On the origin and direction of replication of the Escherichia coli K12 chromosome

PT.Mol. Biob. (1968) 32, 611-629

On the Origin and Direction of Replication of the Escherichia coli K12 Chromosome BEVERLY WOLF, ANITA NEWMAN AND DONALD A. GLASER Virus Laboratory, University of California Berkeley, California, 94720, U.S.A. (Received 18 September 1967, and in revised form 15 November 1967) Replication

of the chromosome of Escherichia

coli K12 bacteria

was studied by

measuring transduction frequencies of generalized transducing phage Pl prepared upon donor bacteria containing DNA pre-labeled with Sbromouracil near the beginning or near the end of the chromosomes. Alignment of the chromosomes for specific labeling was accomplished by amino acid starvation. The phage containing the bromouracil-labeled DNA were isolated by cesium chloride density-gradient ultracentrifugation and were analyzed by transduction experiments using 11 genetic markers distributed nearly uniformly over the genetic map. It was found that chromosome replication terminates in a definite region of the genetic map during amino acid starvation and initiates a new cycle in the same region when amino acids are restored. Three strains (F- DG68, OF- DG75, and Hfr P7201) have their terminus-origin in the region between lys and zyl (54 to 70 minutes on the E. coli genetic map); the clockwise direction of replicat,ion agrees best with the data. Another strain (Hfr DG163) has its terminusorigin in the region between pro and gal (6 to 16 minutes on the map) and counterclockwise replication. There seems to be no uniform rule relating the chromosomal replication pattern with F-factor integration. It cannot be deter-

mined from these studies whether the terminus-origin observed in amino acid starvation experiments is identical to the vegetative origin presumed to exist in normally

growing

cells.

1. Introduction The chromosome of Escherichia coli is thought to be a single circular molecule of double-stranded DNA which replicates semi-conservatively and sequentially along its whole length (Meselson & Stahl, 1958; Cairns, 1963; Bonhoeffer & Gierer, 1963; .Lark, Repko & Hoffman, 1963). Although some evidence indicates that replicat,ion begins at a definite site on the chromosome and proceeds in a definite direction, there is no general agreement on the map location of the origin, the direction of synthesis, or the possible influence of F-factors, physiological variables, or strain differences (Nagata, 1963; Jacob, Brenner & Cuzin, 1963; Vielmetter & Messer, 1964; Berg & Caro, 1967). Our present experiments are similar in principle to the experiments done on Bacillus subtilis by Yoshikawa & Sueoka (1963). Using the technique of transfhrmation after isotopic transfer experiments these investigators were able to demonstrate sequential DNA replication and to construct a genetic map of B. subtilis delineating an origin and terminus. Since general transformation is not successful in h’. coli, the process of generalized transduction was used to genetically analyze specific 611

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segments of labeled bacterial chromosome. The chromosomes of two F- and two Hfr strains of E. coli K12 were labeled with a heavy nucleotide analog either where replication was halted during amino acid starvation or where it resumed just after starvation. The following strategy was used and is shown schematically in Fig. 1. Ends Exponential

-Amino +5-BU

growth

acid (*)

Transducing phage made

Beginnings Exponential growth

-Amino acid + thymine

+ Amino acid + 5-BU (m)

Transducing phage made

FIG. 1. Schematic representation of beginnings and ends experiments. DNA molecules are shown diagrammatically as they exist in cells before and after amino acid starvation, BU labeling, and incorporation into phege co&s to form transducing particles. A complete description of the experiments can be found in the Introduction and experimental procedures section of Materials and Methods.

(a) Allow repletion of replicating ck~onaoeonzerr without starting new cycles of replication by starving for a required ammo acid (Maalee t Hanawalt, 1901; Lark et al., 1963). (b) Label chromosome ends (“ends” experiments) to define the presumed terminus of replication by feeding 5-bromouracil instead of thymine (all strains are thg-) during ammo acid starvation. (c) Label chromosome beginnings (“beginnings” experiments) to define the presumed origin of replication by feeding BUT and the required amino acid to cells whose chromosomes were previously aligned by ammo acid starvation (in the presence of thymine). (d) Prepare generalized transducing phqe PI upon bacteria containing DNA prelabeled with BU in particular regions of the chromosome as described above, t Abbreviation

used; BU. 6-bromourwil.

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The assumption is made that Pl transducing particles incorporate the chromosomal DNA randomly. (e) Isolate high density PI phage containing BU-labeled DNA by C&l densitygradient equilibrium centrifugation. (f) Measure transduction frequencies for hybrid (one strand containing BU instead of thymine) and, when they are present, heavy (two strands containing BU) transducing phage for 11 genetic markers distributed nearly uniformly over the genetic map. Hybrid phage prepared on donors with BUlabeled chromosome ends should transduce markers predominantly near the replication terminus; those from donors with labeled beginnings should transduce markers mainly near the replication origin. The results summarized graphically in Fig. 3 show that both F- and Hfr strains have a unique origin for the initiation of replication of the DNA molecule as defined by amino acid starvation, but the location of the origin on the genetic map and the direction of synthesis appear to be strain dependent, The position of the integrated F factor does not necessarily coincide with that of the origin-terminus.

2. Materials and Methods (a) Bacterial &rake, E. coli K12 Gene symbols correspond to the suggestions of Demerec, Adelberg, Clark & Hartman (1966). The nomenclature used for the arg genes follows that of Maas, Mass, Wiame 85 GlansdorS (1964). (i) Transductional donor8 DC68 = CR34 thy- (from S. Brenner) derivative, = C600 F-, thr-, Zeu-, tonA-, thi-,kZC-, thy-,8tT-.

DG75 = W1485 F- (from W. Messer) derivative, F-, thy-, Zeu-, str+. P7201 Hfr, thr-, Zeu-, met-, thy-, str+ (from S. Brenner). DG163 = Hfr B4 (from P. Broda) derivative, Hfr, met-, (A)-, str+, argA-, thy-. (ii) Tranaductionul recipients X36, F-, argH-, ara-, kac-, gal-, ura-, try-, hk-, purC-, thi-, str-, mal-, xyl-, mtl-, ton- (from S. Brenner). DGSS, isogenic with strain X36 except it is also (Plkc), hr. DGQO, derived from and isogenic with X36, except arga- instead of argH- and (Plkc). AT2002 (from A. J. Clark) F-, proA-, lac-, tax-, gal-, hia-4, xyl-, mtl-, lya-, argE-, thi-, str+ (Mul). DGlll, derived from and isogenic with AT2002, except also aroB- and str-. These latter two strains have about a threefold higher transduction efficiency than DGQO when plated with hybrid transducing phage but are more susceptible to killing by light, infective phage. (b) Bacteriophage Pl vir a (S. Brenner), called Pl in this text, is the phage used for these experiments. It does not form plaques on strain DGQO which is lysogenic for Plkc and is probably a clear or only mildly virulent mutant. It allows some bacterial DNA synthesis to continue after infection (unpublished results) in contrast to the behavior of the PI strain described by Ikeda & Tomizawa (1965). Pl*X36 is Pl grown on strain X36, etc. (0) Media A 500 pg/ml. stock solution of Sbromouracil was prepared weekly. M9 medium contains 0.7% Na,HPO*, 0.3% KHzPO,, 0.1% NH&l, 0.025% MgSO,, 7Ha0, 0.5% NaCl, 3 x low6 M-Fe&, 0.001 y. CaCls, 0.2% glucose. This was supplemented with 100 pg/ml. of DL-amino acids, 10 pg/ml. thymine and 2 pg/ml. thiamine as needed for growth of a particular strain. The presence or absence of thymine or a given amino acid (leucine for P7201, DC68 and DG76; arginine for DG163) is indicated by the following

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shorthand: (+ aa + thy) means the amino acid and thymine are both present, ( -aa + thy) means the amino acid is absent, etc. Thymiue was added with the BU ((10 pg/ml. BU + 2 pg/ml. thy) = (BU + thy)) unless otherwise noted. Supplemented agar pZates contain M9 medium lacking CaCl,, 1.5% agar, 50 pg/ml. shikimic acid and the DL-amino acids histidine, methionine, threonine and leucine; 100 pg/ml. DL-tryptophan; 25 pg/ml. of the L-amino acids arginine, lysine and proline; 15 pg/ml. thymidine, 10 pg/ml. of uracil and hypoxanthine and 1 pg/ml. of thiamine. The appropriate growth factor was left out of the plates to detect cells able to grow independently of the factor. The sugars xylose, galact’ose, or arabinose (at 0.2%) were used instead of glucose when detecting cells able to utilize these sugars. LCTG broth is LC broth (Luria, Adams & Ting, 1960) with 25 pg/ml. thymidine and glucose (0.1% fmal concentration) added; CaCI, is omitted. LCTG agar plates for assay of infective particles is LCTG broth (10 pg/ml. thymidine) + 2x 10M3 M-CaCl, + 1% agar. Adsorption medium is water containing 0.01 M-MgSO,; CaCl, was added so that the &al concentration of this chemical at the time of adsorption of the phage to the cells was 0.005 M. Suspension medium is M9 synthetic medium 0.01 M in MgSO, (-CaCl, -glucose) with 0.002% gelatin. (d) Experimental (i) Growth and @ration

procedures

of cella

Bacteria from an overnight culture were inoculated into M9 supplemented medium (+aa +thy) and allowed to grow 3 to 5 generations to 2 x lOs/ml. Culture media were changed by harvesting the bacteria on a 142 mm sterile Millipore filter (0.65 CL),washing 3 times with pre-warmed supplemented M9 (-aa -thy), and resuspendiig the bacteria in a given fresh pre-warmed medium. About 2 min were required to change the medium. Half of this new suspension was generally used for the ends experiment and the other half for the beginnings experiment. All operations were carried out at 37°C. (ii) Labeling ends of chromosomecr with BU The general procedure of Maal0e & Hanawalt (1961) was followed. The cells from 100 ml. of the 2 x lO*/ml. culture were transferred to supplemented M9 ( -aa +BU +thy) and incubated with shaking for 90 to 150 min (depending upon the experiment). Strain DGl63 was starved for arginine; the other three strains were starved for leucine. The length of the starvation period did not affect the results. After this starvation, 10 ml. of the culture were used to determine the amount of DNAreplication by density-gradient analysis in CsCl using the Beckman model E ultracentrifuge (Meselson & Stahl, 1958), and the cells in the remaining 90 ml. were given 25 pg/ml. thymidine, harvested by centrifugation, and resuspended in 20 ml. of adsorption medium. (iii) Labeling beginnings of chromosomes with BU 100 ml. of culture were treated as above for labeling ends except that completion of replication was done in supplemented M9 medium containing thymine rather than BU ( -aa + thy). The cells were then transferred to supplemented M9 ( + aa +BU +thy) for 45 to 50 min in early experiments, and for one-half generation as determined by cell count in later experiments. (Longer starvation times resulted in longer lag periods.) A sample was then removed for DNA analysis and thymidine was added to the culture as described above to help prevent further utilization of BU. After harvesting by centrifugation, the cells were resuspended in 20 ml. of adsorption medium. (iv) Preparation of Pl from BU-labeled cells After resuspending BU-labeled cells in adsorption medium, Pl*X36 was added at a multiplicity of infection of 4. After a 20-min adsorption period at 37% the infected cells were centrifuged and resuspended in 100 ml. LCTG broth. Infective centers and subsequent phage yield were assayed by plating on strain X36. The culture was shaken at 37’C for 120 to 180 min and then centrifuged to get rid of debris. (Because of longer

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latent periods, a 180-min incubation period was used in ends cultures to attain high yields; transduction patterns did not change using phage harvested after differing incubation times.) Only 1 cycle of phage growth occurs in this period, giving bursts of approximately 100 phageiinfective center. The phage were concentrated by centrifuging the lysate at, 15,000 rev./min for at least 90 min; the pellets were allowed to resuspend overnight at 4°C without shaking in a total volume of approximately 1.7 ml. of suspension medium. (v) Premature initiation experiments Thymine starvation (see Pritchard & Lark, 1964) as well as BU exposure was used t,o induce premature initiation of chromosome1 replication. An exponential culture at 2 x 108/ ml. was transferred to supplemented M9 medium (-/-aa -thy) for 30 min; 10 pg/ml. BU (no supplemental thymine) was then added for 30 min; 25 pg/ml. thymidine was added to minimize further utilization of BU, the cells centrifuged and treated as above for phage preparation. (vi) “Exponential

phage”

lysates

Exponential phage stocks are Pl lysates which have been prepared by infecting cells which had previously been growing exponentially in supplemented M9 medium. The cells were incubated in LCTG broth after infection and treated as above for phage preparation. The unconcentrated lysates were stored over CHCI,. centrz’,ugation (vii) CsCl density-gradient For CsCl density-gradient centrifugation, each sample containing 1.5 ml. of phage suspension was mixed with 1.5 ml. of a saturated CsCl solution (made up in O-01 m-Tris, pH 8.5) to obtain a refractive index of 7 25/~ = 1.3783 f 0.0005. Each preparation, containing between 6 x lOlo and 1 x 10la infective Pl particles, was placed in a lusteroid tube, overlayered with mineral oil and centrifuged at 25,000 rev./min in the SW39 swinging bucket rotor of a Spinco model L2 at 20°C for at least 20 hr. After deceleration, each tube was punctured and l-drop fractions were collected into 1 ml. of Tryptone broth (containing 0.01 M-Mg2+ and saturated with CRC&). The drops were assayed for infective and transducing Pl phage and samples from various fractions of the hybrid region were pooled for transduction experiments.

(viii) Transductions Recipient bacteria were grown to 5 x 108/ml. in LC broth, collected by centrifugation, resuspended at 1 x lo9 cells/ml. in adsorption medium (1.5 x 10ea M-Ca2+) and chilled. Two parts of phage suspended in Tryptone broth (+ 0.01 a%-Mg2+) and 1 part of recipient bacteria were mixed to give a multiplicity of infection of 0.2 or less, incubated at 37°C for 20 min and then 0.1 ml. of this mixture was spread onto each plate containing selective medium. The same dilution of a phage sample was plated for all markers; in this way both multiplicity of infection and total number of bacteria plated remained constant for the sample. As controls, bacteria alone and phage alone were spread onto similar plates. Plates were incubated for about 65 to 70 hr at 37°C. When BU-containing phage were being assayed the experiments were performed in subdued light and the plates were incubated in the dark. Furthermore, the plates with various selective media were randomized before plating and incubating to prevent any bias due to time of plating after attsorpt,ion or local temperature effects in the incubator. (ix) Bacterial cell counts Cell counts were determined by use of a Coulter counter modified with a Tennelec preamplifier, a Tennelec linear amplifier, and a Nuclear Data pulse height analyzer. (x) Mating

controls

Liquid matings essentially according to the procedure of Wollman, Jacob & Hayes (1956) were performed on the Hfr strains used in these experiments to confirm the entry point and direction of transfer established for each strain. The fertility of Hfr strains at the time of the experiment was checked by the replica plate method of Clark (1963).

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3. Results Figure 1 schematically

illustrates

the general plan of the experiment.

(a) Chromosome alignment Starvation of our K12 strains for a requiredaminoacidallows completion of replicating chromosomes, but prevents initiation of new replication cycles. During amino acid starvation there was an increase of only 35 to 55% in the total DNA content of cells for the strains tested as determined by radioactive and analytical ultracentrifuge experiments; unstarved cells showed an increase in DNA content of more than 90% in the same time (unpublished data). These results are consistent with expectations for an exponential culture prevented from initiating new replication cycles (Maabe & Hanawalt, 1961; Lark et al., 1963; Sueoka & Yoshikawa, 1965) but detailed proof of this interpretation depends on the transduction results described below. (b) Isolation of bromouracil-labeled phage Separation of heavy and hybrid transducing phage Pl from light transducing and infectious phage is done by CsCl density-gradient equilibrium centrifugation. Ikeda & Tomizawa (1965) have shown that Pl transducing particles carry bacterial DNA almost exclusively and that those carrying BU-labeled DNA on one or both strands can be separated from light phage by CsCl density-gradient ultracentrifugation. Figure 2 shows typical density distribution profiles of argH + and purC+ transducing phage obtained in beginnings and ends experiments (Fig. 2(a) and (b), respectively). Like Ikeda & Tomizawa (1965), we have found that all of the transducing particles from the same phage stock have essentially the same density no matter what markers they contain. We were, therefore, able to pool fractions from the peak or higher density tail of the hybrid phage band for transduction analyses. (c) Transduction frequencies Transduction frequencies of the hybrid phage are analyzed with respect to the genetic map of the E. wli chromosome (Taylor & Thoman, 1964) using the 11 different genetic markers shown in Fig. 3. If T, is the number of colonies produced by cells transduced for marker i, then the transduction frequency is defined to be

where the sum is taken over all of the n markers tested for that recipient. Transduction frequencies defined in this way can be calculated separately for the hybrid and light fractions. In experiments involving heavy label, only the transduction frequencies for the hybrid fractious were useful. If host DNA synthesis were prevented by Pl infection, the light transducing phage should give results reciprocal to those of the hybrid phage. Unfortunately, the Pl strain that we used allows some bacterial DNA synthesis to continue after infection preventing us from obtaining useful information from the light transducing phage peak or from using data from this peak for normalization purposes. Results obtained with the hybrid phage are unaffected by this continued synthesis of bacterial DNA.

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Drop number Cc) (b) (a) FIQ. 2. Density-gradient pro&s of Pi transducing phage. The procedures used for the preparation, centrifugation and assay of the Pl phage are described in section (d) of Materials and Methods. Infective phage were assayed on strain X36, transducing phage on strain DC88 ----, plaques. -O-O-, argH+ transductants; -O-e--, PVC! + transductants; The horizontal bars indicate where samples were pooled for transduction analyses. (a) Beginnings experiment, strain DG76. 82 drops in the sample. (b) Ends experiment, strain DG75. 81 drops in the sample. (c) Premature initiation experiment, strain DG68. 97 drops in the sample. The arrows show the peakpositions of the light (T-T), hybrid (BU-T), and heavy (BU-BU) transducing particles. The density separation is greater in (c) than in (a) and (b), since in (c) no thymine was added along with the BU during the labeling period.

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FIQ. 3. The genetic-map of E. cc&. The 11 markers used in the transduction experiments and the mating origins of Hfr strains P7201 and DG163 are shown. The map is baaed on the data ofTaylor &Thoman (1964), Signer, Beckwith & Brenner (1965) (for the location of ~TI”), and Pittard & Wallace (1906) (for the location of afoB). Each inner arrow indicates the range of possible locations of the origin of replication and the direction of synthesis of the ohromosome in the strains examined. The length of an arrow reflects the uncertainty of the exact location of origin and terminus and ia not meant to imply that the origin is not a unique site. c - - - +, meana direction is not known; -, strains DC68. DG76 and P7201; -----, strain DG163.

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In Fig. 4 the transduction frequencies for the markers are plotted versus their position on the circular map opened at argti and plotted with the clockwise direction going to the right on the horizontal axis; arga is plotted at both ends to indicate the circularity of the chromosome. Two different recipient strains, one carrying six markers and one carrying seven (xyl- and his- are carried by both) are used for scoring in order to get good coverage of the genetic map and to avoid bias that might result from the biological traits of a single recipient. In preliminary experiments two additional, related recipients were used and gave consistent results. Because the recipient strains are not identical and contain different numbers of markers, their transduction frequencies cannot be compared directly. To facilitate comparison of

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FIG. 4. Transduction frequencies of beginnings and ends experiments. A description of the manner in which the data are compiled and plotted is given in section (c) of Results. Errors shown are standard deviations. The general procedures employed to obtain the transduction frequencies are described in section (d) of Materials and Methods. The specific incubation conditions employed for each strain for ohromosomal alignment and BU labeling of the DNA were as follows. (a) Strain DG75. Beginnings experiment: (-leu +thy) 90 min; (+leu +BU -+-thy) 45 min. Ends experiment: (-leu +BU +thy) 90 min. Total number of colonies counted: (0) 402, (A) 1366, (0) 7206, (A) 8065. (b) Strain DG68. Beginnings experiment: (-leu fthy) 150 min; (fleu +BU +thy) 110 min total incubation (50 min lag + 60 min = l/2 generation). Ends experiment: (-leu +BU j-thy) 160 min. Total number of colonies counted: (0) 5447, (a) 19,811, (0) 1992, (A) 2745. (c) Strain P7201. Beginnings experiment: (-leu $-thy) 90min; (+leu +BU +thy) 45 min. Ends experiment: (-leu + 2 rg thy/ml.) 30 min; at 30 min 10 pg BU/ml. was added for 90 min. The total amino acid starvation time was 120 min. Total number of colonies counted: (0) 1820, (0) 2731, (A) 1141, (0) 5167, (A) 10,343. (0) recipient AT2002. Beginnings experimeut,s 0, A, 0. Ends experiments l , A.

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slopes and peak shapes on the experimental curves, we have adjusted the ordinate scales to superpose the plots by making the values of one of the common markers, his or xyl, coincide. Each curve represents a compilation of the results of several transduction assays of both the peak and tail regions of the same hybrid phage band, since the frequencies from the tail and peak regions were very similar and the transduction results were consistently repeatable from day to day within statistical error. The theoretical curves shown in Fig, 5 are based on the analysis of the age distribution of members of an exponentially growing population (Sueoka & Yoshikawa, 1965; Powell, 1956). To simplify the calculations it was assumed that each cell division cycle time is exactly the mean division time for the whole population with no variation from cell to cell or cycle to cycle. Other detailed assumptions are given in the legend of Fig. 5. These curves should predict the general trend of the experimental results and the positions of maxima and minima. A more realistic calculation would round off the sharp peaks predicted by this simple model and give closer agreement with the data. Comparison of the experimental curves in Fig. 4 with the theoretical unnormalized curves indicates a terminus near lys (54 minutes), an origin somewhere in the region

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FIG. 5. Theoretical curves. Number of BUJ8beled genes per origin81 chromosome, proportional to tmnsduction ----, frequencies if transduction efficiency were the s&me for 8ll markers. (The exponential plot shows the number of genes per chromosome without BU labeling.) -, Normalized tmnsduction frequencies. Assumptions for the computations: (1) each chromosome has one growing point moving with conetent speed end with no time gap between replication cycles; (2) the chromosome popul8tion hse an exponential 8ge distribution before the experiment begins (cf. Powell, 1956); (3) emino acid sterv8tion 8bsolutely blocks initietion of new DNA replic8tion cycles; all chromocomes simultaneously resume normel synthesis in a new cyale when amino acids 8re restored, and (4) the origin and terminus of the cycle coincide. If the exponentiel (or vegetative) origin and starvation terminus were assumed to be et different plecea, the ends curves would have 8 sm811 kink or cusp. The present d8t8 are not 8CCUrah3 enough to detect such cusps. For illustration we have chosen the c&88 in which (8) the beginning8 curves correspond to lebeling 30% of the chromocome with BU; (b) the origin is taken to be at argQ with 8 clockwise direction of synthesis. For comparison with experimental ourves, the origin ten be ehifted end the direction reversed to obtein the best assignment of origin end direotion.

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of lys and xyl(54 minutes to 70 minutes), and a clockwise direction of synthesis based on the rather sharper drop-off on the right side of the ends peak than on the left side. The reciprocity of the beginnings and ends results and the general agreement of the curves in Fig. 4 with the theoretical curves shown in Fig. 5 were surprising, since the raw transduction frequencies of individual markers were expected to be unsystematically variable due to unknown factors affecting transductions. (One such unknown factor may be the reason for the high ara values seen in the ends experiments, since normalization eliminates this effect (see Fig. 7).) A number of special molecular effects could introduce variations in transduction efficiencies that could obscure or alter general trends in these plots of the unnormalized data. Among these effects are host modification and restriction, local variation in recombination probabilities, special configurations of the chromosome in the cell, and possible attachment of particular chromosomal sites to the cell membrane (Hartman, 1963; Inselberg, 1966). Ideally these variations could be eliminated by normalizing the experimental data with data obtained from transducing phage derived from a population of cells with completed chromosomes containing equal numbers of all genes. But effects due to BU would be corrected only if these chromosomes contain hybrid DNA. Because of technical difficulties in preparing such “equigenic” chromosomes with or without BU, we used normal exponential unlabeled cells as donors for the normalizing sample. These samples do not normalize away local effects due to BU. Although we are calling the normalizing samples exponential phage stocks we cannot be certain that the A

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FIG. 6. Transduction frequencies of an exponential ph8ge lysate Pl.DG75. The procedures used for prepsretion 8nd 8eeey of the phege are described in section (d) of Materi8ki and Methods. Errors shown are standard devietions. Total number of colonies counted:

(0) 9546,(A)

5036.

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transducing phage carry bacterial DNA representative of an exponential population because of the unknown effects of the continuation of bacterial DNA synthesis after PI infection. Figure 6 is a plot of transduction frequencies obtained for an exponential phage stock of Pl*DG75. (cl) Normalized transduction frequencies The normalized transduction frequency curves shown in Fig. 7 are obtained by dividing the data shown in Fig. 4 by transduction frequency data obtained from exponential phage stocks prepared from the same strains. For example, the data seen in Fig. 4(a) were divided by the data seen in Fig. 6 to obtain the curves shown in Fig. 7(a). A value of 1 on the ordinate indicates that the transduction frequency of an experimental marker is the same as that of the normalizing exponential marker. The results should be compared with the theoretical curves drawn with solid lines in Fig. 5. The origin should lie to the left of the beginnings peak for strains which replicate clockwise, and to the right of the beginnings peak for strains which replicate counterclockwise. Only the asymmetry of the experimental ends plots, when pronounced, seems able to indicate the direction of replication as clockwise for the three

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FIQ. 7. Normalized transduction frequencies. Errors shown are standard deviations. 0, l assayed on recipient DG90; A, A on DGlll. Beginnings 0, A. Ends 0, A. (a) Strain DG75. Beginnings and ends transduction frequencies (seen in Fig. 4(e) ) were divided by transduction frequencies obtained from an exponential phage lysate Pl.DG75 (seen in Fig. 6). (b) Strain DG68 beginnings and ends transduction frequencies normalized by transduction frequencies obtained from an exponential phage lysste Pl-DG68. (c) Strain P7201 beginnings and ends transduction frequencies normalized by transduction frequencies obtained from an exponential phage lysate Pl.P7201.

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FIG. 7(b) and (0).

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strains shown, but the present evidence is somewhat ambiguous. The terminus lies near the side of steepest slope of the ends peak. The normalized results again indicate a terminus and origin in the region of the chromosome between lys and xyl (54 minutes to 70 minutes). It is reassuring that the normalization procedure does not change the position of the origin or terminus, or the apparent direction of synthesis for these three strains. (One exception is the ambiguity in the interpretation of the normalized DG68 ends experiment, Fig. 7(b).) This relative insensitivity to normalization is a measure of the effectiveness of the BU labeling, density-gradient techniques which allow the isolation in high concentrations of phage carrying specific areas of the chromosome. Normalized transduction frequencies obtained with a second stable Hfr (DG163) are shown in Fig. 8. From this plot we tentatively conclude that there are two possible origins in this strain, one near argE (77 minutes) and one near gal (16 minutes), and that the terminus is near gal (16 minutes) with a counterclockwise approach to the terminus. One origin is fairly near that found for the three strains already described, and the other is near the F-factor of Hfr DG163. We do not mean to imply that both origins necessarily exist or operate simultaneously in a single cell.

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FIG. 8. Normalized transduction frequencies, strain DG163. DC163 beginnings and ends transduction frequencies normalized by transduction frequencies obtained from an exponential phage lysate Pl.DGlB%. 0, l Aasayed on reoipient DGSO; A, A on DGlll. Errors shown are standard deviations. Beginnings 0, A. Ends 0, A. The general procedures used to obtain the transduction frequencies are described in section (d) of Materi& and Methods. The specific incubation conditions employed for strain DC163 for chromosomal alignment and BU-labeling of the DNA were: beginnings experiment: (--erg +thy) 120 min; (+arg +10 pg BU/ml.) 110 min total incubation (70 min lag + 40 min = l/2 generation). Ends experiment: (-arg +BU +thy) 120 min. Strain

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Interpretation of the experimental data depends on using the theoretical curves as a description of the consequences of a simple model of bacterial chromosome replication. We cannot expect the data to fit the theoretical curves extremely closely because the model does not include factors such as the preferential utilization of thymine over BU by the cells, the possible leakiness of the amino acid starvation block of DNA synthesis, non-uniform uptake of BU under different physiological conditions, lags and population heterogeneities in replication rate and responses to experimental treatments, premature initiation of new rounds of DNA synthesis caused by BU (see below), etc. From a radioactive uptake experiment with exponentially growing cells we found t(hat when cells are placed into a medium containing thymine and BU, the rate of incorporation of thymine into DNA is linear whereas there is a lag in the incorporation of the BU (unpublished results). This early preferential uptake of thymine would tend to lower the transduction frequencies of markers close to the origin in beginnings experiments and would make localization of the origin more difficult. On the other hand, preferential thymine uptake in ends experiments would only enhance the difference seen between early markers and late ones. Most of the other effects mentioned above have not been examined experimentally, but they, like those mentioned for the preferential utilization of thymine, would tend only to reduce the sharpness or change the heights of the predicted peaks. Premature cycle initiation could, on the other hand, introduce new peaks.

(e) Prewmture initiation

of replication cycles

Thymine starvation and/or the presence of BU causes premature initiation of DNA replication in E. coli K12 strains. In early experiments we found that when an exponential culture of DG68 was grown in the presence of BU without supplemental thymine, the resultant transducing phage profile contained three peaks-light, hybrid, and heavy. (This heavy peak could be eliminated by using a mixture of BU and thymine, and it was for this reason that most of the beginnings and ends experiments were performed with small amounts of thymine present during the BU labeling period.) Transduction analyses of the heavy peak revealed a pattern similar to the DG68 beginnings experiment. The result was interpreted to mean that BU caused reinit’iation of the chromosome at the origin, resulting in early formation of heavy DNA. Further, “premature initiation experiments” were done by simply adding BU to an exponential culture pretreated with 30 minutes of thymine ‘starvation (based on the thymine starvation results of Pritchard & Lark, 1964). A typical resultant transducing phage profile after CsCl density-gradient ultracentrifugation is shown in Fig. 2(c). The results in Fig. 9 show patterns very similar to the beginnings patterns of the respective strains, indicating that thymine starvation or BU alone initiates new rounds of replication at the same place as beginnings experiments. Premature initiation of DNA synthesis is prevented by amino acid starvation and therefore does not affect ends experiments. Although the beginnings results are consistent with the ends results and with the predictions of the model, we cannot rule out. the possibility that they are largely an artifact caused by BU and unrelated to the preliminary amino acid starvation treatment. However, recent experiments by Abe & Tomizawa (1967) also show that BU causes premature initiation of replication at thr origin of E. coli K12 chromosomes. 40

ti26

B. WOLF,

A. NEWMAN

AND

D. A.

GLASER

(a)

-3

-2

5

Premature htiation Expomnt~al

3

3 -1 1 I SL 0

I o(b)

-2 Premature

initiation

Exponential

-1 +

\ ,o

Genetic

map

FIG. 9. Normalized transduction frequencies of premature initiation experiments. The procedures employed EPB described in section (d) of Materials and Methods. Shown are the transduction frequencies of hybrid phage prepared from premature initiation experiments normalized by transduction frequencies obtained from exponential phage lysates of the same strain. Similar patterns were obtained from heavy phage. Errors shown are standard deviations. The open symbols indicate assays on recipient DG90; the closed symbols on DGI 11. (a) Strains DC68 0, 0. P7201 0, n . DC75 A, A. (b) Strain DG163.

REPLICATION

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1. coli

CHROMOSOME

627

4. Discussion Nagata (1963) has proposed that the origin of replication in vegetative cells of E. eoli is associated with the F-factor and that replication proceeds from the F-factor to the conjugational origin and that F- strains have no specific site where DNA replication consistently starts. Jacob et al. (1963) propose that the F-factor plays no role during vegetative growth and that there is a specific site on the chromosome for all strains. including F- , from which replication begins. Our results show that: (a) two K12 F- strains (Da68 and DG75) and one Hfr strain (P7201) each have a unique origin between lys and xyl (54 to 70 minutes on the Taylor & Thoman (1964) map); (b) the origin and terminus of each strain lie in the same region of the map and could be identical; (c) the clockwise direction of synthesis is favored but not yet conclusively demonstrated; (d) for P7201 the terminus is near but distinguishable from the location of the F factor; (e) Hfr DG163 appears to have an origin and terminus between pro and gal (6 to 16 minutes) in the same region as the F-factor; the direction of synthesis is counterclockwise so that genes are replicated vegetatively in the same order as they are transferred during conjugation, and (f) Hfr DG163 appears to have a second origin which may be activated by BU or by thymine starvation. It lies near xyl(64 to 88 minutes) and could be the same as that found for the other three strains. From these results we conclude that all four strains, including two F- strains, have definite origins and coinciding termini for DNA replication as detlned by an amino acid starvation block. These sites are also the places at which BU or thymine starvation can prematurely initiate new rounds of replication. Each strain seems to possess a definite direction of synthesis, although the evidence for this is less compelling. The conclusion that synthesis proceeds in a clockwise direction for three of the strains tested is based on the relative steepness of the two sides of the peak in an ends experiment. This conclusion is strengthened by the fact that agreement between the location of the origin and terminus is obtained only for the clockwise direction. Similar experiments to those presented here have been performed by Abe & Tomizawa (1967). The results with their strains are in general agreement with the results we get for strains DG68, DG75, and P7201. The origin for their strains is thought to be between his and lys (38 to 54 minutes) and for ours between lys and xyl (54 to 70 minutes). The two regions overlap, but due to the limited precision of the present experiments, it is not yet known whether all of these strains have origins mapping in precisely the same place. Although the origin is near the F-factor in strain P7201, it is distinguishable from it. Nagata’s model would predict xyl near the origin, counterclockwise replication, and argE near the terminus. The latter two conditions are not fuhllled. On the other hand, strain DG163 Hfr does appear to have the site of its terminus near its F factor, but synthesis is in a direction opposite to that predicted by the Nagata model. It is not yet known whether the proximity of the origin to the F-factor is fortuitous in this strain, or whether the F-factor plays a role in determining the origin of replication of the chromosome under the experimental conditions we employed. Strain DG163

628

B. WOLF,

A. NEWMAN

AND

D. A. GLASER

may have an additional origin near that for the other three strains. It is not known whether both origins exist or operate simultaneously in a single cell. Since the ends results for this strain indicate a single terminus, this second origin, found in a beginnings and perhaps in a premature initiation experiment, may well have been induced mainly by BU or by thymine starvation, as no supplemental thymine was added to the BU in either of these experiments. The origins of all of the strains tested seem inducible in this way, and their functioning in the absence of these stimulations is established clearly only by the ends experiments in which initiation of new replication cycles is inhibited by amino acid starvation. Although the ends experiments may be unaffected by BU-induced artifacts, they may be identifying termini imposed by amino acid starvation only, and not origins for undisturbed vegetative chromosomal replication. To answer these questions, experiments must be done which avoid the use of BU and amino acid starvation. The relationship between our experimental findings and the vegetative normal physiological mode of DNA replication is left open for future study. It seems clear, however, that strain differences do exist, and it is probable that more than one site on the chromosome can be involved in the initiation of DNA replication. The authors wish to thank: Miss Miriam Boehlke and Mrs Ruth Ford for their fine technical assistance; Mr Don &gal for computer programming; A. J. Clark and M. Chamberlin for critical reading of the manuscript in advance of publication; S. Brenner, P. Broda, A. J. Clark, N. Fiil, N. Franklin, W. Messer, J. Pittard and A. L. Taylor for providing phage and bacterial strains; and S. Brenner who, more than two years ago, originally suggested the “three- or four-month” experiment which we have been pursuing diligently ever since. This investigation haa been supported in part by the U.S. Public Health Service through research grants GM12624 and GM13244 from the National Institute of General Medical Sciences.

REFERENCES Abe, M. & Tomizawa, J. (1967). Proc. Nat. Acad. Sci., Wash., 58, 1911. Berg, C. M. & Caro, L. G. (1967). J. Mol. Bill. 29, 419. Bonhoeffer, F. & Gierer, A. (1963). J. Mol. Biol. 7, 534. Cairns, J. (1963). J. Mol. BioZ. 6, 208. Clark, A. J. (1963). Genetics, 48, 105. Demerec, M., Adelberg, E. A., Clark, A. J. & Hartman, P. E. (1966). Genetics, 54, 61. Hartman, P. E. (1963). Ii Methodology irz Basic Genetics, ed. by W. J. Burdette, p. 103. San Francisco: Holden Day Inc. Ikeda, H. & Tomizawa, J. (1965). J. Mol. BioZ. 14, 85. Inselburg, J. (1966). Virology, 39, 257. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spr. Harb. Symp. Quant. BioZ. 28, 329. Lark, K. G., Repko, T. & Hoffman, E. J. (1963). Biochim. biophys. Acta, 76, 9. Luria, S. E., Adams, J. N. & Ting, R. C. (1960). Virology, 12, 348. Maaloe, 0. & Hanawalt, P. C. (1961). 3. Mol. BioZ. 3, 144. Maas, W. K., Maas, R., Wiame, J. M. & Glansdorff, N. (1964). J. Mol. BioZ. 8, 359. Meselson, M. & Stahl, F. W. (1968). Proc. Nat. Acud. Sci., Wash. 44, 671. Nag&a, T. (1963). Proc. Nat. Acad. Sci., Wmh. 49, 651. Pittard, G. & Wallace, B. J. (1966). J. Bact. 91, 1494. Powell, E. 0. (1956). J. Gen. Microbial. 15, 492. Pritchard, R. H. & Lark, K. G. (1964). J. Mol. BioZ. 9, 288. Signer, E. R., Beckwith, J. R. & Brenner, S. (1965). J. Mol. Biol. 14, 153.

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H. (1965). Genetics, 52, 747. M. S. (1964). Genetics, 50, 659. W. (1964). Ber. Bunaengeselkwhaft

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