Concerning the Influence of Integrated Episomes on Chromosomal

Proceedings of the National Academy of Sciences Vol. 65, No. 2, pp. 467-474, February 1970

Concerning the Influence of Integrated Episomes on Chromosomal Replication in Escherichia coli Helen Eberle DEPARTMENT OF RADIATION BIOLOGY AND BIOPHYSICS, UNIVERSITY OF ROCHESTER, ROCHESTER, NEW YORK Communicated by Donald A. Glaser, November 28, 1969 Abstract. Two strains of Escherichia coli K12, C600, and DG163, in which the origins and directions of DNA replication had been mapped previously, were made lysogenic for X and Xi434. The origins of replication in these lysogenic strains were located with respect to the prophage location by chromosome alignment and subsequent hybridization of 3H-labeled X vir DNA with newly replicated and "old" DNA. The results obtained are consistent with similar origins of replication observed in the nonlysogenic strains. These findings suggest that DNA replication in these cells is under the control of the E. coli chromosomal replicon and is independent of the presence of the X replicon as predicted by the Jacob, Brenner, and Cuzin model of DNA replication.

Introduction. An essential feature of the Jacob, Brenner, and Cuzin replicon model of DNA replication is a structural gene which codes for a specific diffusible product called the "initiator." The intiator acts at another specific site on the replicon, called the "replicator," to initiate DNA replication. In the cytoplasmic state, episomal DNA such as an F factor or that of a bacteriophage, replicates autonomously as an independent replicon. However, when the episomal DNA becomes integrated into the host chromosome, according to the model, its replication is controlled by the host replicon system.' In this case the origin and direction of replication of the host chromosome should remain the same as that observed before the integration of the episomal DNA. However, early experiments performed by Nagata2 suggested that the origin and direction of replication in Escherichia coli K12 depended on the presence and location of the integrated F factor. Replication appeared to start at the site of the integrated F factor and proceed in a direction opposite of that observed in chromosomal transfer during conjugation. Many other workers have since described a variety of experiments which, in some cases, support Nagata's ob- servations,3-5 and in other cases contradict them.6'0 Because it was suggested that one type of episomal DNA, namely that of the integrated F factor, may influence the replication pattern of the host chromosome, one might ask the general question as to whether the introduction of any type of episomal DNA into the host chromosome will alter the origin or direction of 467 Downloaded by guest on September 24, 2021 468 GENETICS: H. EBERLE PROC. N. A. S.

replication of the host chromosome. We felt it would be useful to test another type of episomal DNA, that of prophage X, for its effect on the replication pattern of the host chromosome. To accomplish this, we took an F- and an Hfr strain of E. coli K12, in which the origins and directions of replication had been previously mapped, and made them lysogenic for X. The origins of replication in these lysogenic strains were located with respect to prophage site by chromosome alignment and subsequent hybridization of 3H-labeled X vir DNA with newly replicated, and old lysogenic DNA. The results of these experiments, described below, suggest that the introduction of the episomal DNA of prophage X into the chromosome of E. coli K12 does not change the DNA replication pattern of the host, which provides additional supporting evidence for the replicon model of DNA replication. Materials and Methods. Bacterial strains: Escherichia coli K12 strains C600 F-, thy-, thr-, leu-, Bi-, lac-; and DG163 Hfr of Broda, met-, arg-, thy- (obtained from Dr. B. Wolf9) were made doubly lysogenic for X and Xi434 by purifying cells from the centers of plaques produced by doubly or sequential single infection with X and Xi434. The colonies were tested for double lysogeny by spotting an ultraviolet induced lysate on C600(X) and C600(Xi434) indicator cells. Growth and labeling of cells: Both K12 double lysogens were grown in M9 medium,1' supplemented with the required amino acids at 20 ug/ml and thiamine at 2 Mg/ml. Thymine was present during periods of radioactive labeling in C600 cultures at 2 and 4 ,g/ml in DG163 cultures, and in overnight cultures 20 ,g/ml was present. During density labeling with 5-bromouracil (at 20 Mg/ml), thymine was also present at 2 and 4 ,ug/ml in C600 and DG163 cultures, respectively. Radioactive thymine, thymine-2- 14C, and thymine-methyl-3H (New England Nuclear) was present at 0.05 and 3 UCi/mi respectively, during periods of radioactive labeling. Experimental procedures were performed at cell densities of 1 to 2 X 101 cells/ml. Culture transfers were accomplished by collection of cells on membrane filters and washing as described by Pritchard and Lark." During sampling, growth was stopped by chilling the cells in medium ice, after which the cells were collected by centrifugation and stored at -10OC until lysed. CsCl density-gradient centrifugation: The cells were thawed at 370 and then sus- pended in 1 ml of 0.15 M NaCl, 0.01 M EDTA (disodium ethylenediaminetetraacetate). The cells were then lysed by the addition of 80 ,ug/ml lysozyme (Worthington) followed by the addition of 100 MLg/ml heated pancreatic ribonuclease A (Sigma) and finally the addition of 100 Mg/ml pronase (Calbiochem). An incubation period of 30 min at 370 for each of the above enzymes was allowed. An alternate method of lysing the cells by the addition of 1 ml of 0.01 M EDTA, 1% SLS (sodium lauryl sulfate) followed by incubation with 100 Ag/ml pronase for 30 min at 370 was also used. The volume of the lysate was then brought to 3.36 ml by the addition of distilled water and the lysate added to 4.36 gm of CsCl to give the desired density. The samples were overlaid with mineral oil and centrifuged at 40,000 rpm in a type 50 rotor in a Spinco model L ultracentrifuge for 48 to 60 hr. Seven drops were collected in the first tube, followed by three drops in each subsequent tube. Each fraction was diluted by the addition of 0.5 ml of distilled water. The radio- activity in the fractions was assayed by spotting 50 or 100 MAl of each fraction on glass fiber filters, drying, and counting. Samples were counted on a Nuclear-Chicago 720 series liquid scintillation system using toluene-liquifluor scintillation fluid. Hybridization: Fractions from the hybrid and light peaks, respectively, were pooled and 6 X SSC (SSC = 0.15 M NaCl, 0.015 11 sodium citrate) added to bring the volume to 5 ml. The samples were heat denatured at 1000 for 10 min and quick cooled. More 6 X SSC was added to bring the total volume to 10.3 ml. The denatured DNA Downloaded by guest on September 24, 2021 VOL. 65, 1970 GENETICS: H. EBERLE 469

was bound to HAWP Millipore membrane filters (0.45 s-pore size) in duplicate 5-ml aliquots. DNA-DNA hybridization with sonicated denatured 'H-labeled X vir DNA (0.028 iAg/sample; 22,000 cpm/Ag) was accomplished by the method of Denhardt.12 The 3H-X vir DNA was prepared and isolated as described by Burgil3 and Mandell and Hershey,'4 but using buffer (0.1 M Tris, 1% SLS; pH 9)-saturated phenol. The percentage of X DNA replicated was calculated from the expression:

% % 3H counts hybridized to lysogenic DNA of hybrid density X DNA replicated = 200- (% 'H counts hybridized to lysogenic DNA of hybrid density) where the per cent 3H counts hybridized to lysogenic DNA of hybrid density = 3H counts hybridized to DNA of hybrid density (3H counts hybridized to DNA of hybrid density) + (3H counts hybridized to DNA of light density) It should be noted that there is a certain amount of homology between the E. coli chromosome and X DNA,25-27 which is not necessarily at the X integration site. In Figure 4A the small amount of X DNA replicated before 35% of the total DNA is replicated may be partly due to this factor, as no corrections were made to subtract for this "noise" level homology. Experiments and Results. Test for induction of prophage by the conditions of the experiment: In order to determine whether the presence of prophage X changes the origin of DNA replication of the host chromosome, two strains of E. coli K12 were made lysogenic for X and Xi434. The strains were made doubly lysogenic in order to have two copies of the genome present for hybridization. In the case of double lysogeny by X and Xi434, the prophage are thought to occupy adjacent sites on the host chromosome"5-"7 in the region between the bacterial genes determining the ability to ferment galactose and the biosynthesis of biotin. 18 In order to correctly interpret the results of the experiments described below, it was necessary to first determine whether the growth conditions imposed on the cells in the course of the experiment (i.e., amino acid starvation and growth in 5- Bromouracil containing medium) would cause induction of the prophage. This was examined by growing the cells under the conditions required by the experiment, removing samples, adding chloroform to lyse the cells, and testing for the presence of phage on E. coli C600 cells. The results listed in Table 1 indicate that the experimental conditions do not induce the production of phage. Test for chromosome alignment by amino acid starvation: The degree of chromosome alignment which could be obtained with these two strains of K12, using the amino acid starvation technique, was determined by a similar method as that described by Lark, Repko, and Hoffman,'9 and Lark.20 An exponential culture was transferred to medium lacking amino acids and incubated for 100 minutes. Amino acids were added back and the origins were labeled with 'H- thymine during a 45-minute period. The culture was shifted to medium containing amino acids and '4C-thymine and grown for three generations. The cells were then transferred to medium lacking amino acids and containing 14C-thymine for 100 minutes in order to complete the round of replication and again align the chromosomes. The culture was then shifted to medium containing amino acids and 5-Bromouracil and samples were taken at intervals. The samples were subjected to CsCl density gradient centrifugation and analyzed for the Downloaded by guest on September 24, 2021 470 GENETICS: H. EBERLE PROC. N. A. S.

TABLE 1. Test for prophage induction by experimental methods. Plaque- Frequency Status of culture forming units/ml (Plaque-forming units)/cell C600(X, Xi434) (1) +AA + thy 5.5 X 106 7.4 X 10-2 (2) -AA + thy (100 min) +AA + BU 0min 2.1 X 104 2.1X10-4 55min 5.6 X 105 4.0 X 10-3 100 min 1.8 X 106 7.3 X 10-3 150 min 1. 3 X 106 2.8 X 10-3 DG163(X, Xi434) (1) +AA + thy 1.2 X 106 1.0 X 10-2 (2) -AA + thy (100 min) +AA + BU 0min 8.0 X 104 8.5 X 10-4 50min 7.5 X 105 7.5 X 10-3 70min 1.6 X 106 1.1 X 10-2 90min 1.8 X 106 1.1 X 10-2 The cells were grown in M9 medium containing amino acids and thymine to a density of about 1 X 108 cells/ml, filtered, washed and transferred to medium lacking amino acids and containing thymine and incubated for 100 min. The cells were then filtered, washed, and transferred to medium containing amino acids and 5-Bromouracil. Samples were taken at the times indicated. Plaque- forming units (PFU) and total viable cells were assayed as described by Campbell.2'

replication of the 3H- and 14C-labeled DNA. The per cent 3H-labeled DNA and "4C-labeled DNA replicated was ascertained by the percentage of the respective counts in DNA of hybrid density. Figure 1 shows the results obtained. Early transfer of the 3H-labeled DNA to hybrid density after reinitiation in 5-Brom- ouracil containing medium is not as dramatic as that exhibited by E. coli 15 T-,20 but preferential transfer of the 3H-labeled DNA is observed, especially with C600 (X, Xi434). DG163(X, Xi434) shows a different pattern of replication of

100-

D 80- 80 C600LX,X 434) 60- 60 -

-44 0 .7040 0 40e-nts16the c e terDT43td ~20 20-

0 C~~~~~~~~~~~~~~~~- 02040 60 80 ~~~~~~~~~~0~~~20 40 60 80 PERCENT TOTAL DNA REPLICATED PERCENT TOTLA DNA REPLICATED

FIG. 1.-Test for chromosome FIG. 2.-Replication of X DNA alignment by amino acid starva- in an exponentially growing tion. The dashed line repre- culture. 0 0600 (X, Xi434), sents the curve expected for A = DG163 (X Xi434). The random replication of the 3H- dashed line represents the curve labeled DNA. A, O = C600 expected for the random replica- (X, Xi434); 0 = DG163 (X, tion of X DNA. Mi434). Downloaded by guest on September 24, 2021 VOL. 65, 1970 GENETICS: H. EBERLE 471

the 3H-labeled DNA. ere appears to be an early burst of replication followed by a leveling off and th n another increase in replication. The significance of this pattern of replication will be discussed below. Test for replication of X DNA in an exponential culture: In an exponentially growing culture, where hromosomes are in all stages of completion, one would expect the prophage DN (like any other given piece of DNA in the chromosome) to be replicated in a random fashion. That is to say, the amount of X DNA of hybrid density should be directly proportional to the amount of total DNA of hybrid density after a shift to 5-Bromouracil containing medium. To test the replication of prophage DNA, cultures were grown exponentially in the presence of 14C-thymine to uniformly label the chromosomes, then transferred to 5-Brom- ouracil containing medium and samples taken at intervals. Newly replicated DNA of hybrid density was separated from old DNA of light density by CsCl density-gradient centrifugation. The amount of total DNA replicated was determined by the percentage of 14C counts in the hybrid band and the amount of X DNA replicated was determined by hybridization with 3H-labeled X vir DNA. The results seen in Figure 2 show experimental values which are less then that expected for random replication for both strains. This may indicate that there is some slight prejudice against hybridization with 5-Bromouracil containing DNA. Replication of prophage X DNA after chromosome alignment. As indicated above, the origins of replication for the two strains of K12 chosen for this study have been previously, mapped.9 C600 has an origin near the arg G locus with a clockwise direction of replication and DG163 has one origin near the gal locus with a counterclockwise direction of replication and another possible origin near the arg E locus.9 Since the prophage DNA is inserted between the gal and the bio loci, one can make predictions, on the basis of the above knowledge, as to when the prophage DNA should be replicated if the origins of replication have not been altered by the presence of the prophage. In the case of C600 one would expect the prophage DNA to be replicated at a time when approximately 50 per cent of the total DNA has been replicated after reinitiation of DNA replication at the origin (see Fig. 3 and Table 2). In the case of DG163 one would expect a

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X85 5 Xlac argE 80 DG 163 0 gal / \ \kX~~~~~~~~i434 FIG.3.-Abbreviated linkage map 7 \ bio of E. coli based on the locations as N given by Taylor and Trotter.22 The 70 20 arrowhead represents the location of the Hfr site and the direction of chromosomal transfer during con- 5 25 pyrF jugation. ,_~~proA Downloaded by guest on September 24, 2021 472 GENETICS: H. EBERLE PROC. N. A. S.

TABLE 2. Distance between markers. Distance* Markers Direction of replication (in per cent of chromosome) Arg G to X Clockwise 51 Arg G to X Counterclockwise 49 Arg E to gal Clockwise 37.5 Arg E to gal Counterclockwise 62.5 Hfr to arg E Clockwise 12.5 Hfr to arg E Counterclockwise 87.5 * Calculated from the Taylor and Trotter revised linkage map of E. coli.22 very early or a very late replication of the prophage DNA, depending upon which side of the prophage the origin near gal is located. If, however, the insertion of the prophage introduced a new origin of replication at the X origin, then one would expect an early replication of the prophage DNA in both strains. The origin of DNA replication was located with respect to the position of the prophage in strains C600 and DG163 by the following experiment. Cells were grown exponentially in the presence of 14C-thymine to label the chromosomes uniformly, then transferred to medium lacking amino acids and containing 14C_ thymine for 100 minutes to complete the round of replication which was in prog- ress and align the chromosomes. The cells were transferred to medium containing amino acids and 5-Bromouracil, and 25 or 50-ml samples were taken at intervals. The samples were lysed and subjected to CsCl density-gradient centrifugation. Fractions from the hybrid and light density peaks were pooled respectively, and the DNA hybridized with 3H-labeled X vir DNA. The percentage of X DNA replicated was determined by hybridization and the percentage of total DNA replicated was determined by the percentage of 14C counts in DNA of hybrid density. The results of these experiments can be seen in Figures 4A and B. In a plot of percentage of X DNA replicated versus percentage of total DNA replicated, C600 shows a pattern of prophage replication in which very little replication occurs until about 45 per cent of the total DNA has been replicated, followed by a rapid increase. This pattern of prophage replication is consistent with an origin about 55 per cent of the chromosome away from the prophage site, which would place it somewhere near the arg G locus (see Table 2). With DG163 there appears to be an early replication of prophage DNA before much total DNA has been replicated. There is then an indication of a halt in prophage replication. followed by a sharp increase in replication at a time when about 43 per cent of the total DNA has been replicated. These results suggest that there is an origin

A80 CU9 (X 4A34) 4) FIG. 4.-Replication of X DNA after 60 you/chromosome. alignment. I4 ,,./^ / 1"" (A) C600(X, Xi434): o, A and L repre- 0 J o-/0sentj I,results~ from different experiments. (B) 1G163(X, Xi434): 0 and A represent results from different experiments. The dashed lines represent the curves O expectedid To Sfork .the. trandom replication of 0 10 t x DNA. PERCENT TOTAL DNA REP(ICATED Downloaded by guest on September 24, 2021 VOL. 65, 1970 GENETICS: H. EBERLE 473

near the prophage site and another origin about 48 per cent of the chromosome length away from the prophage. Discussion. The results described above indicate that there is an origin of DNA replication approximately 55 per cent of the length of the chromosome from the prophage in the lysogenic strain of C600. This would place the origin somewhere near the arg G locus. In the lysogenic strain of DG163 there appears to be one origin very near the prophage, which is near the gal locus. If the direction of replication from the origin near gal is counterclockwise, as the work of Wolf, Newman, and Glaser indicates,9 then the origin must be on the bio side of the prophage in order to get the immediate replication of prophage DNA which we observe. However, the fact that the origin near gal is also very close to the prophage site complicates the interpretation of whether the origin is at the prophage site or on the bio side of the prophage in this strain. We also find indications of two possible origins in DG163. The biphasic curves obtained in both the experiments testing for chromosome alignment by amino acid starvation (Fig. 1) and the replication of prophage DNA after chromosome alignment (Fig. 4B) indicate that there are two possible origins of replication, either of which may be chosen in a random fashion. Although the direction of replication cannot be determined directly by this type of experiment, given that the location of the prophage is near gal, the results with DG163 are consistent with a counterclockwise direction of replication from the origin near gal, and a clockwise direction from an origin 46-48 per cent of the chromosome length from the prophage DNA, or between 64' and 66' on the E. coli map (see Fig. 3). Wolf, Newman, and Glaser have placed this origin closer to the arg E locus.9 If the origin near qal in DG163 were actually at the intergration site of the F factor, and replication were to proceed in a clockwise direction, one would expect no replication of prophage DNA until about 12 per cent of the total DNA had been replicated (Table 2). The results in Figure 4B indicate that the replication of prophage DNA starts at the same time as the replication of total DNA. How- ever, due to the heterogeneity of initiation of replication after amino acid starvation,20 it is not possible to rule out an origin at the F factor site with a clockwise direction of replication. Wolf et al.23 have discussed the possibility that under certain conditions replication may be initiated at sites other than the normal origin, such as at the integrated F factor. Thus, the results described above are consistent with origins of replication similar to or not far removed from those described by Wolf, Newman, and Glaser for these two strains of E. coli K12 when in the nonlysogenic state. These findings suggest that the regulation of initiation of DNA replication in these composite replicons is controlled by the E. coli replicon system.

I wish to thank Drs. Allan M. Campbell and Henry M. Sobell for their encouragement and support during the course of this work. These studies were initiated while H. E. was a postdoctoral fellow of the National Institutes of Health (7-F2-GM-31,275-03) and was supported in part by USPHS grant E-2862, an institutional grant from the American Cancer Society and a contract with the U.S. Atomic Energy Commission at the Uni- versity of Rochester Atomic Energy Project. This paper has been assigned report no. UR49-1147. Downloaded by guest on September 24, 2021 474 0.J1VETICS: H. J9BE9RLE PROC. N. A. S.

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