+ parent over the growth temperatures of region I (Fig. 6(o)).
INITIATION
OF CHROMOSOMAL
0.71
, 32
,
REPLICATION
,
j
,
35 Temperature
,
IN E. COLI
283
, 30
(“Cl
FIG. 6. Summary of effect of growth temperature on chromosomal replication in N167 cells. Figs 1, 4 and 5 were used to compute the ratios of the mutant (&xx+) values for total initiation frequency (a), chromosome copy number (b), and initiation pace (c). In this presentation we have equated cell division frequency with initiation pace and DNA content with chromosome copy
number. The temperature range (31 to 3892) has been divided into two regions: region I corresponds to temperatures between 31°C and 35T and region 11 to temperatures above 3692. Only at growth temperatures above 35°C does the dnaA167 lesion effect this parameter. In summary, the results presented clearly illustrate that the two components of the total initiation frequency of chromosomal replication; initiation pace and the number of replicating chromosomes per cell, can be varied independently of one another in a cl?zuAtS mutant. These results demonstrate the existence of at least two independently regulated cellular components which are involved in the control of chromosomal replication in E. coli. Furthermore, the observation that the initiation pace remains normal over a temperature range which adversely affects the dnaA product (31 to 35°C) suggests that this gene product is not the “pace-maker” or the
284
J. A. FRALICK
regulatory signal which triggers successive rounds of initiation on a chromosome. However, the dnaA gene product, clearly affects the total initiation frequency; t,he higher the temperature, the lower the total initiation frequency, presumably because of decreasing levels of active dnaA gene product’. The actual means by which the product of the d%aA gene exerts its effect, on initiation is unknown. However, the mere number of gene products involved in the initiation and replication of the bacterial chromosome (Wechsler $ Gross, 1971; Geider, 1976; Wechsler, 1978; Alberts & Stermglanz, 1977) indicates that replication is probably carried out by an assemblage of interacting proteins which may form the hypothetical “replication apparatus” (Jacob et al., 1963; Lark $ Lark, 1964,1966; Bleecken, 1971 i Fralick & Lark, 1973; Helmstetter, 1974; Kogoma & Lark, 1975) and it is, therefore, tempting to speculate that the dnaA gene product is a component of this complex, essential for its maturation or structural imegrity. This hypothesis can explain our data as follows. The total initiat)ion frequency of chromosome replication is dependent on two factors; the rat’e at, which initia,tion-replication complexes are matured or assembled, which is influenced by the number of functional dnad proteins in the cell, and the frequency at which these complexes are Driggered, which is controlled by the pace-maker. Thus, at the permissive growth temperatures of 39°C two functional replication apparatuses are assembled and triggered approximately every 55 minutes in both the dnats mutant and the dnaA+ parental strain. However, at temperatures above 31°C the dnaA167 gene product becomes rate limiting for the assembly of functional complexes and hence for the total initiation frequency. Consequently, a decrease in the total initiation frequency is observed with increasing temperature of growth temperatures above 31°C (Fig. 5) until the non-permissive 43°C is reached, at which no functional replicative apparatuses are assembled and thus no new initiation events can occur. The initiation pace of the dnaAtS mutant should not change compared to wild type complex per cell can be assembled more rapidly as long as a single mature replication than the frequency at which a second regulatory component, the pace-maker, triggers it off. Thus, over the 31 to 35°C temperature range the rate at which replication complexes are assembled is falling continuously with increasing temperature, but the frequency at which these complexes are triggered (initiation pace) remains the same as wild type (region I in Fig. 6). L4t 35°C the rate at which a single mature complex is assembled per cell is equal to the rate at which it is triggered, and the chromosome number therefore equals one. Thus, at growth temperatures up to 35”C, the dnaA gene product has not affected the initiation pace, only changing the average number of replicating chromosomes per cell. However, since the number of replicating chromosomes per cell will not fall below one, the rate at which replication complexes are assembled begins to limit the actual rate of initiations per chrosome at growth temperatures above 35°C. Therefore, even though the pace-maker may be signalling for more frequent initiation events, a decrease in the initiation pace is seen. Under these circumstances the dnaA gene product becomes limiting for the initiation pace (region II, Fig. 6). The above explanation is in accord with reports from other laboratories which demonstrate the dnaA gene product to be a trans.acting protein (Gotfried & Wechsler, 1977) which may act as a multimer or in a complex with other proteins as suggested by the partial dominance displayed by some drLaAts alleles (Zahn et al., 1977) and by allele specific suppressor mutations which map outside of the dnaA locus (Wechsler
INITIATION
OF
CHROMOSOMAL
REPLICATION
IN
E. COLI
285
& Zdzienicka, 1975). One prediction that can be made from this interpretation is the possible existence of a class of initiation mutants whose defect lies in the factor which triggers the initiation event (i.e. controls the initiation pace). We are currently searching for such a class of “initiation pace” mutants among the known temperature sensitive, conditionally lethal, initiation mutants of E. coli. I thank Dr B. of this manuscript I also thank Drs stimulating and assistance. This research (GM22653).
M. Olivera,, whose enthusiasm and encouragement have made the writing possible and whose critical suggestions have made it understandable. T. Kogoma, C. Lark, K. G. Lark, T. M. Joys and J. Wechsler for some very and Mr A. M. Davis for his excellent technical valuable discussions, was
supported
by
a grant
from
the
National
Institutes
of Health
REFERENCES 69, I-15. Abe, M. S-: Tomizawa, J. (1971). Genetics, (London), 269, 655-661. Alberts, B. & Sternglanz, R. (1977). Nature Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976). Bacterial. Rev. 40, 1161G7. Bird, R. & Lark, K. G. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 799-808. Bird, R. & Lark, K. G. (1970). J. Mol. Biol. 49, 343-366. Bleecken, S. (1971). J. Theoret. Biol. 32, 81-92. Caro, I,. G. (1970). J. Mol. Biol. 48, 320.-338. Cooper, S. & Helmstetter, C. E. (1968). J. &ZoZ. Biol. 31, 519-540. Donachie, W. D. (1968). Nature (London), 219, 107771079. Fralick, J. -4. & Lark, K. G. (1973). J. Mol. Biol. 80, 459-475. Geider, K. (1976). Current Topics Microbial. Immunol. 74, 55-112. (London), 206, 93. Giles, K. & Myers, A. (1965). Nature Gotfried, F. & Wechsler, J. A. (1977). J. BacterioZ. 130, 963-964. Gross, J. (1972). Current Topics Microbial. Immunol. 57, 39-74. Helmst’etter, C. E. (1974). J. Mol. BioZ. 84, 21-36. Helmstetter, C., Cooper, S., Pierucci, 0. & Revelas, IX. (1968). Cold Spring Harbor Symp. &ant. Biol. 28, 809-822. Hiraga, S. & Saitoh, T. (1974). Mol. Gen. Genet. 132, 49-62. Hiraga, S. 8: Saitoh, T. (1975). AVoZ. Gen. Genet. 137, 239-248. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spring Harbor Xymp. Quant. BioZ. 28, 329. Kogoma, T. & Lark, K. G. (1975). J. Mol. BioZ. 94, 243-256. Lark, C. (1968). &fethods Enzymol. 12, 616-625. Lark, C. & Lark, K. G. (1964). J. Mol. BioZ. 10, 120-136. Lark, K. 0. (1969). Annu. Rev. Biochem. 38, 569-604. Lark, K. G. (1972). J. Mol. BioZ. 44, 217. Lark, K. G. & Lark, C. (1966). J. Mol. BioZ. 20, 9-19. Lark, K. G. & Renger, H. (1969). J. Mol. BioZ. 42, 221-235. Lowry, 0. H., Rosebrough, A., Farr, A. L. & Randall, R. J-. (1951). J. BioZ. Chem. 193, 265-275. Maaloe, 0. & Hanwalt, P. C. (1961). J. Mol. BioZ. 3, 144-155. Maaloe, 0. & Kjeldgaard, PI’. 0. (1966). In Control of Macromolecular Synthesis, pp. 154182, W’. A. Benjamin Co., Inc., New York. Messer, M. (1972). J. Bacterial. 112, 7-12. Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Overath, P., Schrairer, H. U. & Stoffel, W. (1970). Proc. Nat. Acad. Sci., U.X.A. 68, 3180. Pardee, A. B. & Rozengurt, E. (1975). In Biochemistry of Cell JVaZZs and MembraTaes, (Kornbrrg, H. L. & Phillips, D. C., eds), pp. 155-185, University Park Press, Baltimore. Saitoh, T. & Hiraga, S. (1975). Mol. Gen. Genet. 137, 249-261. Sompapac, L. 85 Maalee, 0. (1973). Nature h’ew Biol. 241, 133-135. Sueoka, N. Xs Yoshikawa, H. (1965). Genetics, 52, 747-748.
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Wechsler, J. A. (1978). In NATO ASI: DLNA Synthesis Present and Future (Molineux, I. & Kohiyama, M., eds), Plenum Press, in the press. Wechsler, J. A. & Gross, J. D. (1971). Mol. Gen. Genet. 113, 273-284. Wechsler, J. A. & Zdzienicka, (1975). In DNA Synthesis and its Regulation (Goulian, M. & Hanawalt, P., eds), pp. 602-617, W. A. Benjamin Inc., California. Hartor Synrp. Quad. Biol 33, 843-855 Yoshikawa, H. & Haas, M. (1968). cold Spring Yoshikawa, H., O’Sullivan, A. & Sltcokn, N. (1964). Proc. Nat. Acad. Sci., U.S.A. 52, 973-980. Zahn, G., Tippe-Schindler, R. & Messer, W. (1977). Mol. Gen. Genet. 153, 45-49.
(1978)
122, 271-286
Studies on the Regulation of Initiation of Chromosome Replication in Escherichia coli JOE
A.
PRALICK
Department of Microbiology Texas Tech University Xchool of Medicine Lubbock, Tex. 79409, U.S.A. (Received 4 August 1977, and in revised form I March 1978) The total initiation frequency of chromosome replication in Escherichia coli is dependent on two factors; the timing or time interval between successive initiations on an individual chromosome (initiation pace) and the number of individual chromosomes which are being replicated per cell. We have examined these parameters in a dnaAtS, conditionally-lethal, “initiation mutant” of an E. co& K12 strain growing at different permissive temperatures. Our results indicate that at temperatures between 30 and 35’C the gene product of the drzaA167 allele becomes limiting with respect to the number of replicating chromosomes per cell, which decreases from two at 30°C to one at 35°C. However, over this same temperature range it is clear that cell growth is balanced and the initiation pace, as determined from the growth rate, increases with temperature and is indistinguishable from that of the dnaA + parent. These results demonstrate that one can alter the total initiation frequency independently of the initiation pa,ce, indicating the involvement of at least two cellular components in the regulation of initiation. They also suggest that while the d%aA product may be involved in determining the total number of initiation events which can occur per cell per doubling time it does not control the timing or pace at which successive initiation events are triggered on each chromosome, i.e. it is not the “pace-maker” for initiation.
1. Introduction The initiation of chromosomal replication has been shown to play a central role in the cell cycle of the bacterium Escherichia coli (Yoshikawa et al., 1964; Maalee & Kjeldgaard, 1966; Cooper & Helmstetter, 1968). The rate of DNA synthesis, the amount of DNA per cell and the timing of cell division have all been shown to be tightly coupled to this event (Helmstetter et al., 1968; Yoshikawa & Haas, 1968; Donachie, 1968; Sompayrac & Maalee, 1973). However, although the need for de novo protein and RNA synthesis in the initiation process has been demonstrated (Maalee & Hanawalt, 1961; Lark & Renger, 1969; Lark, 1972; Messer, 1972), the actual means by which the cell regulates this important activity has remained obscure. Theoretically there are two distinct levels at which the control of initiation could exist. On one hand, regulation could be exerted by determining the rate or frequency at which each chromosome is initiated, a parameter which will be defined as the initiation pace. Under balanced growth conditions this would also determine the rate of termination of chromosomal replication and presumably the rate of cell division (Helmstetter et al., 1968; Donachie, 1968; Pardee & Rosengurt,, 1975). It is clea’r from 271 0022-2836/78/1223-7186
$02.00/O
0 1978 Academic
Press Inc. (London)
Ltd.
272
S. A. FRALICK
the work of Cooper & Helmstetter (1968) and of Bird& Lark (1968) that the initiation pace can be altered with nutritional shifts. On the other hand, the initiation of chromosomal replication could also be regulated by determining the number of initiation events which occur per cell per unit time, which we wiI1 define as the total initiation frequency and which is equivalent to the product of the initiation pace and the number of replicating chromosomes per cell. The experimenta approach used in this study has been to examine the initiation pace and the total initiation frequency of a dnaAts mutant growing under physiological conditions at. which the temperature labile gene product is limiting (i.e. at semipermissive temperatures). The resuIts demonstrate that the total initiation frequency can be varied independently of the initiation pace in this mutant, indicating that there are at least two separately regulated cellular components which control the initiation of chromosomal replication in E. coli.
2. Materials and Methods (a) Bacterial
strahs
All bacteria used in these experiments were derivatives of E. co&i K12 strains. The pertinent properties of these strains are given in Table 1. The E. coli strain, designated N167, selected for this study has been well-characterized as a temperature sensitive, conditionally lethal mutant unable to initiate new rounds of chromosomal replication at the non-permissive temperature and whose temperature sensitive lesion maps at the dnaA locus (Abe & Tomizawa, 1971; Hirago & Saitoh, 1974, 1975; Saitoh & Hiraga, 1975). The dnaA gene product of this strain is apparently a thermolabile protein which is converted t.o an inactive state at the restrictive temperature but which, upon a shift back to the permissive temperature, can resume an active conformation in the presence of chloramphenicol (i.e. its activity is thermoreversible) (Hirago $ Saitoh, 1974). In our hands, growing in a glucose/Casamino acid minimal salts mediam the non-permissive temperature for this strain lies between 42 and 43°C. Below 42”C, DNA synthesis can continue for more than a single round of replication. (b) Isotopes and chemicals (0.25 mCi/O.GTi mg) were [MethyZ-3H]thymine (5mCi/0.32 mg) and [methyZ-14C]thymine purchased from New England Nuclear Corp. Oleic acid (sodium salt, 99% pure), Brij 58, all amino acids, thymine and rifampicin were purchased from Sigma Chemical Co. Ilford Nuclear Research Emusion L4 was purchased from Polyscience Inc., Warrington, Penn. (c) Growth of bacteria grown in a New Brunswick gyratory water bath Bacterial cultures were routinely (Aquatherm G86) at 200 revs/min. The temperature was monitored with a telethermometer Springs, Ohio). The precision of temperature (Yellow Springs Instrument Co., Yellow measurement and regulation for the instruments is &O-l deg. C. 1972) supplemented with 0.4% Bacteria were grown in minimal A medium (Miller, glucose as a carbon source and containing 0.1 o/0 Casamino acids (Difco, vitamin-free) plus the required supplements which included : thymine (5, 10 or 20 pg/ml), thiamine (2 pg/ml), methionine (50 pg/ml), tryptophan (50 pg/ml), and oleic acid (IO pg/ml) dissolved in,0.02°/0 Brij 58 (polyoxyethylene 20 cetyl ether), depending on the E. coli strain used. (d) Measurement
of cell growth
Extinction of cultures was measured in an Acta CIII Instruments, Inc.) at a wavelength of 550 nm. Uninoculated turbidity control.
spectrophotometer (Beckman medium was used as a zero
strains
thy, met, dnnA167 thy, met
from
J. Wechsler
J. Wechsler
P. Overath
J. Tomizawa J. Tomizawa
Obtained
coli &ailzs
1
revertant
by Overath
Transductant
Described
of JW150
by Wechsler
using E508 as the donor
& Zdzienicka.
of K1059 (1975)
of N167 which can grow at 43°C
(1971)
et (II. (1970)
Thymine-requiring mutant Transductant of K1059-T
Described
a spontaneous
Described by Abe & Tomizawa Parental strain of N167
Remarks
t Genetic abbreviations are as used by Bachmann et al. (1967). The allele number of the dnu mutations is according to Gross (1972). $ A thy mutant of K1059 was selected for with trimethoprim according to the method of Miller (1972). From these thy mutants low t,hymine requirers were selected. §The dnaA167 allele was co-transduced with bgl marker. SZ:Tmutants of N167 were selected according to Miller (1972).Pl transduction was carried out as described by Miller (1972).
thy, lac, rha, str, dnnA508
F-,
E508
JW154
f&B, fadE, thi, th,y
F-,.fabB, f&E, thi, thy, dnaA167 F-, thy, thr, leu, thi, kzc, tonA, &=, dlzaA508
F-,
HfrH, thy, met, R3 F- , fnbl3, fadE, thi
HfrH, HfrH,
Description?
KA22§
K1059 KlOBY-T%
HMT N167-R
N167
Bacterial
Escherjchia
TABLE
274
J. A. FRALICK
Cell number was measured by counting cells in physiological saline (Cutter Laboratories, Inc.) on a Coulter counter model ZF (Coulter Electronics, Inc.) using a 30 pm orifice, l/aperture setting of l/4, l/amplification setting of 1 and a lower threshold of 5 to 10. On occasion, cells were also count,ed microscopically using a Petroff-Hauser counting chamber. Viable cell counts were determined by colony counts on the appropriate media. (e) Labeling
procedure
Exponentially growing cells were labeled at cell densities ranging from 5 x lo7 to 2 x lo* cells/ml. When [14C]thymine was used, cultures were labeled at a spec. act. of 0.5 PCi (5 pg)/ml. In steady-state labeling experiments the cells were labeled for more than 4 doublings before samples were taken. (f) Measurement
of raclioisotope
incorporation
Incorporation of radioactive precursors, [3H]thymine or [14C]thymino, into acidinsoluble material was measured by collecting 0.05 to 0.1 ml samples on Whatman 3 MM filter discs and washing them by passage through trichloroacetic acid and alcohol (Fralick & Lark, 1973). Dried discs were counted in a Beckman scintillation counter, model LS-230 using 0.5% 2,5-bis-2-[5-tert-butylbenzoxazolyll-thiophene (Research Products International Corp., Elk Grove Village, Ill.) dissolved in toluene as a scintillation cocktail. Under our high voltage settings, gain settings and window settings the counting efficiency for 3H was 14% and that for 14C was 61%. (g) Chemical
estimation
of DNA
and
protein
Exponentially growing cells were harvested in a cell density of 1 x lo8 to 2 x IO8 cells/ml by centrifugation. Samples were washed 3 times by centrifugation and resuspended in 5 ml of ice-cold buffered saline containing 0.01% gelatin (usually at about lo9 cells/ml). Cell numbers were determined at this point with a Coulter counter. Concentrated perchloric acid was then added to a final concentration of 1.0 M and then heated to 90°C for 20 min with agitation. The hydrolyzed samples were chilled in an ice-bath and centrifuged to pellet the acid-insoluble cell components. The supernatant was assayed for deoxyribose by the modified diphenylamine method described by Giles & Myers (1965). Calf thymus DNA wa,s extracted similiarly for use as a standard. A DNA standard curve was constructed for each experiment. The acid-insoluble pellet from the extraction described above was resuspended in 5 ml of 1 Jr-NaOH and left in a stoppered tube overnight at room temperature. Protein analysis of this extract was performed by the method of Lowry et al. (1951). Bovine serum albumin in 1 Jr-NaOH was used as a standard, and a standard curve was run for each experiment. (h) Autoradiography Radioactively labeled cells were fixed in 4% formaldehyde (Caro, 1970). Autoradiographs were prepared a.ccording to Lark (1968). Drops of cell suspension were placed on agar from which impression smears were made. The slides were dried, washed twice in 5% cold trichloroacetic acid, rinsed with water and dried. They were then dipped in 1: 1 dilution of Ilford Emulsion L4, dried and exposed at room temperature for a period of time. Slides were developed in Kodak D-19 developer for 3 min, fixed at room temperature and stained in 0.174 gentian violet for 1 min before examination under oil immersion (1200 x ) using a Zeiss phase-contrast microscope. Grain counts were analyzed as described in the text.
3. Results (a) Effect
of growth
temperature
on the initiation
pace of N167 cells
The time interval between successive DNA initiations in cells growing with balanced, steady-state growth has been shown to be equal in time to the interval between successive divisions (Maalme & Kjeldgaard, 1966; Helmstetter et al., 1968;
INITIATION
OF CHROMOSOMAL
REPLICATION
IS
Pa.rdee & Rozengurt, 1975). Thus the frequency of cell division, stances, can be considered equivalent to the initiation pace, (i) Temperature
E. COLI
“75
under there circum-
effect on cell division
The effect of growth temperature on the rate of cell division in the dnaAts strain, N167, and the dnaA+ parent, HMT, is shown in Figure 1. Note that both of these strains had similar, if not identical, temperature coefficients for cell division at temperatures below 35°C. At growth temperatures above 35”C, however, the rate of cell division for the mutant leveled off and began to decline. Therefore, it appears that the dnaA167 gene product became rate limiting for cell division at temperatures greater than 35°C.
l-6
0
I
I
35
30
Temperature
0
(“Cl
FIG. 1. Effect of growth temperature on division rate in N176 and HMT cells. X167 ( 0) and HMT ( 0) cells were grown at the indicated temperature for more than 5 doublings before determining the exponential cell division coefficient (growth rate). The exponential growth rate was determined with a Coulter counter. Greater than 5 different sampling times were used to caldate the growth rate of each oulture.
(ii) Evidence for balanced growth Figure 2 presents the increase in cell mass (A,,,), cell number (particle count) and DNA content (steady-state [14C]thymine corporation) of Nl67 cells which had been growing at the extremes of the temperature range studied (above 30°C and 38°C). Similar results were obtained at the intermediate growth temperature of 34°C (data not shown). From these data it can be seen that N167 cells can grow with balanced,
276
J. A. FRALICK
(b)
I
I
I
1
I
20
40
60
80
100
120
Time (min)
2. Growth characteristics of N167 cells growing at 30°C and 38°C. Cultures of Nl67 cells were grown in glucose/Casamino acid medium (see Materials and Methods) containing [l%]thymine (0.5 @iI5 pg per ml) for 4 doublings before starting the experiment. Samples were removed at the time intervals indicated and the rate of increase in absorbance (-u-C-), particle number (-O-O-) and [‘T!]thymine incorporation (--A-A-) was determined at 30°C (a) and 38°C (b). FIG.
exponential growth at temperatures as high as 38°C. Therefore, it appears that the product of the dnaA167 gene becomes limiting with respect to the initiation pace only at growth temperatures above 35°C. (b) Efj’ect of growth temperature (i) DNA
on chromosome
copy number of N167 cells
content
It has been calculated from the amount of acid-insoluble [14C]thymine incorporation in Figure 2 that the amount of DNA per cell in N167 cells growing at 38°C is approximately one-half that of N167 cells cultured at 30°C (13,000 cts/min per IO* cells and 23,000 cts/min per 10’ cells, respectively). One artifact which could lead to this result would be that the specific activity of the DNA of these cells was temperature dependent. As shown in Table 2, the specific activity of the DNA in N167 cells
1 2 3
1 2 3
30
38
1 2 3
1 2 3
Experiment number
38
30
Growth temperature (“Cl
15.2 15.8 14.5
14.7 13.7 13.9
6.69 7.1 6.8
12.6 14,5 13.7
DNA/cell (pg x 10-y
2146 -.
2288 -
2344 --
-
2292
Specific activity7 of DNA (cts/min per pg)
3.6 3.7 3.4
3.5 3.2 3.3
1.6 1.7 1.6
2.9 3.4 3.2
Chromosome$ equivalents/cell
2.5 2.6 2.4
2.4 2.2 2.6
I.09 1.16 1.09
2.0 2.3 2.2
Heplicating § chromosome equivalents/cell
on chromosome composition
2
355 396
325 330
406 355
327 326
Protein/cell (g x 10-y
0.044 0.036
.0.042 0.042
0.018 0.019
0.044 0.042
DNA/protein ratio
t N167 and HMT ~011sgrowing at 30°C and 38OC were harvested by oentrifugation at a population density of approx. 2 x lo* cells/ml and analyzed for DNA and protein as described in Materials and Methods. In this experiment the cells were grown for more than 4 doublings in media containing [‘*C]thymiuo (0.1 @/ 5 pg per ml). Cell protein was not determined. $ Chromosome equivalents were calculated by assuming the mass of a unit chromosome to be 4.22 x lo-l5 g which corresponds to a molecular weight of 2.5 x 109 daltons for the E. csli chromosome. 8 For comparative purposes the value of 1.44 unit chromosomes per replicating chromosomes was used in this calculation. This value was obtained from the equation of chromosomal replication (Sueoka & Yoshikawa, 1965) assuming the average number of replicative forks (pairs of replicative forks) per chromosome and the replicative position distance (HP-distance) to be one (Table 3) in cells growing at both temperatures.
HMT
N167
Strain
Effect of growth tenxpernture
TABLE
J. A. FRALICK
278
and the dnaA+ parent, HMT, was not affected by growth temperature. However, the actual amount of DNA per cell, and the DNA to protein ratio in the dnaAts mutant growing at 38”C, was decreased by a factor of two compared to the same cells growing at 30°C. This temperature dependent difference was not observed with the dnaA+ strain. That this temperature effect on DNA content in N167 cells was representative of the majority of the population of the culture can be seen from the autoradiographic analysis of Figure 3. The effects of intermediate growth temperatures on the cellular DNA content in N167 and HMT cells is illustrated in Figure 4. Note that the relative amount of DNA per cell for HMT remained relatively constant over the temperature range examined. The dnaA167 mutant, however, demonstrated a decrease in DNA content with increasing growth temperatures between 31°C and 35°C. These results suggest that the dnaA167 gene product becomes limiting for the amount of DNA on a per cell basis at temperatures above 31°C.
3(
Ln = 9 z
2(
B E z’ IC
0123456705
,
Groins /ccl I
FIG. 3. Autoradiographio N167 cells were grown for more per ml) at 38°C (a) and 30°C (b). Materials and Methods (exposure
histograms of N167 cells growing at 30°C and 38°C. than 4 doublings in the presence of [3H]thymine (100 @i/5 p*g Autoradiographs of these cells were prepared as described in time 17 days) and grains were counted.
(ii) Chromosome composition To determine if the temperature dependent difference seen in the DNA content of forks per chromoN167 cells had been due to a difference in the number of replicative some or to a difference in the number of replicating chromosomes per cell, the increment in DNA synthesized at the restrictive temperature by N167 cells which had been growing at 30, 34 and 38°C was measured. The underlying assumption to this experiment was that new rounds of chromosomal replication could not commence after transfer to the restrictive temperature and that existing replication forks would progress to the chromosome terminus (Abe & Tomizawa, 1971). The amount of DNA synthesized at the non-permissive temperature, therefore, would be a measure of the relative number and position of replication forks on the chromosome (Sueoka &
INITIATION
OF CHROMOSOMAL
REPLICATION
Temperature
IN
E. COLI
279
PC)
FIG. 4. Effect of growth temperature on the DNA content of N167 and HMT cells. and HMT (- X-X-) cells were grown at the inCultures of N167 (0, q , A, XjJ, -----) dicated temperatures for more than 4 doubling3 in the presence of [l%]thymine (0.5 $3/B pg per ml) before being assayed for acid-insoluble radioactivity. Cell number was determined with a Coulter counter. Eash symbol type represents a series of independent experiments. Each data point is the average of 6 or more different determinations.
Yoshikawa, 1965). Table 3 presents the results. The calculated increase in DNA, as measured by [14C]thymine steady-state labeling, was essentially the same for all three cultures and is consistent with only one pair of replicating forks per chromosome (39:/, residual increase) (Sueoka & Yoshikawa, 1965). These results are significently different from the 84 and 137% residual increase expected from chromosomes concontaining two and three pairs of replication forks, respectively. Similar results were obtained by blocking new rounds of initiation with the addition of rifampicin (data not shown). Therefore, it appears that the temperature sensitivity of the c&A167 gene product does not affect the number of replication forks per chromosome or the rate of polynucleotide chain elongation per se but more likely affects the number of chromosomes per cell. This explanation is also in agreement with the data of Table 4, which indicate that the amount of DNA per cell in N167 cells growing at 38°C was compatible with a single replicating chromosome that had only one pair of replication forks (Sueoka & Yoshikawa, 1965), while the same cells growing at 30°C contained
J. A. FRALICK
280
TABLE 3 Residzcal increase in the amount of DNA
synthesized at 43°C
Growth temperature before shift to 43°C
Residual increase in DNA synthesized at 43OC
30 34 38
43.4&&S 38.316.1 38.2*&S
Cultures of N167 were grown at the indicated temperatures in media containing [l%]thymine (0.5 @i/B pg per ml). At a titer of 5 x 10’ to 10s cells/ml, they were placed at 43°C and the incorporation of thymine measured. The residual increase in DNA was measured as the difference between the initial radioactivity. These values represent mean and standard deviation from a minimum of 4 independent experiments.
TABLE 4 Effect of growth temperature on the DNA/protein ratio of dnaA initiation mutants and their derivatives
Strain?
Relevant genotype
N167
dn,aAtS
N167-R$
R3
HMT
dnaA +
KA22
dnaAts
K1059-T
dnaA +
E508
dnaAtS
JW154
dnaAt”
Growth
temperature (“C) 38 30 38 30 38 30 38 30 38 30 38 30 38 30
DNA/protein ratio
DNA/protein DNA/protein
0.017 0.041 0.045 0.043 0.048 0.042 0.013 0.036 0.040 0.038 0.02 0.044 0.0125 0,0325
Cultures of the above strains were grown at 30°C or 38°C to a cell density 2 x lo* to 3 x lOa and then harvested by centrifngation. The DNA and proteins and assayed as described in Materials and Methods. t For description of these strains, see Table 1. f Ts+ reversion of the dnaA167 mutation.
at 38°C at 30cc
0.4 1.05 1.1 0.33 1.04 0.45 0.38
of approximately were precipitated
DNA for two such replicating chromosomes. These results therefore suggest that the dnaA167 gene product becomes limiting for the number of replicating chromosomes per cell (chromosome copy number) at temperatures above 30°C.
enough
(c) Effect of growth temperature on total initiation frequency in N16Y cells The total initiation frequency (TIP) is equivalent to the product of the initiation (IP) a.nd the number of replicating chromosomes per cell (N) (i.e. TIF = IP x N). If one assumed that the growth rate (under balanced growth conditions)
pace
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was equivalent to the initiation pace and that the DNA content (as determined from [14C]thymine steady-state incorporation) was directly proportional to the number of replicating chromosomes per cell, then one could estimate the total initiation frequency for N167 and HMT cells growing at different growth temperatures from the data of Figures 1 and 4. Such calculations have been plotted in Figure 5. Note that while the t’otal initiation frequency for HMT cells increased in a linear fashion with increasing temperatures between 31 and 38”C, it decreased for N167 cells growing over this same temperature range. In fact, unlike the effect of growth temperature on initiation pace or DNA content per cell, this decrease in the calculated total initiation frequency for N167 cells is linear over the entire temperature range examined. lt appears, therefore, that the dnaA167 product becomes limiting for the tot,al initiation frequency at growth temperatures great’er than 31°C. I
I
30
I
I
/
I
I
I
,
I
35 Temperature
PC)
5. Effect of growth temperature on the total initiation
frequency of N167 and HMT cells. The values for this Figure were derived from the data of Figs 1 and 4 using the equation: Total initiation frequency = initiation pace x chromosome copy number. In this calculation we have assumed the cell division frequency to be equivalent to initiation pace and that the radioactivity of N167 cells growing at 38°C to be equivalent to one replicating chromosome per cell on the average (see Table 2). FIG.
(d) EJect of growth tempembure on another dnaAtS mutant In an attempt to determine whether the temperature dependent effect seen in A-167 cells was characteristic of the dnaAtS phenotype in general or to the N167 strain in particular, the DNA to protein ratio in an independently derived E. coli K12 dnaAtS mutant and its derivatives were examined. The results are summarized in Table 4. It can be seen that there was little or no temperature dependence of this ratio in the parental HMT and K1059-T cells or in a spontaneous revertant of the dnaAtS phenotype (Nl67-R). However, all of the dnaAts mutants tested show a
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decreased DNA to protein ratio at 38°C. From these results it may be concluded that the temperature dependent effects observed with N167 cells were not unique to their specific dnaAts allele nor to the genetic background with which it was associated.
4. Discussion It is an accepted view that’ the bacterial cell regulates DNA synthesis by controlling the initiation frequency of chromosomal replication (Maaloe & Kjeldgaard, 1966; Helmstetter Q Cooper, 1968; Lark, 1968; Bleecken, 1971; Sompayrac & Maalee, 1973 ; Pardee & Rozengurt, 1975). However, there are at least two aspects of initiation frequency which may or may not be subject to the same control mechanism(s). The first aspect deals with the individual chromosome. How often will each chromosome be initiated? Under balanced growth conditions the bacterial cell must be able to monitor its growth rate and accurately adjust the timing between successive initiation events so that new rounds of chromosomal duplication occur once, and only once, each time the cell mass and/or cell number doubles. This aspect of initiation frequency has been termed the initiation pace, which is equiva’lent to the pace at which cells divide under steady-state growth conditions. The second aspect deals with the total number of initiation events that’ take place per cell division. This factor also controls the average number of replicating chromosomes that are present in a cell during balanced growth (chromosome copy number). We have termed this aspect of initiation frequency the total initiation frequency, which is equal to the product of the initiation pace and the number of replicating chromosomes per cell. We have examined the effects of growth temperature on these aspects of initiation in a dnaAts mutant and its dnaA+ parent. The results of this study are summarized in Figure 6 and can be described as follows. (1) The dnaA167 gene product becomes limiting for t’he total initiation frequency at growth temperature greater than 31°C. This is illustrated in Figure 6(a), in which the effect of growth temperature on the derived values for the total initiation frequency is given. Note that the overall initiation frequency for this mutant diminishes linearly over this temperature range, indicating that the dnaA167 gene product, or some fraction of its total concentration, becomes temperature labile at growth temperatures above 31°C and that the magnitude of t’his effect increases with increasing growth temperature. (2) The dnaA167 gene product becomes limiting for the cells’ chromosome composition at growth temperatures above 31°C. This can be seen in Figure 6(b), in which the amount of DNA per cell has been equated to the number of replicating chromosomes per cell (Fig. 4 and Table 2). From this graph it can be seen that the average number of replicating chromosomes per cell in the dnaAts mutant decreases with increasing temperatures over the temperature range of region I (31 to 35”C), after which it levels off to equal approximately one-half that of the parental dnaA+ strain growing at the same temperature. Quantitatively, N167 cells growing at 30°C contain enough DNA for two replicating chromosomes, while those growing at 38°C contain only enough DNA for one (Table 2). At both of these growth temperatures, growth is balanced and each chromosome contains only one pair of replication forks (Table 3). (3) The initiation pace, as determined by the frequency of cell division under balanced growth conditions (Figs 1 and 2), remains normal for the dnaAts mutant compared to the dnaA+ parent over the growth temperatures of region I (Fig. 6(o)).
INITIATION
OF CHROMOSOMAL
0.71
, 32
,
REPLICATION
,
j
,
35 Temperature
,
IN E. COLI
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, 30
(“Cl
FIG. 6. Summary of effect of growth temperature on chromosomal replication in N167 cells. Figs 1, 4 and 5 were used to compute the ratios of the mutant (&xx+) values for total initiation frequency (a), chromosome copy number (b), and initiation pace (c). In this presentation we have equated cell division frequency with initiation pace and DNA content with chromosome copy
number. The temperature range (31 to 3892) has been divided into two regions: region I corresponds to temperatures between 31°C and 35T and region 11 to temperatures above 3692. Only at growth temperatures above 35°C does the dnaA167 lesion effect this parameter. In summary, the results presented clearly illustrate that the two components of the total initiation frequency of chromosomal replication; initiation pace and the number of replicating chromosomes per cell, can be varied independently of one another in a cl?zuAtS mutant. These results demonstrate the existence of at least two independently regulated cellular components which are involved in the control of chromosomal replication in E. coli. Furthermore, the observation that the initiation pace remains normal over a temperature range which adversely affects the dnaA product (31 to 35°C) suggests that this gene product is not the “pace-maker” or the
284
J. A. FRALICK
regulatory signal which triggers successive rounds of initiation on a chromosome. However, the dnaA gene product, clearly affects the total initiation frequency; t,he higher the temperature, the lower the total initiation frequency, presumably because of decreasing levels of active dnaA gene product’. The actual means by which the product of the d%aA gene exerts its effect, on initiation is unknown. However, the mere number of gene products involved in the initiation and replication of the bacterial chromosome (Wechsler $ Gross, 1971; Geider, 1976; Wechsler, 1978; Alberts & Stermglanz, 1977) indicates that replication is probably carried out by an assemblage of interacting proteins which may form the hypothetical “replication apparatus” (Jacob et al., 1963; Lark $ Lark, 1964,1966; Bleecken, 1971 i Fralick & Lark, 1973; Helmstetter, 1974; Kogoma & Lark, 1975) and it is, therefore, tempting to speculate that the dnaA gene product is a component of this complex, essential for its maturation or structural imegrity. This hypothesis can explain our data as follows. The total initiat)ion frequency of chromosome replication is dependent on two factors; the rat’e at, which initia,tion-replication complexes are matured or assembled, which is influenced by the number of functional dnad proteins in the cell, and the frequency at which these complexes are Driggered, which is controlled by the pace-maker. Thus, at the permissive growth temperatures of 39°C two functional replication apparatuses are assembled and triggered approximately every 55 minutes in both the dnats mutant and the dnaA+ parental strain. However, at temperatures above 31°C the dnaA167 gene product becomes rate limiting for the assembly of functional complexes and hence for the total initiation frequency. Consequently, a decrease in the total initiation frequency is observed with increasing temperature of growth temperatures above 31°C (Fig. 5) until the non-permissive 43°C is reached, at which no functional replicative apparatuses are assembled and thus no new initiation events can occur. The initiation pace of the dnaAtS mutant should not change compared to wild type complex per cell can be assembled more rapidly as long as a single mature replication than the frequency at which a second regulatory component, the pace-maker, triggers it off. Thus, over the 31 to 35°C temperature range the rate at which replication complexes are assembled is falling continuously with increasing temperature, but the frequency at which these complexes are triggered (initiation pace) remains the same as wild type (region I in Fig. 6). L4t 35°C the rate at which a single mature complex is assembled per cell is equal to the rate at which it is triggered, and the chromosome number therefore equals one. Thus, at growth temperatures up to 35”C, the dnaA gene product has not affected the initiation pace, only changing the average number of replicating chromosomes per cell. However, since the number of replicating chromosomes per cell will not fall below one, the rate at which replication complexes are assembled begins to limit the actual rate of initiations per chrosome at growth temperatures above 35°C. Therefore, even though the pace-maker may be signalling for more frequent initiation events, a decrease in the initiation pace is seen. Under these circumstances the dnaA gene product becomes limiting for the initiation pace (region II, Fig. 6). The above explanation is in accord with reports from other laboratories which demonstrate the dnaA gene product to be a trans.acting protein (Gotfried & Wechsler, 1977) which may act as a multimer or in a complex with other proteins as suggested by the partial dominance displayed by some drLaAts alleles (Zahn et al., 1977) and by allele specific suppressor mutations which map outside of the dnaA locus (Wechsler
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& Zdzienicka, 1975). One prediction that can be made from this interpretation is the possible existence of a class of initiation mutants whose defect lies in the factor which triggers the initiation event (i.e. controls the initiation pace). We are currently searching for such a class of “initiation pace” mutants among the known temperature sensitive, conditionally lethal, initiation mutants of E. coli. I thank Dr B. of this manuscript I also thank Drs stimulating and assistance. This research (GM22653).
M. Olivera,, whose enthusiasm and encouragement have made the writing possible and whose critical suggestions have made it understandable. T. Kogoma, C. Lark, K. G. Lark, T. M. Joys and J. Wechsler for some very and Mr A. M. Davis for his excellent technical valuable discussions, was
supported
by
a grant
from
the
National
Institutes
of Health
REFERENCES 69, I-15. Abe, M. S-: Tomizawa, J. (1971). Genetics, (London), 269, 655-661. Alberts, B. & Sternglanz, R. (1977). Nature Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976). Bacterial. Rev. 40, 1161G7. Bird, R. & Lark, K. G. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 799-808. Bird, R. & Lark, K. G. (1970). J. Mol. Biol. 49, 343-366. Bleecken, S. (1971). J. Theoret. Biol. 32, 81-92. Caro, I,. G. (1970). J. Mol. Biol. 48, 320.-338. Cooper, S. & Helmstetter, C. E. (1968). J. &ZoZ. Biol. 31, 519-540. Donachie, W. D. (1968). Nature (London), 219, 107771079. Fralick, J. -4. & Lark, K. G. (1973). J. Mol. Biol. 80, 459-475. Geider, K. (1976). Current Topics Microbial. Immunol. 74, 55-112. (London), 206, 93. Giles, K. & Myers, A. (1965). Nature Gotfried, F. & Wechsler, J. A. (1977). J. BacterioZ. 130, 963-964. Gross, J. (1972). Current Topics Microbial. Immunol. 57, 39-74. Helmst’etter, C. E. (1974). J. Mol. BioZ. 84, 21-36. Helmstetter, C., Cooper, S., Pierucci, 0. & Revelas, IX. (1968). Cold Spring Harbor Symp. &ant. Biol. 28, 809-822. Hiraga, S. & Saitoh, T. (1974). Mol. Gen. Genet. 132, 49-62. Hiraga, S. 8: Saitoh, T. (1975). AVoZ. Gen. Genet. 137, 239-248. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spring Harbor Xymp. Quant. BioZ. 28, 329. Kogoma, T. & Lark, K. G. (1975). J. Mol. BioZ. 94, 243-256. Lark, C. (1968). &fethods Enzymol. 12, 616-625. Lark, C. & Lark, K. G. (1964). J. Mol. BioZ. 10, 120-136. Lark, K. 0. (1969). Annu. Rev. Biochem. 38, 569-604. Lark, K. G. (1972). J. Mol. BioZ. 44, 217. Lark, K. G. & Lark, C. (1966). J. Mol. BioZ. 20, 9-19. Lark, K. G. & Renger, H. (1969). J. Mol. BioZ. 42, 221-235. Lowry, 0. H., Rosebrough, A., Farr, A. L. & Randall, R. J-. (1951). J. BioZ. Chem. 193, 265-275. Maaloe, 0. & Hanwalt, P. C. (1961). J. Mol. BioZ. 3, 144-155. Maaloe, 0. & Kjeldgaard, PI’. 0. (1966). In Control of Macromolecular Synthesis, pp. 154182, W’. A. Benjamin Co., Inc., New York. Messer, M. (1972). J. Bacterial. 112, 7-12. Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Overath, P., Schrairer, H. U. & Stoffel, W. (1970). Proc. Nat. Acad. Sci., U.X.A. 68, 3180. Pardee, A. B. & Rozengurt, E. (1975). In Biochemistry of Cell JVaZZs and MembraTaes, (Kornbrrg, H. L. & Phillips, D. C., eds), pp. 155-185, University Park Press, Baltimore. Saitoh, T. & Hiraga, S. (1975). Mol. Gen. Genet. 137, 249-261. Sompapac, L. 85 Maalee, 0. (1973). Nature h’ew Biol. 241, 133-135. Sueoka, N. Xs Yoshikawa, H. (1965). Genetics, 52, 747-748.
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Wechsler, J. A. (1978). In NATO ASI: DLNA Synthesis Present and Future (Molineux, I. & Kohiyama, M., eds), Plenum Press, in the press. Wechsler, J. A. & Gross, J. D. (1971). Mol. Gen. Genet. 113, 273-284. Wechsler, J. A. & Zdzienicka, (1975). In DNA Synthesis and its Regulation (Goulian, M. & Hanawalt, P., eds), pp. 602-617, W. A. Benjamin Inc., California. Hartor Synrp. Quad. Biol 33, 843-855 Yoshikawa, H. & Haas, M. (1968). cold Spring Yoshikawa, H., O’Sullivan, A. & Sltcokn, N. (1964). Proc. Nat. Acad. Sci., U.S.A. 52, 973-980. Zahn, G., Tippe-Schindler, R. & Messer, W. (1977). Mol. Gen. Genet. 153, 45-49.
