Salmonella typhimurium - Journal of Bacteriology - American Society

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Apr 3, 1973 - typhimurium map) and ilv (122 min) to a terminus in purE-trpB region ..... 1.63. 2.12 tions in MM or NB before adding chloram- phenicol (CAP) ...
JOURNAL OF BACTERIOLOGY, JUlY 1973, p. 168-176 Copyright © 1973 American Society for Microbiology

Vol. 115, No. 1 Printed in U.S.A.

Bi-Directional Chromosomal Replication in Salmonella typhimurium T. FUJISAWA' AND A. EISENSTARK2 Division of Biology, Kansas State University, Manhattan, Kansas 66502

Received for publication 3 April 1973

Transducing frequencies of phage P22 lysates prepared from Salmonella typhimurium exponential cultures in minimal and nutrient broth media were compared. The assumption is that cells grown in a minimal medium will have one replication fork per replication unit, but cells in nutrient broth will have multiple replication forks; therefore, the frequency of genetic markers near the origin of replication will be higher in the nutrient broth culture. Analysis of transduction showed a gradient of marker frequencies from the highest (the cysG-ilv region) to the lowest (purE-trpB region) in both clockwise and counter clockwise directions. This supports our previous observation that chromosome replication proceeds bidirectionally from the origin between cysG (109 min on S. typhimurium map) and ilv (122 min) to a terminus in purE-trpB region (20 to 53 min). Since this method avoids possible artifacts of other methods, the results are assumed to reflect the sequence of chromosome replication in exponentially growing cells. Evidence for the existence of multiple replication forks in nutrient broth-grown cells was supported by the following: (i) the marker frequency data fitted the assumption of multiple replication fork formation; (ii) residual deoxyribonucleic acid increase after inhibition of protein synthesis to complete a round of chromosome synthesis which was 44% in cells grown in a minimal medium and 82% in those in nutrient broth; (iii) segregation patterns of the 3H-thymidine-labeled chromosome strands during subsequent growth in nonradioactive medium were studied by autoradiography, and the number of replication points per chromosome per cell was estimated as 5.6 for the nutrient broth culture and 2.5 for the minimal medium culture. These data support a model of symmetrical and bidirectional chromosome replication. In Escherichia coli, the chromosome is a single-circular deoxyribonucleic acid (DNA) molecule (7) that replicates sequentially and semiconservatively (6, 18, 25). Many experiments have been performed to determine the genetic site where chromosome replication is initiated and the direction in which a replication point(s) proceeds. Although earlier studies (1-3, 9-11, 17, 27, 34, 35) suggested that chromosome replication proceeds from a fixed position between 45 and 75 min on the chromosome map of E. coli (32) in a clockwise direction, the possibility of bidirectional replication has been raised in E. coli (9) and in Salmonella typhimurium (26). Recently, Masters and Broda ' Present address: University of Califomia-Irvine, California College of Medicine, Environmental Interactions, Irvine, Calif. 92664. 2Present address: Division of Biological Sciences, Tucker Hall, University of Missouri-Columbia, Columbia, Mo. 65201.

(24), Bird et al. (5), Yahara (36), Jonasson (20), and Prescott and Kuempel (28) have presented further evidence of bidirectional replication in E. coli by various techniques. We have investigated the genetic position of origin and the direction of chromosome replication in an F- strain of S. typhimurium LT2 by marker frequency analysis via phage P22 transduction. The data suggest that replication proceeds simultaneously from a fixed origin between cysG (109 min) and ilv (122 min) on the genetic map of S. typhimurium (29) in two directions to the terminus near trp (53 min). These data are compatible with those obtained by Nishioka and Eisenstark (26) in S. typhimurium. In marker frequency analysis, it is assumed that multiple replication forks are formed in a replication unit of a circular chromosome of rapidly growing cells, while only a single replication fork per replication unit exists in slowly 168

CHROMOSOMAL REPLICATION IN S. TYPHIMURIUM

VOL. 115, 1973

growing cells, and evidence will be presented to show that this is the case.

MATERIALS AND METHODS Bacteria and phage. Strain KSU1619 (Iys6) of S. typhimurium LT2 F- was used as the donor in transduction. Recipient strains used in transduction are listed in Table 1. For 3H-thymidine uptake experiments and autoradiography, strain KSU2426 (thy-arg69-phe) was used. A mutant of phage, P22 (L4), kindly supplied by H. 0. Smith, was used in

transduiction. Media. Minimal medium (MM) was made by mixing equal volumes of 2X-minimal salts solution and water supplemented with 0.4% glucose. 2X-minimal salts solution contained 21 g of K2HPO4, 9 g of KH2PO4, 2 g of (NH4)2SO4, 1 g of sodium citrate, and 0.1 g of MgSO4-7H2O, dissolved in 1 liter of water. Nutrient broth (NB) was prepared by dissolving 8 g of dehydrated NB (Difco) and 5 g of NaCl in 1 liter of TABLE 1. Recipient strains used in transduction Cistron

KSU

and

strain

allele no.

no.

argA 160 argE13 aroD5 aroD86 cysBlO cysB23 cysC389 cysE199 cysG385 cysJ299 hisB200 hisB210 ilvA120 ilvC130 ilvD150 leu-550

5160 5012 5209 5291 5409 5422 5739 5570 5735 5652 6468 6469 7237 7236 7238 7367 7996 8061 8087 8072 8062 8226 8516 8560 8564 4779 9267 9007 8578 2641 9435 8153 2641 2430 9816

lys-17 metB630 metC90 metE740 metF640 metF232 phe-50 proA2 proB7

purE242 serA272

serBlO trpA8 trpB2 trpB2 trpD7 tyr-40 thy-341 val-15 a

UV, ultraviolet irradiation.

Mutational

origin 2-aminopurine 2-aminopurine Spontaneous Diethylsulfate Uva

UV X rays 2-aminopurine X rays Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Nitrosoguanidine 2-aminopurine Spontaneous Spontaneous Spontaneous Spontaneous 2-aminopurine Spontaneous UV UV Diethylsulfate Diethylsulfate UV UV UV UV UV

Diethylsulfate Nitrosoguanidine Diethylsulfate

169

water. Minimal agar was made by mixing equal volumes of 2X-minimal salts solution and 2X-agar supplemented with 0.4% glucose. 2X-agar contained 18 g of Ionagar (Colab Laboratories) per liter. Buffer. Phosphate-buffered saline (PBS) was prepared as described previously (26). Cell number count. Cell number was measured by a Coulter Counter model F with a 30-am aperture. NaCl solution, Abbo-liter (Abbott Laboratories), or 0.1 N HCl was used for the suspending electrolyte. Cell growth. An overnight culture was diluted in a fresh medium with 1: 100 dilution and was incubated with aeration at 37 C. A Coulter counter was used for monitoring cell growth. When cells were transferred from one medium to the other, they were filtered through a membrane filter ( 0.45 Am pore size; Millipore Corp.). Cells were washed on the filter with prewarmed medium without glucose, and subsequently suspended in the desired medium. This procedure was always completed within 2 min. Preparation of donor phage. Three milliliters of overnight culture of KSU1619 was diluted in 300 ml of a fresh medium (MM or NB). When the titer reached 1.5 x 10' to 2.5 x 10' cells/ml, phage P22 was added with a multiplicity of infection (MOI) of 5. After 15 min of adsorption, cells were centrifuged and the pellet was suspended in 100 ml of a fresh medium. The cells were allowed to grow for either 35 min in NB or for 55 min in MM and then chloroform was added. Only one cycle of phage growth occurred in this period. The lysates were centrifuged twice to remove debris. The supernatant titers ranged from 5 x 1010 to 2 x 1011 plaque-forming units (PFU)/ml. Transduction procedure. Recipient cells were grown overnight and diluted in NB to adjust the titer to 109 cells/ml. A series of 0.3 ml of bacterial suspension and 0.1 ml of phage suspension were mixed and tubes were incubated at 37 C without shaking. MOI was always 1.5. After 30 min, 2.5 ml of minimal soft agar (MSA) was added to each tube and plated immediately on the appropriate screening-agar plate. MSA contained 5 g of NaCl and 6 g of lonagar in 1 liter of water. Plates were incubated at 37 C for 48 h and the number of transductants was determined. Isotope incorporation and radioactivity assay. Bacterial DNA was labeled with 3H-thymidine (specific activity 10 uCi/mmol: New England Nuclear Corp.) in the presence of 10 glg of cold thymidine per ml and 1.5 mmol of uridine to prevent the induction of thymidine phospholylase in both NB and MM. Twotenth milliliter samples of bacterial culture were treated with the same volume of 10% ice-cold trichloroacetic acid and allowed to stand in an ice bath for at least 40 min. One-tenth milliliter of carrier DNA (0.1 mg of highly polymerized salmon sperm DNA per ml; Mann Research Laboratories) was added to each sample before adding trichloroacetic acid. After incubation with trichloroacetic acid, samples were collected on membrane filters (Reeve Angel) and washed twice with ice-cold 5% trichloroacetic acid. Filters were dried and placed in glass counting vials containing 5 ml of organic scintillation fluid. Radioactivity was measured in a Beckman liquid scintillation counter.

FUJISAWA AND EISENSTARK

170

J. BACTERIOL.

exists per chromosome when cells are grown in a rich medium (4). Figure 1A illustrates that the replication points proceed bidirectionally from replication origin to terminus. For better visualization, this is presented in linear form, omitting the opening at the origin in the circular chromosome. In the figure, the replication unit is defined as the chromosome region within which replication proceeds from origin to terminus. As may be seen in Fig. 1A(a), for NB, frequencies near the replication origin should be about four times greater than genetic markers near the terminus. On the other hand, in cells grown in MM, only one replication point per replication unit exists, and marker frequency near the origin should be about twice as great as a marker near the terminus [Fig. 1A (b) ]. Thus, assuming that bacterial genes are randomly incorporated into protein coats, the transduction frequency of markers would reflect the frequency of genes existing in the original cell populations grown in different media. Theoretical curves of marker frequency calculated from equation 1 (31) are shown in Fig. 1B. In the RESULTS equation g0(x) = 20(1-x), g,(x) is the frequency of Experimental design for determination of a given marker, x is the relative marker posigenes near origin and terminus via transduc- tion, (assigning position 0 to the origin of the tion data. More than one replication point replication and 1 to the terminus, 0 s x s 1),

Autoradiography. Exponentially growing cells of KSU2426 in NB (supplemented with 10 tsg of thymidine per ml and 1.5 mmol of uridine) or an MM (supplemented with 10 Ag of thymidine per ml, 1.5 mmol of uridine, 20 ,ug of L-arginine and L-phefiylalanine per ml) were pulse labeled with 3H-thymidine (specific activity 10 Ci/mmol) for 3 or 5 min, respectively. Labeling time was one-tenth of the generation time in each medium. At the end of labeling, cells were harvested and washed with PBS by centrifugation and then were suspended in a prewarmed fresh medium without radioactive material. A portion of the culture was removed at one-generation intervals up to the sixth generation. Samples were washed thoroughly with PBS to remove free 3H-thymidine and then were suspended in water. Cells (105) were spread on clean glass slides and were air-dried. After drying, slides were dipped in 5% ice-cold trichloroacetic acid for 3 min and subsequently in 95% ethanol for 3 min. Completely dried slides were dipped in the autoradiographic emulsion of Kodak NTB2 at 45 C. After 10 to 21 days of exposure, slides were developed with Microdol X (Kodak) for 3 min, in 1% acetic acid for 10 s, and in Rapid Fixer (Kodak) for 1 min. Grain counts were made by phase contrast microscopy.

(a) Nutrient Broth t repl. pt

/

2d

2d rept

repipt

x3

\ \

terminus

(b) Minimal medi

Replication unit

Replication unit 0

Conniei)

0.5

1 0.5 (terminus) Position of Markers (x)

(0

o

(oriqin)

' 1)

FIG. 1. A, Schematic representation of a bidirectional replication model of circular chromosome in NB and MM. The circular chromosome is opened at the origin end to exhibit a linear structure. Replication proceeds in two directions from the origin toward the common terminus. The replication unit is defined as the chromosome region within which replication proceeds from origin to terminus. When cells are grown in NB (a), a seccnd round of replication is initiated before the first round is terminated. Thus, dichotomous replication forks 4re formed. In the hypothetical steady state, the second initiation occurs when the first replication point (1st repl. pt.) reaches about the mid-point of the replication unit. In cells growing in a minimal medium (b), only one replication point per replication unit exists. B, Graphic representation of marker frequency curves in different media. Theoretical curves of marker frequency in an exponentially growing population in NB (a) and MM (b) are shown. Calculations are based on equation (1) in the text. In NB (a) the replication origin has four dosages of a gene but two for MM (b).

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CHROMOSOMAL REPLICATION IN S. TYPHIMURIUM

and 0 is the number of replication position. The replication position is defined as the position of a set of equivalent replication points on a replication unit in the chromosome. Although the assumption is made that phage P22 incorporates and transduces bacterial genes at random, no data are available to prove it. There is considerable variation of transduction frequency between markers. Therefore, in order to have the transduction frequency for a particular gene reflect the proper relationship to the transduction frequency of all of the other markers, data were normalized as follows. The relative frequency of transductants for each marker was calculated as shown in equation 2. f1 = -n Ti in which f1 is the relative frequency Ti/y, of gene i, Ti is the number of transductants for gene i, and n is the number of markers used in the experiments. fi is calculated for transductions carried out by using phage lysates prepared in NB and MM, and designated f1NB and fiMM, respectively. The ratio, f1NB/f1MM should be proportional to the ratio of marker frequency, g2(x)/g,(x), where it is assumed that there are three replication points per replication unit at any stage of exponential growth in NB and one replication point per replication unit in cells growing in a minimal medium. When the ratios, fiNB/fiMM, are calculated, the highest ratio should represent the marker closest to the replication origin and the lowest ratio should represent the marker closest to the terminus. Also, there should be a gradient of ratio values between origin and terminus. Transduction data. Table 2 shows the number of transductants for each marker (Ti) obtained from NB and MM lysates, respectively. These are the results of a single, typical experiment, but experiments were repeated up to 10 times with consistent results. The sum of transductants for all markers (Z1 n Ti) obtained by NB and MM lysates were 10.406 and 13.588, respectively. In Table 3, normalized values of f1, f1NB/f1MM are shown. The ratios of f1NB/ftMM are plotted in Fig. 2 against a schematic abscissa of the relative positions of genes in which hisB (62 min) is arbitarily chosen to be at one end of the genetic map and the region from hisB to aroD (73 min) is repeated at the other end. In Fig. 2, the genes between argE (102 min) and metF (128 min) have high ratios with cysG being the highest. On the other hand, the genes between purE (19 min) and trpB have low ratios. Furthermore, the ratios decrease gradually from the top of the curve (cysG) to the bottom (purE-trpB region) in a symmetrical fashion (Fig. 2). This indicates that replication proceeds simultaneously from the region be-

TABLE 2. Number of transductants obtained from NB and MM lysates (typical experiment; five repetitions of same experiment gave comparable results) Recipient markers

KSU strain no.

argA argE aroD

5160 5012 5209 5291 5409 5422 5739 5570 5735 5652 6468 6469 7237 7236 7238 7367 7996 8061 8087 8072 8062 8226 8516 8560 8564 4779 9267 9007 8578 2641 9435 8153 2641 2430 9816

cysB cysC

cysE cysG cysJ hisB ilvA ilvC ilvD leu lys metB metC metE metF phe proA proB purE serA serB trpA trpB

trpD tyr thy val

No. of transductants NB

Min

72 116 193 365 339 236 252 85 193 243 214 1175 1063 562 960 219 350 225 154 68 276 83 383 140 147 273 122 272 112 222 645 113 294 185 55

42 78 263 439 573 380 203 108 106 369 338 1956 604 465 680 343 435 131 136 56 176 53 535 205 188 628 113 311 368 463 1945 403 313 148 34

tween cysG and ilv to around trp in both directions. The ratio for cysE has always been far outside of the best fitted curve. The reason for this is not known, but possible explanations will be offered later. Evidence for existence of multiple replication forks. Transduction data suggested that multiple replication forks were formed when cells were grown exponentially in NB at 37 C. As may be seen in Fig. 2, there was a several-fold increase in the ratio of transductants when cells were grown fast in NB. Measurement of residual DNA synthesis after inhibition of protein synthesis suggested the presence of multiple replication forks in a chromosome of rapidly growing cells. KSU2426 cells were labeled with 3H-thymidine for four genera-

FUJISAWA AND EISENSTARK

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TABLE 3. Normalized values obtained from transduction data fiNB and f1MM; the relative frequency of transductants for gene i was obtained by using NB lysates and MM lysates, respectively Recipient

KsUri

markers

sri no.

argA argE aroD

5160 5012 5209 5291 5409 5422 5739 5570 5735 5652 6468 6469 7237 7236 7238 7367 7996 8061 8087 8072 8062 8226 8516 8560 8564 4779 9267 9007 8578 2641 9435 8153 2641 2430 9816

cysB cysC

cysE cysG cysJ

hisB

ilvA ilvC ilvD leu lys

metB metC metE metF

phe proA proB purE serA serB trpA trpB trpD

NB fNB 0.00692

0.0111 0.0185 0.0351 0.0326 0.0227 0.0242 0.00817 0.0185 0.0234 0.0206 0.113 0.1021 0.0540 0.0923 0.0210 0.0336 0.0216 0.0148 0.00653 0.0265 0.00798 0.0368 0.0135 0.0141 0.0262 0.0117 0.0261 0.0108 0.0213 0.0620

0.0109

f

MM

0.00309 0.0057 0.0194 0.0323 0.0422 0.0280 0.0149 0.00794 0.00780 0.0272 0.0249 0.144 0.0445 0.0342 0.0500 0.0252 0.0320 0.00964

fINB fIMM 2.24 1.95 0.95

1.09 0.77 0.81 1.62 1.03 2.37 0.86 0.827 0.785 2.29 1.58 1.85 0.83

1.05 2.24 0.0100 1.48 0.00412 1.58 2.04 0.0130 0.00390 2.05 0.93 0.0394 0.89 0.0151 1.02 0.0138 0.57 0.0462 1.41 0.0083 1.14 0.0229 0.40 0.0271 0.63 0.0341 0.43 0.143 0.37 0.0297 0.023 0.0283 0.0109 1.63 0.00250 2.12

J. BACTERIOL.

replication forks exist in rapidly growing cells. The average number of replication points per chromosome was also estimated by autoradiography, assuming that each cell contains one replicating chromosome. The doubling time of the donor strain, KSU1619, used in transduction was 50 and 30 min at 37 C in MM and NB, respectively. For autoradiography, a thyminerequiring strain, KSU2426, was used which has the same doubling time as KSU1619 in MM and in NB, respectively. Exponentially growing cells of KSU2426 were pulse labeled with 3H-thymidine for 5 min in MM + 10 jig of thymidine per ml or 3 min in NB. After washing, cells were transferred to nonradioactive medium and samples were removed at one-generation intervals. Autoradiographs were prepared as described in Materials and Methods and were examined for the percentage of labeled cells in each generation. Figure 5 shows the segregation patterns of the labeled cells during subsequent growth in cold medium. Unlabeled cells were segregated between the first and second generation in MM and between the second and third generation in NB. Extrapolation to zero generation indicates that there were 2.5 labeled strands (per replication unit) in a chromosome of slowly growing cells and 5.6 labeled strands in a chromosome of rapidly growing cells. Since there are two labeled strands per replication point and because replication is bidirectional (there are two replication units per chromosome), these results indicated that each cell contains one chromosome, with an average of 2.5 and 5.6 replication points in MM and NB, respectively.

DISCUSSION The existence of a distinct origin and terminus of chromosome replication is supported for S. typhimurium LT2 F- strain KSU1619 by tions in MM or NB before adding chloram- data presented in Fig. 2. The comparison of phenicol (CAP) (150 Ag/ml), which inhibits a relative marker frequency in cells growing in new round of DNA replication but allows the NB to that in MM by transduction via phage growing points already initiated to proceed P22 showed the ratios gradually decreasing, toward the replication terminus (21). DNA starting from one point and proceeding in two synthesis was measured by uptake of 3H-thymi- directions. The interpretation is that the origin dine. As shown in Fig. 3, DNA synthesis con- lies between cysG and ilv, and that replication tinued at a decreased rate and then stopped proceeds in two directions toward a terminus after the addition of the drug. The level of the close to trp. The ratio for cysE is the only one residual increase of DNA was 44% higher than which deviated significantly from the best fitted that at zero time (time when the drug was curve of normalized marker frequency. The added) in MM (Fig. 3A) and 82% in NB (Fig. reason for this is not understood, but in E. coli 3B). These values are compatible with the the number of transductants for the xyl marker theoretical values of DNA increase in the ab- is irregular and very low (10), and Caro and sence of protein synthesis, (39 and 84% when 0 Berg (10) explained this on the assumption that = 1 and 2, respectively) (31). These findings in xyl is close enough to the origin so that chromoFig. 3 support the view that multiple sets of some discontinuity could occur and interfere tyr

thy val

0.0283 0.0178 0.00529

VOL. 115, 1973

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CHROMOSOMAL REPLICATION IN S. TYPHIMURIUM

Lwd-

One Genome Terminus

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