Proceedings of the National Academy of Sciences Vol. 65, No. 3 pp. 601-608, March 1970

Origin and Direction of Replication of the of E. coli B/r Millicent Masters

MEDICAL RESEARCH COUNCIL, MICROBIAL RESEARCH UNIT, DEPARTMENT OF MOLECULAR , EDINBURGH UNIVERSITY, EDINBURGH, SCOTLAND Communicated by Arthur B. Pardee, July 3, 1969 Abstract. The origin and direction of replication of the E. coli B/r chromo- some has been determined by comparing frequencies in Pl-transducing lysates prepared on cultures growing at different rates. The gene frequencies found are consistent with the idea that replication of the chromosome is di- chotomous in rapidly growing B/r. The origin was found to be between 40 and 55 min on the E. coli genetic map with replication proceeding in a clockwise direction. Markers near the origin behaved anomalously.

The chromosome of E. coli is a single circular molecule of DNA which replicates sequentially along its length.'-3 Since the demonstration by Yoshikawa and Sueoka4-5 that the B. subtilis chromosome replicates sequentially from a genet- ically fixed origin, many attempts have been made to determine whether this is also true for the of E. coli. Although some early work6-7 indicated that the presence and position of such an origin might be dependent on the integration of the F factor, later work has supported the idea that the E. coli chromosome is always replicated sequentially in a particular direction from a fixed origin whose position may be strain dependent, but probably does not de- pend on the integration of the F factor.8-'5 The majority of strains which have been studied are estimated to have an origin lying between 40 and 70 min on the genetic transfer map of E. coli and to replicate their DNA in a clockwise direction. These findings were based on experiments which involved the measurement of numbers of replicated at particular times in the bacterial . They were measured either directly through incorporation into transducing phage, or indirectly by monitoring events in the which might be expected to reflect changes in . None of these methods, however, allows precise determination of the . In the experiments where numbers of genes were measured di- rectly,'0 12, 13 amino-acid starvation was used to allow chromosome replication to terminate in the absence ofr einitiation. Bromouracil was used either to label the DNA synthesized during termination or was added subsequently, in the pres- ence of amino acids, to label origins synthesized during reinitiation. Unfor- tunately, all do not necessarily terminate during this treatment.'2' 13 They may t hen terminate after the amino acids are returned to the medium, and 601 Downloaded by guest on September 24, 2021 602 GENETICS: M. MASTERS PROC. N. A. S.

bromouracil added to label origins will label some ends as well. Also, bromo- has been shown to cause the initiation of extra rounds of DNA replication at the time of its addition. 10 The experiments which measured gene dosage effects base their conclusions on the validity of the assumption that the inducibility of an doubles im- mediately after replication of the corresponding gene.8' 9, 14, 15 They depend, furthermore, on a correlation between the times at which changes of rate occur in the synthesis of and the time of initiation of new rounds of DNA syn- thesis. The position of the replicative origin cannot be obtained more accurately than this correlation can be made. We therefore thought it would be worthwhile to attempt to determine the origin and direction of replication of the chromosome of E. coli B/r by a method which allows one to use undisturbed cells in balanced growth and should in theory permit precise location of the origin. This method is based on the finding of Yoshikawa et al."6 that rapidly grow- ing cultures of B. subtilis have more than one DNA replication point per chromosome. Thus, such fast-growing populations are relatively richer in markers replicated early than are slowly growing cultures where each chromosome has no more than one replication point. This was demonstrated by extracting transforming DNA from cultures growing at different rates and showing that the transforming activity for origin markers compared with that for terminus markers was relatively higher in rapidly growing cultures than in slowly growing ones. Assuming that dichotomous replication also occurs in the chromosomes of rapidly growing E. coli, we reasoned that if generalized transducing phage were prepared on E. coli growing at different rates, we should be able to demonstrate the same variation in transducing frequencies that Yoshikawa et al. found in the transforming DNA of B. subtilis. We would expect markers located near the origin of replication to be relatively more frequent in lysates prepared on rapidly growing cultures than in lysates prepared on slowly growing ones. Therefore, if a gradient in relative frequencies was obtained for a number of markers in the two lysates, and it correlated with the map positions of the mark- ers studied, we could (1) determine precisely the origin and direction of replica- tion of the chromosome, and (2) confirm by genetic means our assumption that dichotomous replication occurs in rapidly growing E. coli cells. This has already been proposed by Cooper and Helmstetter17 to explain the pattern of DNA syn- thesis during the cell cycle of E. coli B/r. In fact, a gradient in marker frequencies was obtained consistent with the existence of dichotomous replication in rapidly growing cultures. In addition we localized the origin of replication of E. coli B/r to between 40 and 55 min on the genetic map. Attempts to locate it more precisely gave unexpected results which will be discussed in this paper. Materials and Methods. Strains and mapping: All strains used in the experiments were derived from B/r/1 which was obtained from Dr. S. Glover. The thy was of spontaneous origin and was selected with the aid of .18 The other markers were selected one at a time after UV irradiation and penicillin selection,'9 in the order ara-, xyl-, cys B-, his-. This multiple was used as a parent for the separate selection of tyr A-, gua -, and nic B - strains. Downloaded by guest on September 24, 2021 VOL. 65, 1970 GENETICS: M. MASTERS 603

Phage Plbt was obtained from Dr. E. Engelsberg. Mapping was done by interrupted mating using either AC 2524, an Hfr derived from B/r by Dr. H. Boyer21 (from whom it was obtained) or B/r/1 infected with F-15 (F-15 was obtained from Dr. N. Willetts) as male parent. The matings were done by diluting overnight cultures of the parent 1 -- 10 into cultures containing males (ODuo = 0.5) growing exponentially on Oxoid broth. Samples taken periodically were diluted 1 .-- 3 (F- X F-15 crosses) or 1 -- 20 (F- X Hfr crosses), agitated for 30 sec using a vortex mixer and plated on selective media. AC 2524 transfers in the order cys B, his, thy A. F-15 mobilizes the chromosome for transfer in the opposite direction beginning at thy A. Preparation of transducing lysates: Broth lysates were prepared from B/r/1 growing exponentially on L-broth (tryptone 10 gm/liter, extract 5 gm/liter, NaCl 10 gm/ liter, pH adjusted to 7.2). The generation time of B/r/1 in broth is 21 min as measured by GD increase at 540 mu. Cells were allowed at least three generations of exponential growth before addition of phage to ensure that chromosome configurations were in the state normal for the growth rate. Cells (ODw0 = 0.3) were then infected with phage P1 added at a multiplicity of 1-2. The infecting P1 had previously been grown on a multiply mutant parent to ensure that all of the markers subsequently scored originated during the cycle of growth on broth. After 15-min adsorption in the presence of 0.0025 M Ca++, cells were washed free of Ca++ and free phage by filtration on a membrane filter and re- suspended in broth. Cultures were aerated until lysis began (after approximately 1 ) and then CHC13 was added to complete lysis and to sterilize the preparations. Debris was removed by 10-min centrifugation at low speed. Minimal lysates were prepared from B/r/1 which was grown on a Tris-buffered low- medium in order to prevent precipitation of the Ca++ required for phage adsorption. This medium contained: 0.25 gm/liter MgSO4.7H20, 0.6 gm/liter NaCl, 1 gm/liter NH4Cl, 0.1% Tris, 10-2 M phosphate, 0.2% glucose, at a pH of 7.4. The generation time on this medium is 43 min, exactly the same as that on a phosphate-buf- fered salts medium and twice that of the broth-grown cells. Otherwise, the procedure for lysate preparation was the same as for broth cultures. Transduction: Recipient bacterial cultures inoculated from single clones were grown to stationary phase in L-broth containing Ca++; 0.5-ml-aliquots of cells were mixed with 2 X 108 and adsorption allowed to take place for 20 min at 370C. The mixture was then diluted into 43 ml of 0.6% agar and 2.5-ml aliquots were layered onto o minimal plates. Transductants were coun- ted after 40-hr incubation at 37°. In order ara to ensure comparability of results, a broth and a minimal lysate were always used in each transduction experiment and the ratios of transductants in the two lysates deter- mined. Results. (1) The location of the re- X l plicative origin between cys B and thy A: The first series of experiments was designed to locate the origin of replica- tion approximately, as well as to test the validity of the assumptions under- thy-A lying the method. B/r thy A - ara- grA his xyl- cys B- was used in these experi- ments as these four markers are fairly FIG. 1.-Map of E. coli showing the posi- well spar ed around the chromosome tions of the markers used in these experi- (Fig. 1). Broth and minimal lysates ments. Taken from Taylor and Trotter." Downloaded by guest on September 24, 2021 604 GENETICS: M. MASTERS PROC. N. A. S.

1.6 1.6 1A1 ; T 1A 1.2B/ 1.


0.8 0.8

0.6 I I I 0.6 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 thy-A xyl era cyst- thy-A tys-B his

FIG. 2.-Marker frequency ratios in P1 lysates prepared on broth- and minimal-grown cul- tures of E. coli B/r. The frequency ratios are plotted as the ordinate and calculated as de- scribed in the text. The abscissa represents the genetic map of E. coli broken at thy A, and the markers scored in each experiment are plotted at their relative map positions. Solid circles represent experimental values and open circles theoretical values. Each experimental point represents the average of 4-6 experiments using several different lysates. Standard deviations from the average are also shown. (A) Experiments in which thy A +, xyl+, ara+ and cys B+ recombinants were scored; (B) experiments in which thy A +, cys B+, and his + recombinants were scored.

phage Plbt prepared on prototrophic B/r were used to transduce the multiple mutant. The relative frequency of transductants for each of the markers A, B, C, . . . (defined as the fraction of the sum of all transductants for all markers represented by the marker under consideration) in the broth lysate (BA, BB, ...) and in the minimal lysate (MA, MB, . . .) was calculated. The relative frequency for each marker in the broth lysate was divided by that in the minimal lysate to give a broth to minimal ratio (BA/MA, BB/MB, .. .). These ratios are a function of the particular series of markers used in a given experiment and have no absolute significance outside of the experiment in which they were derived. A marker with a high Broth/Minimal frequency ratio would be near the origin of replication and one with a low ratio would be near the terminus. It was found (Fig. 2a) that the ratio was highest for and lowest for cysteine, while intermediate values were obtained for the other markers. In other experiments (data not shown) a leu-ara+ strain prepared from the multiple mutant by trans- duction with phage prepared on a leu- donor was used as the recipient. The B/M ratio obtained for leucine was the same as that obtained for arabinose in the experiments above, as would be expected for linked markers. These experiments suggest that the original assumption, namely, that the average number of replica- tion forks per chromosome is greater in broth than in minimal cells, is correct. It also indicated that the origin of replication was between the thy A and cys B loci, and that replication proceeded in a clockwise direction. A theoretical curve was calculated by the same procedure used to calculate the experimental curve. In place of the number of transductants found experi- mentally, their theoretical frequencies were used. The theoretical frequencies were calculated using the equation derived by Sueoka and Yoshikawa2' which Downloaded by guest on September 24, 2021 VOL. 65, 1970 GENETICS: M. MASTERS 605

gives the frequency of a marker at a given position on the chromosome, as a func- tion of the average number of replication points on the chromosome. To make this calculation we assumed that the broth chromosome has two replication points and the minimal chromosome one, as suggested by the results of Helmstetter and Cooper.22 We also assumed that thy A was located at the origin. In fact, the shape of the curve is not dependent on the point chosen as the origin, provided it is between cys B and thy A. As can be seen, the experimental ratios are close to the theoretical ones.


1.4 FIG. 3.-Marker frequency ratios in P1 lysates on broth- and minimal- 1.2 grown cultures (see Fig. 2) showing the behavior of markers near the /M origin. A, lysates scored on thy A- 1.0 cys B- his- nic B- recipients; 0, recipients as above but gua, nic B +; 0, recipients as above but 0.8 tyr Agua+nic B4.I 0.6 I I 0 0.2 OA 0.6 0.8 1 nic-B thy-A cys-B his guu tyr -A

(2) The location of the replicative origin between his and thy A: Now that the origin had been located between cys B and thy A, we tried to localize it further by selecting a mutant for a marker between them. A mutant was selected for his, a which is about half way between cys B and thy A, and the same sort of experiment repeated. Histidine proved to be a late marker (Fig. 2b), and its - tive frequency in broth compared with minimal lysates was close to that pre- dicted on the assumptions used above. (3) Behavior of markers between his and thy A: In order to locate the origin more precisely still, we proceeded to isolate for markers located be- tween his and thy A. The first such isolated was tyr A. Its behavior in the trans- duction analysis was neither that of an origin nor that of a terminus marker but rather that which might be expected from a marker located at about 80 minutes on the coli map (i.e., about one third of the way between origin and terminus (Fig. 3). We therefore considered the possibility that strain B might have a transposi- tion for this marker, although no differences have yet been found between the order of markers on the B and K12 maps.23 Interrupted mating experiments were performed using AC 2524, an Hfr derived from B/r, as donor. This strain donates in a clockwise direction starting with the cys B marker.23 Tyr+ recombinants first appeared after his+ but before thy+ Downloaded by guest on September 24, 2021 606 GENETICS: M. MASTERS PROC. N. A. S.

1200 A. 18 B.


1000 15

THY 800 12 TYR

0 600 9

FG 400 6 z z4 200 3 0

0 20 40 60 80 0 20 40 60


FIG. 4.-Interrupted mating experiments confirming the location of tyr A between thy A and his. The female strain was the tyr A - recipient used in the transduction experiments. (A) B/r/1 X AC 2524; (B) B/r/1 X B/r/1 F-15.

ones (Fig. 4). This is consistent with a map position for tyr+ at 47 min as previ- ously reported. Since it might be argued that the map of our strain of B/r might be different from that of AC 2524, we infected our strain with F-15 which pro- motes oriented transfer in a counterclockwise direction from thymine. The F-15 episome does not carry the tyr A marker. When interrupted mating experi- ments were done using B/r F-15 as donor tyr+ recombinants appeared early, before those for his+ (Fig. 4). Therefore, tyr A is not transposed in our strain but is in the region previously reported. We then proceeded to isolate two other markers between his and thy A in an effort to determine whether this unanticipated behavior was peculiar to the tyr locus or whether all the markers in the same region behaved in this fashion. Mutants for gua and nic B, previously located at 48 and 45.5 min, respectively, were isolated and their positions confirmed to be in this region by interrupted mating experiments. They too were transferred between his and thy A. Their relative frequencies in broth and minimal grown phage lysates were thein determined. The results obtained were similar to those reported above for tyr A. The ratio of the frequencies of the markers in broth lysates to those in minimal lysates was that characteristic of markers located near the middle of the chromo- some rather than at one of its extremes. Furthermore, the ratios obtained for the three markers are not the same but increase in the order nic B < qua < tyr A. Discussion. The results reported here support the hypothesis that there are twice as many replication forks in E. coli B/r growing with a division time of 20 minutes as there are in B/r growing with a division time of 40 minutes. The results, obtained with the five markers spaced over the bulk of the genetic map, indicate Downloaded by guest on September 24, 2021 VOL. 65, 1970 GENETICS: M. MASTERS 607

that the point of origin of chromosome replication is between 40 and 55 minutes on this map. This is in reasonable agreement with the less precise results ob- tained by enzyme-induction studies in synchronously dividing cultures of this strain.9' 14, 15 The origin we have found is particularly close to that obtained by Cerda-Olmeda et al.1' for several other strains of E. coli. They located the origin by measuring the change in frequency of mutation induced by nitrosoguanidine at the replication fork during synchronous growth. The behavior of the markers close to the origin is puzzling. Several possible explanations have occurred to us which have not been tested. These are: (a) Replication does not begin in every cell at a unique site but rather occurs with a certain probability within an "origin region" and proceeds in a unique direction. Since in some cells a given marker would be near the origin and in others near the terminus, in the mixed population it would seem to be somewhere near the mid- dle. The region within which replication can originate represents at least 5 per cent of the length of the chromosome. (b) Markers near the origin of replication in broth-grown cells are less accessi- ble to incorporation into transducing phage than similar markers in minimal- grown cells. This might be the result of a particularly complex configuration at the origin of a dichotomously replicating chromosome. (c) The markers in the anomalous region are replicated twice, both at the beginning and at the end of replication. Closure of the new chromosome into a ring might then proceed by a recombination process which results in the loss of the doubly replicated piece. If the extra copies were to remain available for in- corporation into phage during most of the cell cycle, the result could be an in- creased B/M ratio for markers in the doubly replicated region. In experiments where beginnings and ends of the chromosomes were labeled, both (a) and (c) would predict that markers near the origin would appear as members of both classes. Wolf et al.'2 find that markers near the origin of K12 strains do appear in both classes. However, this might also be attributed to in- complete termination during amino acid starvation in their experiment. Hypothesis (a) predicts that a gradient in B/M would be obtained for markers near the origin, which would vary between the values expected for origin and terminus markers. The value obtained for a given marker would depend on the probability of replication initiating to the left or right of it. In fact, such a gradient is obtained: Bit/TIM > Bgua Bnicn(See Fig. 3.) Ittir /MguaMncM> If hypothesis (b) were correct and the origin were actually between his and nic, a gradient would also be expected; the lowest values of B/M would be obtained for markers closest to the origin. However, one does not necessarily expect the ratios to be greater than those obtained for terminus markers. A gradient could also be consistent with hypothesis (c) if the length of the re- gion which was replicated twice was variable. I am grateful to Mr. Derek Harland for excellent technical assistance; to Drs. W. Donachie and J. Scaife for helpful discussions during the course of this investigation; Downloaded by guest on September 24, 2021 608 GENETICS: M. MASTERS PRoc. N. A. S.

to the above and to Drs. J. Gross and M. Monk for critical reading of the manuscript and to Dr. Claire Berg for an introduction to phage P1 methodology. 1 Cairns, J., J. Mol. Biol., 6, 208 (1963). 2 Meselson, M., and F. W. Stahl, these PROCEEDINGS, 44, 671 (1958). 8 Lark, K. G., T. Repko, and E. J. Hoffman, Biochim. Biophye. Acta, 76, 9 (1963). 4 Yoshikawa, H., and N. Sueoka, these PROCEEDINGS, 49, 559 (1963). 6 Ibid., 806 (1963). 6 Nagata, T., these PROCEEDINGS, 49, 551 (1963). 7 Nishi, A., and T. B1oriuchi, J. Biochem., 60, 338 (1966). 8 Donachie, W. D., and M. Masters, Genet. Reg., 8, 119 (1966). 9 Donachie, W. D., and M. Masters, in The Cell Cycle-Gene Enzyme Interaction, ed. Padilla, Whitson, and Cameron (New York, London: Academic Press, 1969), p. 37. 10 Abe, M., and J. Tomizawa, these PROCEEDINGS, 58, 1911 (1967). 11 Cerd&Olmedo, E., P. C. Hanawalt, and N. Guerola, J. Mol. Biol., 33, 705 (1968). 12 Wolf, B., A. Newmian, and D. Glaser, J. Mol. Biol., 32, 611 (1968). 13 Caro, L. G., and C. M. Berg, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 33 (1968), p. 559. 14 Helmstetter, C., J. Bacteriol., 95, 1634 (1968). 15Pato, M. L., and D. Glaser, these PROCEEDINGS, 60, 1268 (1968). 16Yoshikawa, H., A. O'Sullivah, and N. Sueoka, these PROCEEDINGS, 52, 973 (1964). 17 Cooper, S., and C. E. Helmstetter, J. Mol. Biol., 31, 519 (1968). 18Okada, T., K. Yanagisawa, and F. J. Ryan, Z. Vererbungslehre, 92, 403 (1961). 19 Gorini, L., and H. Kaufman, Science, 131, 604 (1960). 20 Taylor, A. L., and C. D. Trotter, Bacteriol. Rev., 31, 332 (1967). 21 Sueoka, N., and H. Yoshikawa, Genetics, 52, 747 (1965). 22 Helmstetter, C. E., and S. Cooper, J. Mol. Biol., 31, 507 (1968). 23 Boyer, H., J. Bacteriol., 91, 1767 (1966). 24 Clark, D. J., and 0. Maalje, J. Mol. Biol., 23, 99 (1967). Downloaded by guest on September 24, 2021