- Email: [email protected]
Chromosome replication in Escherichia coli
J. Mol. Biol. (1967) 29, 419431
Chromosome Replication in Escherichia coli I. Lack of Influence of the Integrated F Factor CLAIRE M. BERGS AND LUCIEN G. CAROM
Biobgy Division, Oak Ridge National Laboratory Oak Ridge, Tenn. 37830, U.X.A. Microbial
Medical Research Council Genetics Research Unit, Hammersmith Hospital London, W.12, England (Received 24 June 1967)
Therelative gene frequency inisogenic Hfr strains of Escherichia co.5 K12 has been assayed by Pl transduction in order to evaluate the influence of the integrated F factor on the origin and direction of chromosome replication. Three strains with different sites of integration of the F factor were studied. No significant differences in transduced gene frequency were found in either a one-cycle lysate made on exponential cultures of the strains or in the transducing particles of hybrid density obtained from a lysate made on cells previously pulse-labeled with 5-bromouracil. We conclude that, in these strains, the integrated F factor does not affect the origin or the direction of chromosome replication.
1. Introduction In studying chromosome replication in Escherichia co& a most important question is the determination of whether each round of replication starts from a fixed site on the genetic map. In 1963, Nagata approached this problem by considering the yield of the prophages X and 424 produced after induction of doubly lysogenic strains of E. wli K12. The F- and two Hfi- strains studied gave different time sequences for the relative yield of the two phages during one cycle of synchronized growth. On this basis, Nagata proposed that in Hfr strains chromosome replication starts at the site of integration of the F factor and proceeds in the direction opposite to that of the sequence of transfer during conjugation. He also proposed that the chromosome of F- cells starts its replication at random from any site on the continuous circular structure. We have investigated the first aspect of the Nagata model, the role of the integrated F factor in chromosome replication. To this end we have analyzed the state of replication of the chromosomes in several strains of E. coli K12. The method of analysis is similar to that used for Bacillus subtilis by Yoshikawa 6 Sueoka (1963), who measured
t Present address: Institut de Biologic Mol&ulaire, Universite de Genkwe, Switzerland. Reprint requests should be sent to Oak Ridge National Laboratory. $ Present address: Oak Ridge National Laboratory, Oak Ridge, Term. 37830, U.S.A. 419
420
C. M. BERG
AND
L.
G. CAR0
the relative frequency per chromosome of various genetic markers using transformation with extracted DNA. They argued that, in an exponential population, the probability of occurrence of a marker located at the origin of chromosome replication should be twice as high as that of a marker located at the end, with a continuous gradient of probability in between. Instead of transformation, we are using generalized transduction with the bacteriophage PI (Lennox, 1955) as the method of sampling chromosomal markers. We infect exponential liquid cultures of E. coli K12 strains with Pl. The progeny phage from a one-cycle lysate is then used to transduce as many as seven different markers into a multiply marked recipient. Since the efficiency of transduction is different for each marker, marker frequencies cannot be established directly. Instead, we calculate the proportion of transductants for each marker relative to the total number of transductants in any one experiment. We then compare, two by two, the values obtained for strains which would be expected to give different gradients of marker frequency if the origin and the direction of chromosome replication were determined by the position and orientation of F. Our results demonstrate that the position and orientation of the integrated F factor in these strains have no effect on the origin and direction of replication.
2. Materials and Methods (a) Media L broth, L soft agar (0.65% agar) and L agar (1.2% agar) (Lennox, 1955), supplemented with 2.5 x 10-e M-calcium chloride and with 20 pg thymine/ml., were used for growing cells and for phage assays. All phage lysates were made in this medium regardless of the previous growth conditions of the cells. Before Isbeling with 5-bromouracil, the cultures were grown inM9 (glucose-salts) medium (Adams, 1959) supplemented with 0.5% Casamino acids (Difco) and 5 pg thymine/ml. Transductants were selected on appropriately supplemented medium E agar (1.5%) overlaid with unsupplemented medium E soft agar (0.65%) (Vogel & Bonner, 1956). Supplements, purchased from Calbiochem, were used at the following concentrations, in ag/ml. : thymine, 5; adenine, 40; thiamine-HCI, 2 ; L-proline, 30; L-tryptophan, 20; L-lysine-HCl 85; L-methionine 10; and L-leucine 20. The carbon sources were glucose, lactose or arabinose at 0.5% final concentration. Streptomycin sulfate was added to give 200 pg/ml. (b) Strains The strains
of E. co.% used are listed
in Table
1.
(c) Phage stocks A clear plaque mutant was selected from a strain of Plkc obtained from Roy Curtiss. This phage, Pl.L4, had the following advantages over Plkc: higher titers in one-cycle liquid lysates, up to 2 x lOlo phages/ml. ; good yield of transducing particles; increased stability in cesium chloride solutions and low calcium requirement. PlaL4 stocks were prepared by the confluent plate lysis method (Adams, 1959) on x 478, a strain isogenic with the recipient strain x 484; thus the transducing potential of unadsorbed carry-over Assays were made on LC35 phage was eliminated. Titers of 1 to 2 x 1O1l were obtained. with a IO-min adsorption period before plating. (d) Trur~sductio~r The recipient of the recipient
used throughout was x 484, a Plkc-lysogenic were grown from a number of single-colony
derivative isolates.
of x 478. Cultures The one with the
CHROMOSOME
REPLICATION TABLE
Xtraim
strain
Designation
IN E. COLI
421
1 used
Relevant characteristics
source
Ref.
x 354
F-
pro, - lac deletion
cortias
x 473
F-
B1- lac,- proa- ade=- try-
(W1485) TV945
,?(434
F-
lya- metE- ara- leu- strrP B,- la,- pro,- ade,- trylye- metE- ara- lezl-
LC617 LC35
FF-
met E. coli C, met- arg- try- ade
LC618 x 416 x 493
Hfr H Hfr C Hfr OR11
met- (A, 424)
x 562
Hfr OR11
leu- thy-
W1485 P”) x 493
x 629
Hfr IOR 3
leu- thy-
x 562
(1967) Bera & Curtisa (1967)
x 618
F’ ORF 8
leu- thy-
x 562
Berg & Curt&s
CB 03
Hfr TOR 36
leu- thy-
x 618
Berg & Curtiss (1967)
LC615
F’ ORF 8 partial diploid F’ ORF 8 partial diploid
Berg & Curtisa (1967)
x 473
strr (P&c) 58-161 Arber Hayes Nagata
prototroph
Gross & Cero (1966) Nagata (1963) Berg & Curt& (1967) Berg & Curt&s
(1967)
LC616
x618 x ,y 354 x 618 x x 354
Symbols for the genetic markers: B1, leu, thy, pro, ade, try, lys, met, arg, requirement for thiamine, leucine, thymine, proline, adenine, tryptophan, lysine, methionine and arginine, respectively; am, gal, Zac,inability to utilize arabinose, galactose, lactose; strS /str’, sensitivity and resistance to streptomycin; (A), (424), (Plkc), lysogeny for the phages h, 424, Plkc, respectively. Note: All strains are E. co&i K12 except for LC35 (E. coli C). All K12 strains used are SU,,+ (amber suppressor). lowest number of revertants was selected and maintained as a stationary broth culture at 4°C. This procedure was repeated at 2- to 3-month intervals. For transduction assays, an exponential broth culture of this x 484 stock grown to 1 to 2 x 10s cells/ml. was infected with Pl*L4 at multiplicities of 3 to 5 phages/cell and incubated without aeration at 37°C for 30 min. The adsorption period was terminated by spreading or, more commonly, by diluting the mixture with medium E soft agar at 45% and plating equal samples onto 4 plates of each selective medium. The plates were incubated at 37°C for either 30 hr (spreading method) or 39 hr (agar method). (e) Experimental
procedure.
(i) Pl lysates Cultures of the host strains were grown in broth to a concentration of 2 x lOa cells/ml. They were then infected with Pl*L4 at a multiplicity of infection of 3 to 5 in broth. After 60 min of vigorous aeration at 37’C, the culture was chloroformed, treated with DNase (Worthington) (10 pg/ml.), and the cell debris removed by centrifugation. The infectivity and the transducing activity for various markers were tested as described above. The phage titers varied between approximately 2 x lo9 and 2 x lOlo, and the transducing activity between 10e4 and 10m5 of the infectious titer, depending on the
422
C. M.
marker, infection.
the
strain
5-Bromouracil
(ii)
tested,
BERG
and the growth
AND
L. G. CAR0
conditions
of the
bacterial
culture
prior
to
pdses
Cultures were grown in MS supplemented with C&amino acids and thymine to a concentration of 2 x lo* oells/ml. They were washed on an S 8, S membrane filter and collected in M9 with Casamino acids. After 5-min aeration at 37”C, BUt was added to give 10 pg/ml. The labeling period was usually 15 min. After this time the cells were washed by filtration and collected in a small volume of M9 with C&amino acids. They were aerated 5 min, diluted 5-fold with broth and infected with Pl. Lysates were made as described above. In control experiments it was found that the strain x 562 (Hfr OR1 1) and its derivatives 2.5% of the total thy-mine content contain a pool of thymine equivalent to approximately of the cell. A starvation period of 5 min, such as that used before and after BU labeling, is sufficient to exhaust this pool completely. The generation time in BU, measured under the conditions of the experiment by incorporation of 5-[6-3H]bromouracil (Amersham), is 50 min. This incorporation of tritiated BU is approximately exponential for at least 30 min. The generation time in thymine is 35 min. (iii)
Density gradient
Density
gradients
were formed
by mixing
1.5 ml. of the lysate
2.0 ml. of a cesium 15 to 18 hr at 30,000 rev./min in a SW39 rotor in a Spinco model L ultracentrifuge. The fractions, collected with a Buchler fraction collector, consisted of 5 drops each (400 drops total) takeninto 1 ml. of broth. A first assay of transducing activity for one or two markers (ade or met) was made in order to localize the hybrid and the light-density bands and to determine the approximate number of transducing particles in each. The transducing activity for all the selected markers was assayed separately for three or four tubes in the hybrid band, one or three tubes in the light band and for the original lysate. Controls showed little difference in the positions in the gradient of transducing activity for the markers used. No transducing particles were ever found in the position expected for heavy (BU-BU) density.
chloride solution of density 1.78 g/ml. The mixture
with
was centrifuged
3. Results and Discussion (a) Discussiwl
of method
The theory of marker-frequency analysis has been considered in detail by Sueoka $ Yoshikawa (1965). We shall summarize here some of their conclusions in relation to our experimental system. We assume that in an exponentially growing culture of bacteria the chromosomes replicate without interruption (van Tubergen, 1959; Schaechter, Bentzon & Maaloe, 1959) asynchronously, and at a constant rate of displacement of a single replication point (Cairns, 1963aJ; Bonhoeffer & Gierer, 1963; Bleecken, Strohbach & Sarfert, 1966). Under such conditions, the probability density function for the position of replication points is : f(z) = In 2.2l-’
(1) (x = 0) to the end (x = 1)
where x represents a position measured from the origin of the chromosome. This function has the values f(0) = 2 In 2 at the origin and f(1) = In 2 at the end. Therefore, the replication point has approximately twice as high a probability of being in a region near the origin than of being in a region of equal length at the end of the chromosome, with a gradient of intermediate values at points in between. This arises, of course, from the fact that the termination of each round of replication (one growing point at the end) gives rise to two new replicating structures (two growing points at the origin). t Abbreviation
used; BU, 5-bromouracil.
CHROMOSOME
REPLICATION
IN
E.
COLI
433
One can easily derive from equation (1) the average number of copies per chromosome for a genetic marker placed at distance x from the origin: g(x) = 2 l-2.
(2) This function differs only by a constant (In 2) from the distribution function of replication points and follows, therefore, a similar gradient. The gradients for the probability density of growing-point positions and for the relative marker frequency both depend on the origin and direction of replication being fixed. In the case of a randomly distributed origin, both functions would have the same value for all values of 5. Sueoka & Yoshikawa (1965) have also shown that, if there are several growing points running in the same direction, both functions show a steeper gradient than for a single growing point. Consider an Hfr strain, (m), with two markers A and Z placed, respectively, at the origin and at the end of transfer. Under Nagata’s model, Z would be at the origin of replication and A at the end of replication. Their expected transduction frequencies would be: g, (A)=g(l)=K,
andg,
(.Z)=g(O)=2K,
where KA and K, represent the efficiency of transduction for each of the two markers and each is assumed to be constant among isogenic strains. The ratio of the two values is :
It is obvious that the maximum expected change in relative marker frequencies will occur for a strain (n) in which the direction of transfer, and therefore the postulated direction of replication, has been reversed. We would then get: g,(A) = g(O) = 2K, g,(z)
= g(l) = Kz and
Comparing the two strains (m) and (n) : -= Qn
Q,
4,
Intermediate markers would give lower values, but the difference between the two strains should be easily detectable. For strains with the same origin and direction of replication, Q,/Q, would, of course, be equal to one. (b) Lysates on non-isogenic strains In a preliminary experiment, we compared the marker frequencies obtained in PI lysates made on exponential cultures of three non-isogenic strains: Hfr H, Hfr C, and 58-161 F-. If the position of F determined the origin and direction of replication, a number of predictions on the expected relative marker frequencies could be made. Table 2 shows that the differences between the strains do not correspond with these predictions. Thus, since they are closely linked, lac and ade should behave as a unit in Hfr H and Hfr C ; met should be much higher in Hfr H than in Hfr C ; and lys should 28
424
C. M.
BERG
AND
L.
G. CAR0
TABLE 2
Relative transductant frequencies in lysates of non-isogenic strains
Host strain
lac
Hfr H Hfr C F- 58-161
7.2 8.7 16.7
P”
ade
try
lYS
met
ara
5.8 10.3 6.0
11.8 12.0 11.0
7.0 12.0 7.1
25.5 6.8 18.5
22.8 26.9 24.9
19.9 23.3 15.9
Transductant colonies counted 7395 807 10,674
One-cycle lysates of Pl.L4 were made on exponential cultures of the three strains indicated. The phage yield on Hfr C was about 10 times less than on the other strains. This may be due to t,he fact that the strain was lysogenic for X and 424.
be higher, rather than nearly four times lower, in Hfr C than in Hfr H. Neither this low value of lys in Hfr C nor some other features of the marker frequencies can be explained on the basis of a simple replication scheme. It is likely that the differences observed are due to variations in the genetic background of the three strains affecting transduction efficiency. It is clear, therefore, that isogenic strains should be used. (c) Isogenic strains Three isogenic Hfi strains were used throughout the rest of this work. Hfr OR11 is a derivative of W1485. Hi? IOR was derived from Hfi OR11 by an inversion of the F Zacregion. Hfr TOR36 was derived, also from IIfr ORll, by forming a primary (haploid) F’ strain containing the region F lac pro ade on the F’ and re-integrating it, by a reciprocal exchange, into the chromosome. The final result is that the sequence F Eat pro ade has been deleted from its original location and re-integrated in the order pro ade F lac to the left of ara. The position of the relevant markers and their order of
FIG. 1. Isogenic Hfr strains. Map of three isogenic strains used in this work. Note that in IOR3, produced by an inversion of the F Zac region, F has almost the same location as in its parental strain ORll, but that the direction of transfer is reversed (Berg 8: Curtiss, 1967). TOR36 was produced by deleting the region F lac pro ade from OR 11 and inserting it between met and ara.
CHROMOSOME
REPLICATION
E. COLI
IN
425
transfer in mating are shown, for the three strains, in Fig. 1. The isolation and properties of these and related strains have been described (Berg & Curtiss, 1967). Autoradiographic experiments (Caro & Berg, unpublished data) have shown that in these strains DNA synthesis takes place during more than 95% of the cell cycle and involves, generally, all nuclear regions present in each cell. This synthesis is exponential during exponential growth of the cells. It involves large structures which segregate, without significant fragmentation, in the pattern expected for E. coli chromosomes (Forro & Wertheimer, 1960; van Tubergen & Setlow, 1961; Lark & Bird, 1965). In a control experiment, the strain x 562 was grown in BU for 30 minutes under conditions identical to that used in BU labeling experiments (see later). The proportion of transducing particles found in the heavy, hybrid and light bands was analyzed for five markers (tie, aru, met, Zys, try). For all five markers, the proportion found in the heavy band (replicated twice) was less than 4%. The proportion of transducing activity in the hybrid band (replicated once) was nearly the same for all markers, varying between 60 and 69%. It seems, therefore, that the pattern of replication in this strain is normal and is not grossly affected by BU, at least for times TABLE 3
Reproducibility
of transductant frequencies
ade
Lysate
1 2 3
4.8 4.6 4.4
13.3 12.4 12.2
6.6 6.6 6.6
lY8
met
20.5 20.3 19.4
29.0 29.1 29.4
kU
25,s 27~0 28.0
Colonies counted
13,620 11,196 11,340
Relative transductant frequencies (percentage of total transductant colonies counted) for 3 one-cycle lysates, made on 3 separate cultures of x 493 (Hfr ORll). As in all other experiments, lysates were made nearly simultaneously for all three cultures. Assays for transduction were also made on x 484, simultaneously.
TABLE 4
Effect of nzultiyplicz’ty of infection of Pl-L4 on transductant frequencies
m.0.i.t
ade
lY8
met
ara
Transductant colonies counted
Total phage plated x 10-s
10 3.5 1.0 0.3 0.1
20.8 20.9 18.2 20.3 20.1
26.6 28.4 28.7 25.5 24.9
26.4 25.4 26.6 26.7 26.8
26.2 25.4 26.5 27.5 28.2
4286 6832 3519 3572 3760
2.0 3.5 2.0 2.8 2.0
Portions of an exponential culture of x 484 were infected with Pl.L4 at multiplicities varying from 0.1 to 10 phages/cell. Platings for transductants were done by the top agar layer method at dilutions such that the number of phages plated was comparable. The relative transductant frequencies (percentage of total transductant colonies counted) for four markers show no detectable effect of multiplicity of infection. t Multiplicity
of infection.
426
C. M.
BERG
AND
I,.
G.
CAR0
twice as long as that of the BU labeling used in section (f). DNA replication, in the three strains, seems therefore to follow the conditions set at the beginning of section (a) for the application of the probability density function: g(z) = 2l-“. Should the origin and direction of replication be determined by the integration of P on the chromosome, we could, therefore, expect strong differences in the gradient of marker frequencies in the three Hfr strains studied. Control experiments, shown in Tables 3 and 4, indicate that Pl transduction is reproducible enough to detect such clifferences if they occur. A further conditionis that transductant frequency should be proportional to marker frequency. This point is examined in the next section. (d) Response to gene dosage To test that transductant frequency responds to gene dosage, Pl*L4 lysates were made on exponential cultures of a series of strains constructed for this purpose and all derived from W1485. In these strains the markers pro, and ode, are present on the chromosome (Hfr OR11 and x 354), on the F’ factor in a primary F’ (ORF8, a strain in which the markers on the F’ factor are deleted from the chromosome; see Berg & Curtiss, 1967) and on both the chromosome and the episome in two secondary (partially diploid) F’ strains (LC615 and LC616). The marker lac found on the chromosome in OR11 is deleted in x 354 and is present only on the F’ factor in the other strains. There is evidence that an F’ strain carries only one F’ factor per chromosome (Cuzin, 1966; Jacob, Brenner & Cuzin, 1963). If the production of transducing particles for a given marker is directly proportional to the number of copies of that marker, we expect that a partially diploid strain would produce between 1.5 and 3 times more transductants for the duplicated markers, depending on the respective time of replication of the markers on the chromosome and on the F’ factor. TABLE
5
Response of tra~~,~Jucta~ttfrequency Strain
Hfr OR11 F-x354 F’ ORFS (haploid) F’ LC-615 (diploid) F’ LC-616 (diploid)
Genotype lac
Pro
ade
Location markers
+ +
-I-/+
+ Chromosome + Chromosome + Episome
+
+ +
+ -
+ +
+ Episome + Chromosome + Episome + Chromosome
lac
of
T+L+M+A
to gene dosage Pro T+M+L+A
ade T+M+L+A
0.094 0 0.10
0.087 0.066 0.097
o-19 0.17 0.22
0.11
0.15
0.35
0.12
0.17
0.37
Pl.L4 lysates were made on exponential cultures of the five strains shown: Hfr OR11 (x562) is as shown on Fig. 1; F- x354 contains a deletion of the Zac gene; F’ ORF8 (x618) is a primary F’, derived by a single reciprocal exchange on the chromosome of the Hfr OR1 1, in which the markers Zac, pro and ade have become incorporated into an F’ factor, leaving a corresponding deletion on the chromosome; F’ LC-615 and LC-616 have been formed by introducing the F’ factor from ORF8 into F- x354. They are therefore diploid for pro and ade, haploid for Zac, which is present only on the F’ factor and haploid for the other markers, which are located on the chromosome. The transductant frequencies for Zac, pvo and ade are compared with a base line formed by the sum of the frequencies for four chromosomal markers; try, Zya, met and ara (T + L + M + A). The frequency of pro transductants is depressed in x354, presumably because the normally cotransduced Zac locus is deleted in this strain.
CHROMOSOME
REPLICATION
IN
E. COLI
427
The results in Table 5 show that the relative transductant frequencies for lac, pro,, and ade, are raised only slightly when the marker is located on the F’ factor instead of the chromosome, but that the relative transductant frequencies for pro, and ade, are nearly doubled in those strains which are diploid for these two markers. There is, therefore, a definite response of transductant frequency to gene dosage. It is interesting to note that, if this response was shown to be linear, the data would be consistent with the notion that the F’ factor replicates shortly before the lac pro ode region on the chromosome. (e) Lysates on isogenic strains Pl lysates were made by one cycle of infection in exponential cultures of the three isogenic Hfr strains. The relative transductant frequencies found for five markers are shown in Table 6. There is little difference between the three strains. If the origin was TABLE
6
Relative trawductant frequencies in lysates of isogenic Hjr strains
Host
strain
OR11 IOR TOR36
ade
t TY
lY8
met
ara
16.3 19.3 15.6
13.5 11.5 11.8
15.3 14.7 15.1
31.2 31.4 33.8
23.8 23.1 23.6
Tranaductant colonies counted 2331 2847 2728
One-cycle lysates of PlmL4 were made on exponential cultures of the three isogenic Hfr streins shown in Fig .l . The relative transductant frequencies for five markers are expressed as 8 percentage of total transductant colonies counted.
controlled by F, a clear difference should occur between strains OR11 and IOR in which F is integrated in nearly the same position but with opposite orientation. We can take ara as a fixed point and calculate the values of the ratios of the relative proportions of each marker to ara for the two strains. Figure 2 shows that the experimental points give, for this ratio, a constant value of approximately 1.0, as would be expected if the two strains had the same origin (or origins) and the same direction (or directions) of replication. The results show, therefore, no influence of the integrated F factor on the origin and direction of replication. Although a definite response of Pl transduction frequency to gene dosage has been demonstrated (Table 5), several possible occurrences could invalidate the conclusions drawn from this type of experiment. These are : (1) that Pl transducing particles are formed only in cells which have been infected just after completion of a round of DNA replication; (2) that a cell infected with Pl can complete one round of DNA replication but cannot initiate a new round ; (3) that Pl can pick up DNA from only one of the two newly replicated branches of each chromosome and from the non-replicated portion. In all these cases, the chromosome would appear to the phage as a linear, fully replicated structure and would show no gradient of marker frequencies. To avoid these difficulties the experimental design was modified as shown in the next section.
428
C. M.
BERG
L.
of transductent
G. CAR0
I !Ys
I fry
OL Oode
FIG. 2. Comparison
AND
frequencies
I F
I met
with predictions
for F-determined
origin.
The transductant frequencies for four markers, a&, try, Zya and met, relative to that of ara taken ES reference, for a lysate made on an exponential culture of OR11 were compared to those for IOR3. The Nag&a model predicts, for the ratio of the two, a gradient going from approximately 3.33 for ode to 1.20 for met. This gradient is calculated from the position of the marker and its presumed frequency given by g(z) = 2i-‘, assuming that F is the origin and that the direction of replication is opposite to that of transfer in conjugation (- - -). If, on the other hand, the two strains had the same origin and direction of replication, the expected value for the ratio would be 1.0 for allmarkers (- - - -). The data fit better this latter model. Data from Table 6. The abscissa shows the transfer sequence of markers for IOR and their map position relative to F.
(f) Lysates on isogenic strains after labeling with 5-bromouracil Exponential cultures of the three Hfr strains were labeled with BU during approximately one-third of the generation time. Pl lysates were made on these cultures by one cycle of growth. The transducing particles of hybrid density were separated from the light ones and from the infectious particles in a cesium chloride density-gradient and were used to assay the relative marker frequencies. This experiment bypasses or answers the objections raised above. In case (1) only ends of chromosomes would be labeled, and a clear difference between the strains would be predicted if F determined the origin. Case (2) would not affect the results since we are now sampling DNA made exclusively before infection. Case (3) would not affect the results either : the expected gradient in BU-labeled markers arises now, ISO-
I
4
I
I
Tube number
FIQ. 3. Density
profile
of ode transducing
particles
after pulse of BU.
Distribution in a cesium chloride density-gradient of ode-transducing particles from a onecycle Pl.L4 lysate made, in light medium, on a culture of Hfr OR1 1 previously labeled for 16 min with BU. The total number of fractions in the gradient was 80. Arrows indicate the tubes selected for transductant frequency analysis in the hybrid density band.
CHROMOSOME
REPLICATION
IN
429
E. COLI
not from the probability density function of marker frequencies, g(x), but from that of growing-point positions, f(z). As we have seen, the two functions are identical, except for a constant. The function f(x), and therefore the gradient of marker frequencies in the BU band, will be the same whether one or both branches of the newly replicated portion of the chromosome are picked up. The density distribution profiles for ode-transducing activity were analyzed for all three strains (Fig. 3). The proportion found in the hybrid density band was 44% of the total activity in the gradient for Hfr ORll, 44% for IOR3, and 43% for TOR36, showing that ade had been replicated in equal proportion in the three strains. The marker frequencies, for five markers, averaged over three tubes in the hybrid band, are shown in Table 7. Each tube was assayed separately. No significant differences in relative marker frequencies were found between them, showing that density differences between markers are not suf%ent to affect the results. Here again the expected TABLE
Relative transductant frequencies
Host strain
ode
OR11 IOR TOR36
28.7 30.8 27.0
7
from hybrid density Pl band after 5-bromouracil
11.2 9.7 6.2
lYS
met
am
Transductant coloniee counted
6.2 6.0 6.3
33.4 34.7 40.9
20.6 18.9 19.7
6416 7330 11,617
Exponential cultures of the three isogenic Hfr strains shown in Fig. 1 were labeled with E pulse of BU, as described in the text. A one-cycle PlmL4 lysats was made, in light medium, on all three strains shortly thereafter. The transducing particles of hybrid density were separated from the light ones and from the infectious progeny phrages on a cesium chloride density-gradient. The reletive transducing frequencies shown are each the combined data from three tubes in the hybrid density peak (see Fig. 2). Due to the difllculties inherent to the experimental design, the d&a are more scattered than those of Table 6. They do not, however, exhibit the strong differences expected for an F-determined origin and direction of replication.
:
‘\
+I 3,0\ G-ie ‘\ ‘60 *\ + 2.0‘\ z 0 x F ,o o_---Q-------,----;;---=~, ~ ;I0 5-l
LJ
Oode
-\
-\
I
I
fry
‘YS
-1
I me+
I
F
Fm. 4. Comparison of hybrid density transductant frequencies with predictions of an F-determined origin. Same calculations as in Fig. 2, for the data of Table 7 for Hfr OR11 and IOR3; - - -, F-determined origin; - - - -, fixed or variable origin. Here again the results favor a model in which the two strains have similar origins and directions of replication.
430
C. M.
BERG
AND
L. G. CAR0
differences between strains fail to appear. If, as in the previous section, we plot the ratios of the relative proportions of each marker to ara for the two strains OR1 1 and IOR (Fig. 4), it is clear that the experimental points do not fit the distribution predicted by an F-determined replication. Instead, these ratios all have a value close to 1.0, as expected if the two strains did not differ in origin or direction of replication. (g) Conclusions The BU pulse experiments agree completely with the total lysate experiments in showing no differences in marker frequencies between three isogenic Hfr strains. The differences found between the marker frequencies in a straight lysate (Table 6) and in the BU-labeled band (Table 7) could be explained by some selective inactivation of the markers by BU. They could also reflect a small amount of aberrant replication induced by BU itself or by the experimental procedure. Although this would complicate the interpretation of the results, it is difficult to imagine how such a replication could fail to reflect the differences in marker frequencies that would be produced by different origins and directions of replication in the normal state. The two types of experiments concur, therefore, in showing that the position and direction of integration on the chromosome of the F factor have no detectable effect on the origin and direction of replication in our strains. Nagata’s data (1963) showed that, during the division-cycle of two Hfr strains, with opposite directions of chromosome transfer, the relative yield of two prophages, h and 424, changed abruptly at fairly well-defined times and that the time sequence of these events was reversed in the two strains. This result did not provide a complete demonstration of the model which he proposed, since (1) it was neither demonstrated conclusively that DNA synthesis was synchronized nor that the initiation of DNA replication coincided with that of the cell life-cycle, and (2) it was not shown that the phage yield was directly proportional to the number of copies of the prophage present in the cell at the time of induction. Nagata’s conclusions have, however, been supported by some unpublished evidence of Vielmetter & Messer (1964 ; Messer, personal communication) based on the relative frequency of appearance of sectored and unsectored mutant colonies after 32P-labeling of various Hfr strains. On the other hand, we have studied the replication occurring during treatment and recovery from amino acid starvation (unpublished data) and have found no change in the direction of replication due to a different orientation of the integrated F factor. Other workers have obtained similar results with amino acid starvation (B. Wolf, A. Newman & D. A. Glaser, personal communication) and exposure to phenethyl alcohol (J. Tomizawa, personal communication). These discrepancies could be explained in two ways: (1) the integration of the F factor either affects chromosome replication in some strains but not in others or only under certain experimental conditions, or (2) the observations of Nagata were due to a difference of origin in the strains he used, which happened fortuitously to conform to the model. This second explanation may find some support in that we have found recently that distantly related strains of E. co& K12 have different directions of replication and, perhaps, different origins. These results will be the subject of a subsequent paper. The present data do not completely rule out the possibility that F determines the origin of replication. They could be explained, in that case, by postulating that chromosome replication can proceed, with equal probability, in opposite directions.
CHROMOSOME
REPLICATION
IN
E.
COLI
431
Results to be published subsequently have however, indicated that replication is unidirectional. It seems, thus, that there is no necessary relation between the position and orientation of the integrated F factor and the origin and direction of chromosome replication. We are grateful to Dr W. Hayes for the hospitality given in his laboratory and to many of our colleagues in Oak Ridge and in London, and more particularly to R. Curtiss, J. D. Gross, J. A. Shapiro, and C. M. Steinberg for invaluable discussions and suggestions. The assistsnce of D. J. Atkinson was of great valuain the experiments performed in London. This research was jointly sponsored by a Postdoctoral Fellowship of the North Atlantic Treaty Organization to one of us (C. M. B.), a Senior Postdoctoral Fellowship of the National Science Foundation to the other (L. G. C.), the Medical Research Council of Great Britain and the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. REFERENCES Adams, M. H. (1959). In Bacteriophage, p. 446. New York: Interscience Publishers, Inc. Berg, C. M. & Curtiss, R. (1967). Genetics, 56, 503. Bleecken, S., Strohbach, G. & Sarfert, E. (1966). 2. allg. MikrobioZ. 6, 121. Bonhoeffer, F. & Gierer, A. (1963). J. Mol. Biol. 7, 534. Cairns, J. (1963a). J. Mol. Biol. 6, 208. Cairns, J. (19638). Cold. Spr. Had. Symp. Quant. Biol. 28, 43. Cuzin, F. (1966). Ph.D. thesis, Faculte des Sciences, Universite de Paris. Forro, F.. Jr. & Wertheimer, S. A. (1960). Biochim. biophys. Acta, 40, 9. Gross, J. D. & Care, L. G. (1966). J. Mol. BioZ. 16, 269. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spr. Had. Symp. Quunt. Biol. 28, 329. Lark, K. G. & Bird, R. E. (1965). Proc. Nut. Acad. Sci., Wash. 54, 1444. 1, 190. Lennox, E. S. (1955). Pirology, Nagata, T. (1963). Proc. Nat. Acad. Sci., Wash. 49, 551. Schaechter, M., Bentzon, M. W. & Maalee, 0. (1959). Nnture, 183, 1207. Sueoka, N. & Yoshikawa, H. (1965). Genetics, 52, 747. van Tubergen, R. P. (1959). Ph.D. thesis, Yale University. van Tubergen, R. P. & Setlow, R. B. (1961). Biophys. J. 1, 589. Vielmetter, W. & Messer, W. (1964). Ber. Bunsengesellschaft, 68, 742. Vogel, J. H. & Banner, D. M. (1956). J. Biol. Chem. 218, 97. Yoshikawa, H. & Sueoka, N. (1963). Proc. Nut. Acad. Sci., Wash. 49, 559.
Chromosome Replication in Escherichia coli I. Lack of Influence of the Integrated F Factor CLAIRE M. BERGS AND LUCIEN G. CAROM
Biobgy Division, Oak Ridge National Laboratory Oak Ridge, Tenn. 37830, U.X.A. Microbial
Medical Research Council Genetics Research Unit, Hammersmith Hospital London, W.12, England (Received 24 June 1967)
Therelative gene frequency inisogenic Hfr strains of Escherichia co.5 K12 has been assayed by Pl transduction in order to evaluate the influence of the integrated F factor on the origin and direction of chromosome replication. Three strains with different sites of integration of the F factor were studied. No significant differences in transduced gene frequency were found in either a one-cycle lysate made on exponential cultures of the strains or in the transducing particles of hybrid density obtained from a lysate made on cells previously pulse-labeled with 5-bromouracil. We conclude that, in these strains, the integrated F factor does not affect the origin or the direction of chromosome replication.
1. Introduction In studying chromosome replication in Escherichia co& a most important question is the determination of whether each round of replication starts from a fixed site on the genetic map. In 1963, Nagata approached this problem by considering the yield of the prophages X and 424 produced after induction of doubly lysogenic strains of E. wli K12. The F- and two Hfi- strains studied gave different time sequences for the relative yield of the two phages during one cycle of synchronized growth. On this basis, Nagata proposed that in Hfr strains chromosome replication starts at the site of integration of the F factor and proceeds in the direction opposite to that of the sequence of transfer during conjugation. He also proposed that the chromosome of F- cells starts its replication at random from any site on the continuous circular structure. We have investigated the first aspect of the Nagata model, the role of the integrated F factor in chromosome replication. To this end we have analyzed the state of replication of the chromosomes in several strains of E. coli K12. The method of analysis is similar to that used for Bacillus subtilis by Yoshikawa 6 Sueoka (1963), who measured
t Present address: Institut de Biologic Mol&ulaire, Universite de Genkwe, Switzerland. Reprint requests should be sent to Oak Ridge National Laboratory. $ Present address: Oak Ridge National Laboratory, Oak Ridge, Term. 37830, U.S.A. 419
420
C. M. BERG
AND
L.
G. CAR0
the relative frequency per chromosome of various genetic markers using transformation with extracted DNA. They argued that, in an exponential population, the probability of occurrence of a marker located at the origin of chromosome replication should be twice as high as that of a marker located at the end, with a continuous gradient of probability in between. Instead of transformation, we are using generalized transduction with the bacteriophage PI (Lennox, 1955) as the method of sampling chromosomal markers. We infect exponential liquid cultures of E. coli K12 strains with Pl. The progeny phage from a one-cycle lysate is then used to transduce as many as seven different markers into a multiply marked recipient. Since the efficiency of transduction is different for each marker, marker frequencies cannot be established directly. Instead, we calculate the proportion of transductants for each marker relative to the total number of transductants in any one experiment. We then compare, two by two, the values obtained for strains which would be expected to give different gradients of marker frequency if the origin and the direction of chromosome replication were determined by the position and orientation of F. Our results demonstrate that the position and orientation of the integrated F factor in these strains have no effect on the origin and direction of replication.
2. Materials and Methods (a) Media L broth, L soft agar (0.65% agar) and L agar (1.2% agar) (Lennox, 1955), supplemented with 2.5 x 10-e M-calcium chloride and with 20 pg thymine/ml., were used for growing cells and for phage assays. All phage lysates were made in this medium regardless of the previous growth conditions of the cells. Before Isbeling with 5-bromouracil, the cultures were grown inM9 (glucose-salts) medium (Adams, 1959) supplemented with 0.5% Casamino acids (Difco) and 5 pg thymine/ml. Transductants were selected on appropriately supplemented medium E agar (1.5%) overlaid with unsupplemented medium E soft agar (0.65%) (Vogel & Bonner, 1956). Supplements, purchased from Calbiochem, were used at the following concentrations, in ag/ml. : thymine, 5; adenine, 40; thiamine-HCI, 2 ; L-proline, 30; L-tryptophan, 20; L-lysine-HCl 85; L-methionine 10; and L-leucine 20. The carbon sources were glucose, lactose or arabinose at 0.5% final concentration. Streptomycin sulfate was added to give 200 pg/ml. (b) Strains The strains
of E. co.% used are listed
in Table
1.
(c) Phage stocks A clear plaque mutant was selected from a strain of Plkc obtained from Roy Curtiss. This phage, Pl.L4, had the following advantages over Plkc: higher titers in one-cycle liquid lysates, up to 2 x lOlo phages/ml. ; good yield of transducing particles; increased stability in cesium chloride solutions and low calcium requirement. PlaL4 stocks were prepared by the confluent plate lysis method (Adams, 1959) on x 478, a strain isogenic with the recipient strain x 484; thus the transducing potential of unadsorbed carry-over Assays were made on LC35 phage was eliminated. Titers of 1 to 2 x 1O1l were obtained. with a IO-min adsorption period before plating. (d) Trur~sductio~r The recipient of the recipient
used throughout was x 484, a Plkc-lysogenic were grown from a number of single-colony
derivative isolates.
of x 478. Cultures The one with the
CHROMOSOME
REPLICATION TABLE
Xtraim
strain
Designation
IN E. COLI
421
1 used
Relevant characteristics
source
Ref.
x 354
F-
pro, - lac deletion
cortias
x 473
F-
B1- lac,- proa- ade=- try-
(W1485) TV945
,?(434
F-
lya- metE- ara- leu- strrP B,- la,- pro,- ade,- trylye- metE- ara- lezl-
LC617 LC35
FF-
met E. coli C, met- arg- try- ade
LC618 x 416 x 493
Hfr H Hfr C Hfr OR11
met- (A, 424)
x 562
Hfr OR11
leu- thy-
W1485 P”) x 493
x 629
Hfr IOR 3
leu- thy-
x 562
(1967) Bera & Curtisa (1967)
x 618
F’ ORF 8
leu- thy-
x 562
Berg & Curt&s
CB 03
Hfr TOR 36
leu- thy-
x 618
Berg & Curtiss (1967)
LC615
F’ ORF 8 partial diploid F’ ORF 8 partial diploid
Berg & Curtisa (1967)
x 473
strr (P&c) 58-161 Arber Hayes Nagata
prototroph
Gross & Cero (1966) Nagata (1963) Berg & Curt& (1967) Berg & Curt&s
(1967)
LC616
x618 x ,y 354 x 618 x x 354
Symbols for the genetic markers: B1, leu, thy, pro, ade, try, lys, met, arg, requirement for thiamine, leucine, thymine, proline, adenine, tryptophan, lysine, methionine and arginine, respectively; am, gal, Zac,inability to utilize arabinose, galactose, lactose; strS /str’, sensitivity and resistance to streptomycin; (A), (424), (Plkc), lysogeny for the phages h, 424, Plkc, respectively. Note: All strains are E. co&i K12 except for LC35 (E. coli C). All K12 strains used are SU,,+ (amber suppressor). lowest number of revertants was selected and maintained as a stationary broth culture at 4°C. This procedure was repeated at 2- to 3-month intervals. For transduction assays, an exponential broth culture of this x 484 stock grown to 1 to 2 x 10s cells/ml. was infected with Pl*L4 at multiplicities of 3 to 5 phages/cell and incubated without aeration at 37°C for 30 min. The adsorption period was terminated by spreading or, more commonly, by diluting the mixture with medium E soft agar at 45% and plating equal samples onto 4 plates of each selective medium. The plates were incubated at 37°C for either 30 hr (spreading method) or 39 hr (agar method). (e) Experimental
procedure.
(i) Pl lysates Cultures of the host strains were grown in broth to a concentration of 2 x lOa cells/ml. They were then infected with Pl*L4 at a multiplicity of infection of 3 to 5 in broth. After 60 min of vigorous aeration at 37’C, the culture was chloroformed, treated with DNase (Worthington) (10 pg/ml.), and the cell debris removed by centrifugation. The infectivity and the transducing activity for various markers were tested as described above. The phage titers varied between approximately 2 x lo9 and 2 x lOlo, and the transducing activity between 10e4 and 10m5 of the infectious titer, depending on the
422
C. M.
marker, infection.
the
strain
5-Bromouracil
(ii)
tested,
BERG
and the growth
AND
L. G. CAR0
conditions
of the
bacterial
culture
prior
to
pdses
Cultures were grown in MS supplemented with C&amino acids and thymine to a concentration of 2 x lo* oells/ml. They were washed on an S 8, S membrane filter and collected in M9 with Casamino acids. After 5-min aeration at 37”C, BUt was added to give 10 pg/ml. The labeling period was usually 15 min. After this time the cells were washed by filtration and collected in a small volume of M9 with C&amino acids. They were aerated 5 min, diluted 5-fold with broth and infected with Pl. Lysates were made as described above. In control experiments it was found that the strain x 562 (Hfr OR1 1) and its derivatives 2.5% of the total thy-mine content contain a pool of thymine equivalent to approximately of the cell. A starvation period of 5 min, such as that used before and after BU labeling, is sufficient to exhaust this pool completely. The generation time in BU, measured under the conditions of the experiment by incorporation of 5-[6-3H]bromouracil (Amersham), is 50 min. This incorporation of tritiated BU is approximately exponential for at least 30 min. The generation time in thymine is 35 min. (iii)
Density gradient
Density
gradients
were formed
by mixing
1.5 ml. of the lysate
2.0 ml. of a cesium 15 to 18 hr at 30,000 rev./min in a SW39 rotor in a Spinco model L ultracentrifuge. The fractions, collected with a Buchler fraction collector, consisted of 5 drops each (400 drops total) takeninto 1 ml. of broth. A first assay of transducing activity for one or two markers (ade or met) was made in order to localize the hybrid and the light-density bands and to determine the approximate number of transducing particles in each. The transducing activity for all the selected markers was assayed separately for three or four tubes in the hybrid band, one or three tubes in the light band and for the original lysate. Controls showed little difference in the positions in the gradient of transducing activity for the markers used. No transducing particles were ever found in the position expected for heavy (BU-BU) density.
chloride solution of density 1.78 g/ml. The mixture
with
was centrifuged
3. Results and Discussion (a) Discussiwl
of method
The theory of marker-frequency analysis has been considered in detail by Sueoka $ Yoshikawa (1965). We shall summarize here some of their conclusions in relation to our experimental system. We assume that in an exponentially growing culture of bacteria the chromosomes replicate without interruption (van Tubergen, 1959; Schaechter, Bentzon & Maaloe, 1959) asynchronously, and at a constant rate of displacement of a single replication point (Cairns, 1963aJ; Bonhoeffer & Gierer, 1963; Bleecken, Strohbach & Sarfert, 1966). Under such conditions, the probability density function for the position of replication points is : f(z) = In 2.2l-’
(1) (x = 0) to the end (x = 1)
where x represents a position measured from the origin of the chromosome. This function has the values f(0) = 2 In 2 at the origin and f(1) = In 2 at the end. Therefore, the replication point has approximately twice as high a probability of being in a region near the origin than of being in a region of equal length at the end of the chromosome, with a gradient of intermediate values at points in between. This arises, of course, from the fact that the termination of each round of replication (one growing point at the end) gives rise to two new replicating structures (two growing points at the origin). t Abbreviation
used; BU, 5-bromouracil.
CHROMOSOME
REPLICATION
IN
E.
COLI
433
One can easily derive from equation (1) the average number of copies per chromosome for a genetic marker placed at distance x from the origin: g(x) = 2 l-2.
(2) This function differs only by a constant (In 2) from the distribution function of replication points and follows, therefore, a similar gradient. The gradients for the probability density of growing-point positions and for the relative marker frequency both depend on the origin and direction of replication being fixed. In the case of a randomly distributed origin, both functions would have the same value for all values of 5. Sueoka & Yoshikawa (1965) have also shown that, if there are several growing points running in the same direction, both functions show a steeper gradient than for a single growing point. Consider an Hfr strain, (m), with two markers A and Z placed, respectively, at the origin and at the end of transfer. Under Nagata’s model, Z would be at the origin of replication and A at the end of replication. Their expected transduction frequencies would be: g, (A)=g(l)=K,
andg,
(.Z)=g(O)=2K,
where KA and K, represent the efficiency of transduction for each of the two markers and each is assumed to be constant among isogenic strains. The ratio of the two values is :
It is obvious that the maximum expected change in relative marker frequencies will occur for a strain (n) in which the direction of transfer, and therefore the postulated direction of replication, has been reversed. We would then get: g,(A) = g(O) = 2K, g,(z)
= g(l) = Kz and
Comparing the two strains (m) and (n) : -= Qn
Q,
4,
Intermediate markers would give lower values, but the difference between the two strains should be easily detectable. For strains with the same origin and direction of replication, Q,/Q, would, of course, be equal to one. (b) Lysates on non-isogenic strains In a preliminary experiment, we compared the marker frequencies obtained in PI lysates made on exponential cultures of three non-isogenic strains: Hfr H, Hfr C, and 58-161 F-. If the position of F determined the origin and direction of replication, a number of predictions on the expected relative marker frequencies could be made. Table 2 shows that the differences between the strains do not correspond with these predictions. Thus, since they are closely linked, lac and ade should behave as a unit in Hfr H and Hfr C ; met should be much higher in Hfr H than in Hfr C ; and lys should 28
424
C. M.
BERG
AND
L.
G. CAR0
TABLE 2
Relative transductant frequencies in lysates of non-isogenic strains
Host strain
lac
Hfr H Hfr C F- 58-161
7.2 8.7 16.7
P”
ade
try
lYS
met
ara
5.8 10.3 6.0
11.8 12.0 11.0
7.0 12.0 7.1
25.5 6.8 18.5
22.8 26.9 24.9
19.9 23.3 15.9
Transductant colonies counted 7395 807 10,674
One-cycle lysates of Pl.L4 were made on exponential cultures of the three strains indicated. The phage yield on Hfr C was about 10 times less than on the other strains. This may be due to t,he fact that the strain was lysogenic for X and 424.
be higher, rather than nearly four times lower, in Hfr C than in Hfr H. Neither this low value of lys in Hfr C nor some other features of the marker frequencies can be explained on the basis of a simple replication scheme. It is likely that the differences observed are due to variations in the genetic background of the three strains affecting transduction efficiency. It is clear, therefore, that isogenic strains should be used. (c) Isogenic strains Three isogenic Hfi strains were used throughout the rest of this work. Hfr OR11 is a derivative of W1485. Hi? IOR was derived from Hfi OR11 by an inversion of the F Zacregion. Hfr TOR36 was derived, also from IIfr ORll, by forming a primary (haploid) F’ strain containing the region F lac pro ade on the F’ and re-integrating it, by a reciprocal exchange, into the chromosome. The final result is that the sequence F Eat pro ade has been deleted from its original location and re-integrated in the order pro ade F lac to the left of ara. The position of the relevant markers and their order of
FIG. 1. Isogenic Hfr strains. Map of three isogenic strains used in this work. Note that in IOR3, produced by an inversion of the F Zac region, F has almost the same location as in its parental strain ORll, but that the direction of transfer is reversed (Berg 8: Curtiss, 1967). TOR36 was produced by deleting the region F lac pro ade from OR 11 and inserting it between met and ara.
CHROMOSOME
REPLICATION
E. COLI
IN
425
transfer in mating are shown, for the three strains, in Fig. 1. The isolation and properties of these and related strains have been described (Berg & Curtiss, 1967). Autoradiographic experiments (Caro & Berg, unpublished data) have shown that in these strains DNA synthesis takes place during more than 95% of the cell cycle and involves, generally, all nuclear regions present in each cell. This synthesis is exponential during exponential growth of the cells. It involves large structures which segregate, without significant fragmentation, in the pattern expected for E. coli chromosomes (Forro & Wertheimer, 1960; van Tubergen & Setlow, 1961; Lark & Bird, 1965). In a control experiment, the strain x 562 was grown in BU for 30 minutes under conditions identical to that used in BU labeling experiments (see later). The proportion of transducing particles found in the heavy, hybrid and light bands was analyzed for five markers (tie, aru, met, Zys, try). For all five markers, the proportion found in the heavy band (replicated twice) was less than 4%. The proportion of transducing activity in the hybrid band (replicated once) was nearly the same for all markers, varying between 60 and 69%. It seems, therefore, that the pattern of replication in this strain is normal and is not grossly affected by BU, at least for times TABLE 3
Reproducibility
of transductant frequencies
ade
Lysate
1 2 3
4.8 4.6 4.4
13.3 12.4 12.2
6.6 6.6 6.6
lY8
met
20.5 20.3 19.4
29.0 29.1 29.4
kU
25,s 27~0 28.0
Colonies counted
13,620 11,196 11,340
Relative transductant frequencies (percentage of total transductant colonies counted) for 3 one-cycle lysates, made on 3 separate cultures of x 493 (Hfr ORll). As in all other experiments, lysates were made nearly simultaneously for all three cultures. Assays for transduction were also made on x 484, simultaneously.
TABLE 4
Effect of nzultiyplicz’ty of infection of Pl-L4 on transductant frequencies
m.0.i.t
ade
lY8
met
ara
Transductant colonies counted
Total phage plated x 10-s
10 3.5 1.0 0.3 0.1
20.8 20.9 18.2 20.3 20.1
26.6 28.4 28.7 25.5 24.9
26.4 25.4 26.6 26.7 26.8
26.2 25.4 26.5 27.5 28.2
4286 6832 3519 3572 3760
2.0 3.5 2.0 2.8 2.0
Portions of an exponential culture of x 484 were infected with Pl.L4 at multiplicities varying from 0.1 to 10 phages/cell. Platings for transductants were done by the top agar layer method at dilutions such that the number of phages plated was comparable. The relative transductant frequencies (percentage of total transductant colonies counted) for four markers show no detectable effect of multiplicity of infection. t Multiplicity
of infection.
426
C. M.
BERG
AND
I,.
G.
CAR0
twice as long as that of the BU labeling used in section (f). DNA replication, in the three strains, seems therefore to follow the conditions set at the beginning of section (a) for the application of the probability density function: g(z) = 2l-“. Should the origin and direction of replication be determined by the integration of P on the chromosome, we could, therefore, expect strong differences in the gradient of marker frequencies in the three Hfr strains studied. Control experiments, shown in Tables 3 and 4, indicate that Pl transduction is reproducible enough to detect such clifferences if they occur. A further conditionis that transductant frequency should be proportional to marker frequency. This point is examined in the next section. (d) Response to gene dosage To test that transductant frequency responds to gene dosage, Pl*L4 lysates were made on exponential cultures of a series of strains constructed for this purpose and all derived from W1485. In these strains the markers pro, and ode, are present on the chromosome (Hfr OR11 and x 354), on the F’ factor in a primary F’ (ORF8, a strain in which the markers on the F’ factor are deleted from the chromosome; see Berg & Curtiss, 1967) and on both the chromosome and the episome in two secondary (partially diploid) F’ strains (LC615 and LC616). The marker lac found on the chromosome in OR11 is deleted in x 354 and is present only on the F’ factor in the other strains. There is evidence that an F’ strain carries only one F’ factor per chromosome (Cuzin, 1966; Jacob, Brenner & Cuzin, 1963). If the production of transducing particles for a given marker is directly proportional to the number of copies of that marker, we expect that a partially diploid strain would produce between 1.5 and 3 times more transductants for the duplicated markers, depending on the respective time of replication of the markers on the chromosome and on the F’ factor. TABLE
5
Response of tra~~,~Jucta~ttfrequency Strain
Hfr OR11 F-x354 F’ ORFS (haploid) F’ LC-615 (diploid) F’ LC-616 (diploid)
Genotype lac
Pro
ade
Location markers
+ +
-I-/+
+ Chromosome + Chromosome + Episome
+
+ +
+ -
+ +
+ Episome + Chromosome + Episome + Chromosome
lac
of
T+L+M+A
to gene dosage Pro T+M+L+A
ade T+M+L+A
0.094 0 0.10
0.087 0.066 0.097
o-19 0.17 0.22
0.11
0.15
0.35
0.12
0.17
0.37
Pl.L4 lysates were made on exponential cultures of the five strains shown: Hfr OR11 (x562) is as shown on Fig. 1; F- x354 contains a deletion of the Zac gene; F’ ORF8 (x618) is a primary F’, derived by a single reciprocal exchange on the chromosome of the Hfr OR1 1, in which the markers Zac, pro and ade have become incorporated into an F’ factor, leaving a corresponding deletion on the chromosome; F’ LC-615 and LC-616 have been formed by introducing the F’ factor from ORF8 into F- x354. They are therefore diploid for pro and ade, haploid for Zac, which is present only on the F’ factor and haploid for the other markers, which are located on the chromosome. The transductant frequencies for Zac, pvo and ade are compared with a base line formed by the sum of the frequencies for four chromosomal markers; try, Zya, met and ara (T + L + M + A). The frequency of pro transductants is depressed in x354, presumably because the normally cotransduced Zac locus is deleted in this strain.
CHROMOSOME
REPLICATION
IN
E. COLI
427
The results in Table 5 show that the relative transductant frequencies for lac, pro,, and ade, are raised only slightly when the marker is located on the F’ factor instead of the chromosome, but that the relative transductant frequencies for pro, and ade, are nearly doubled in those strains which are diploid for these two markers. There is, therefore, a definite response of transductant frequency to gene dosage. It is interesting to note that, if this response was shown to be linear, the data would be consistent with the notion that the F’ factor replicates shortly before the lac pro ode region on the chromosome. (e) Lysates on isogenic strains Pl lysates were made by one cycle of infection in exponential cultures of the three isogenic Hfr strains. The relative transductant frequencies found for five markers are shown in Table 6. There is little difference between the three strains. If the origin was TABLE
6
Relative trawductant frequencies in lysates of isogenic Hjr strains
Host
strain
OR11 IOR TOR36
ade
t TY
lY8
met
ara
16.3 19.3 15.6
13.5 11.5 11.8
15.3 14.7 15.1
31.2 31.4 33.8
23.8 23.1 23.6
Tranaductant colonies counted 2331 2847 2728
One-cycle lysates of PlmL4 were made on exponential cultures of the three isogenic Hfr streins shown in Fig .l . The relative transductant frequencies for five markers are expressed as 8 percentage of total transductant colonies counted.
controlled by F, a clear difference should occur between strains OR11 and IOR in which F is integrated in nearly the same position but with opposite orientation. We can take ara as a fixed point and calculate the values of the ratios of the relative proportions of each marker to ara for the two strains. Figure 2 shows that the experimental points give, for this ratio, a constant value of approximately 1.0, as would be expected if the two strains had the same origin (or origins) and the same direction (or directions) of replication. The results show, therefore, no influence of the integrated F factor on the origin and direction of replication. Although a definite response of Pl transduction frequency to gene dosage has been demonstrated (Table 5), several possible occurrences could invalidate the conclusions drawn from this type of experiment. These are : (1) that Pl transducing particles are formed only in cells which have been infected just after completion of a round of DNA replication; (2) that a cell infected with Pl can complete one round of DNA replication but cannot initiate a new round ; (3) that Pl can pick up DNA from only one of the two newly replicated branches of each chromosome and from the non-replicated portion. In all these cases, the chromosome would appear to the phage as a linear, fully replicated structure and would show no gradient of marker frequencies. To avoid these difficulties the experimental design was modified as shown in the next section.
428
C. M.
BERG
L.
of transductent
G. CAR0
I !Ys
I fry
OL Oode
FIG. 2. Comparison
AND
frequencies
I F
I met
with predictions
for F-determined
origin.
The transductant frequencies for four markers, a&, try, Zya and met, relative to that of ara taken ES reference, for a lysate made on an exponential culture of OR11 were compared to those for IOR3. The Nag&a model predicts, for the ratio of the two, a gradient going from approximately 3.33 for ode to 1.20 for met. This gradient is calculated from the position of the marker and its presumed frequency given by g(z) = 2i-‘, assuming that F is the origin and that the direction of replication is opposite to that of transfer in conjugation (- - -). If, on the other hand, the two strains had the same origin and direction of replication, the expected value for the ratio would be 1.0 for allmarkers (- - - -). The data fit better this latter model. Data from Table 6. The abscissa shows the transfer sequence of markers for IOR and their map position relative to F.
(f) Lysates on isogenic strains after labeling with 5-bromouracil Exponential cultures of the three Hfr strains were labeled with BU during approximately one-third of the generation time. Pl lysates were made on these cultures by one cycle of growth. The transducing particles of hybrid density were separated from the light ones and from the infectious particles in a cesium chloride density-gradient and were used to assay the relative marker frequencies. This experiment bypasses or answers the objections raised above. In case (1) only ends of chromosomes would be labeled, and a clear difference between the strains would be predicted if F determined the origin. Case (2) would not affect the results since we are now sampling DNA made exclusively before infection. Case (3) would not affect the results either : the expected gradient in BU-labeled markers arises now, ISO-
I
4
I
I
Tube number
FIQ. 3. Density
profile
of ode transducing
particles
after pulse of BU.
Distribution in a cesium chloride density-gradient of ode-transducing particles from a onecycle Pl.L4 lysate made, in light medium, on a culture of Hfr OR1 1 previously labeled for 16 min with BU. The total number of fractions in the gradient was 80. Arrows indicate the tubes selected for transductant frequency analysis in the hybrid density band.
CHROMOSOME
REPLICATION
IN
429
E. COLI
not from the probability density function of marker frequencies, g(x), but from that of growing-point positions, f(z). As we have seen, the two functions are identical, except for a constant. The function f(x), and therefore the gradient of marker frequencies in the BU band, will be the same whether one or both branches of the newly replicated portion of the chromosome are picked up. The density distribution profiles for ode-transducing activity were analyzed for all three strains (Fig. 3). The proportion found in the hybrid density band was 44% of the total activity in the gradient for Hfr ORll, 44% for IOR3, and 43% for TOR36, showing that ade had been replicated in equal proportion in the three strains. The marker frequencies, for five markers, averaged over three tubes in the hybrid band, are shown in Table 7. Each tube was assayed separately. No significant differences in relative marker frequencies were found between them, showing that density differences between markers are not suf%ent to affect the results. Here again the expected TABLE
Relative transductant frequencies
Host strain
ode
OR11 IOR TOR36
28.7 30.8 27.0
7
from hybrid density Pl band after 5-bromouracil
11.2 9.7 6.2
lYS
met
am
Transductant coloniee counted
6.2 6.0 6.3
33.4 34.7 40.9
20.6 18.9 19.7
6416 7330 11,617
Exponential cultures of the three isogenic Hfr strains shown in Fig. 1 were labeled with E pulse of BU, as described in the text. A one-cycle PlmL4 lysats was made, in light medium, on all three strains shortly thereafter. The transducing particles of hybrid density were separated from the light ones and from the infectious progeny phrages on a cesium chloride density-gradient. The reletive transducing frequencies shown are each the combined data from three tubes in the hybrid density peak (see Fig. 2). Due to the difllculties inherent to the experimental design, the d&a are more scattered than those of Table 6. They do not, however, exhibit the strong differences expected for an F-determined origin and direction of replication.
:
‘\
+I 3,0\ G-ie ‘\ ‘60 *\ + 2.0‘\ z 0 x F ,o o_---Q-------,----;;---=~, ~ ;I0 5-l
LJ
Oode
-\
-\
I
I
fry
‘YS
-1
I me+
I
F
Fm. 4. Comparison of hybrid density transductant frequencies with predictions of an F-determined origin. Same calculations as in Fig. 2, for the data of Table 7 for Hfr OR11 and IOR3; - - -, F-determined origin; - - - -, fixed or variable origin. Here again the results favor a model in which the two strains have similar origins and directions of replication.
430
C. M.
BERG
AND
L. G. CAR0
differences between strains fail to appear. If, as in the previous section, we plot the ratios of the relative proportions of each marker to ara for the two strains OR1 1 and IOR (Fig. 4), it is clear that the experimental points do not fit the distribution predicted by an F-determined replication. Instead, these ratios all have a value close to 1.0, as expected if the two strains did not differ in origin or direction of replication. (g) Conclusions The BU pulse experiments agree completely with the total lysate experiments in showing no differences in marker frequencies between three isogenic Hfr strains. The differences found between the marker frequencies in a straight lysate (Table 6) and in the BU-labeled band (Table 7) could be explained by some selective inactivation of the markers by BU. They could also reflect a small amount of aberrant replication induced by BU itself or by the experimental procedure. Although this would complicate the interpretation of the results, it is difficult to imagine how such a replication could fail to reflect the differences in marker frequencies that would be produced by different origins and directions of replication in the normal state. The two types of experiments concur, therefore, in showing that the position and direction of integration on the chromosome of the F factor have no detectable effect on the origin and direction of replication in our strains. Nagata’s data (1963) showed that, during the division-cycle of two Hfr strains, with opposite directions of chromosome transfer, the relative yield of two prophages, h and 424, changed abruptly at fairly well-defined times and that the time sequence of these events was reversed in the two strains. This result did not provide a complete demonstration of the model which he proposed, since (1) it was neither demonstrated conclusively that DNA synthesis was synchronized nor that the initiation of DNA replication coincided with that of the cell life-cycle, and (2) it was not shown that the phage yield was directly proportional to the number of copies of the prophage present in the cell at the time of induction. Nagata’s conclusions have, however, been supported by some unpublished evidence of Vielmetter & Messer (1964 ; Messer, personal communication) based on the relative frequency of appearance of sectored and unsectored mutant colonies after 32P-labeling of various Hfr strains. On the other hand, we have studied the replication occurring during treatment and recovery from amino acid starvation (unpublished data) and have found no change in the direction of replication due to a different orientation of the integrated F factor. Other workers have obtained similar results with amino acid starvation (B. Wolf, A. Newman & D. A. Glaser, personal communication) and exposure to phenethyl alcohol (J. Tomizawa, personal communication). These discrepancies could be explained in two ways: (1) the integration of the F factor either affects chromosome replication in some strains but not in others or only under certain experimental conditions, or (2) the observations of Nagata were due to a difference of origin in the strains he used, which happened fortuitously to conform to the model. This second explanation may find some support in that we have found recently that distantly related strains of E. co& K12 have different directions of replication and, perhaps, different origins. These results will be the subject of a subsequent paper. The present data do not completely rule out the possibility that F determines the origin of replication. They could be explained, in that case, by postulating that chromosome replication can proceed, with equal probability, in opposite directions.
CHROMOSOME
REPLICATION
IN
E.
COLI
431
Results to be published subsequently have however, indicated that replication is unidirectional. It seems, thus, that there is no necessary relation between the position and orientation of the integrated F factor and the origin and direction of chromosome replication. We are grateful to Dr W. Hayes for the hospitality given in his laboratory and to many of our colleagues in Oak Ridge and in London, and more particularly to R. Curtiss, J. D. Gross, J. A. Shapiro, and C. M. Steinberg for invaluable discussions and suggestions. The assistsnce of D. J. Atkinson was of great valuain the experiments performed in London. This research was jointly sponsored by a Postdoctoral Fellowship of the North Atlantic Treaty Organization to one of us (C. M. B.), a Senior Postdoctoral Fellowship of the National Science Foundation to the other (L. G. C.), the Medical Research Council of Great Britain and the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. REFERENCES Adams, M. H. (1959). In Bacteriophage, p. 446. New York: Interscience Publishers, Inc. Berg, C. M. & Curtiss, R. (1967). Genetics, 56, 503. Bleecken, S., Strohbach, G. & Sarfert, E. (1966). 2. allg. MikrobioZ. 6, 121. Bonhoeffer, F. & Gierer, A. (1963). J. Mol. Biol. 7, 534. Cairns, J. (1963a). J. Mol. Biol. 6, 208. Cairns, J. (19638). Cold. Spr. Had. Symp. Quant. Biol. 28, 43. Cuzin, F. (1966). Ph.D. thesis, Faculte des Sciences, Universite de Paris. Forro, F.. Jr. & Wertheimer, S. A. (1960). Biochim. biophys. Acta, 40, 9. Gross, J. D. & Care, L. G. (1966). J. Mol. BioZ. 16, 269. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spr. Had. Symp. Quunt. Biol. 28, 329. Lark, K. G. & Bird, R. E. (1965). Proc. Nut. Acad. Sci., Wash. 54, 1444. 1, 190. Lennox, E. S. (1955). Pirology, Nagata, T. (1963). Proc. Nat. Acad. Sci., Wash. 49, 551. Schaechter, M., Bentzon, M. W. & Maalee, 0. (1959). Nnture, 183, 1207. Sueoka, N. & Yoshikawa, H. (1965). Genetics, 52, 747. van Tubergen, R. P. (1959). Ph.D. thesis, Yale University. van Tubergen, R. P. & Setlow, R. B. (1961). Biophys. J. 1, 589. Vielmetter, W. & Messer, W. (1964). Ber. Bunsengesellschaft, 68, 742. Vogel, J. H. & Banner, D. M. (1956). J. Biol. Chem. 218, 97. Yoshikawa, H. & Sueoka, N. (1963). Proc. Nut. Acad. Sci., Wash. 49, 559.