DNA cycle of Myxococcus xanthus

J. Mol. Biol.

(1970) 49, 609-619

DNA

Cycle of Myxococcus xanthus

DAVID ZUSMAN AND EUGENE ROSEXBERQ Department University

of Bacteriology and Institute of Molecular Biology, of California, Los Angeles, Calif. 90024, U.S.A. (Received 21 November 1969)

xanthw, The rate of DNA synthesis during the division cycle of Myxococcus growing on a defined medium has been determined. Non-synchronized exponential phase cultures were pulse-labeled with radioactive thymidine and then prepared for quantitative autoradiography. Measurements of both cell size and grain number were performed on 2400 cells. Analysis of the distribution of grains in each of 20 size groups showed that: (a) cells do not synthesize any DNA during 20% of the division cycle; (b) those cells that are synthesizing DNA are doing so at a constant rate independent of cell size or cell age; and (c) the DNA cycle extends from 0.02 to 0.81 generation, septum formation occurs at 0.90 generation, and physical separation of sister cells at I.0 generation. Chemical analysis of the average DNA content of M. xanthw growing in the defined medium yielded 14 x lObe pg DNA per cell, corresponding to 1.7 + 0.2 chromosomes/cell. Thus, the rate of DNA synthesis reported here, 2.5 x lo4 base pairs/minute, is the rate of synthesis of one replicating chromosome.

1. Introduction The relationship between chromosome replication and cell division in bacteria has been the subject of several recent reviews (Helmstetter, Cooper, Pierucci & Revelas, 1969a,b; Lark, 1969). Most of the work reported is based prin1968; Helmstetter, cipally on the Eecherichia coli system using synchronized populations. On the basis

of these studies, a model was oonstructed to account for the co-ordination between chromosome replication and cell division during exponential growth (Cooper & Helmstetter, 1968). The present investigation was undertaken as part of a continuing study of macromolecular synthesis during exponential growth and morphogenesis in the bacterium Myxococcus xanthus (Rosenberg, Katarski $ Gottlieb, 1967; Bacon & Rosenberg, 1967; Zusman & Rosenberg, 1968). Using a simple and direct experimental approach on cells growing exponentially, it was possible to get quantitative data on the rate of DNA synthesis as a function of cell size. Analysis of the distribution of grains within size groups yielded t,he relationship between chromosome replication and the cell cycle. The basic experiment used in these studies was to pulse label exponential nonsynchronized cultures of M. zanthus with radioactive thymidine and then prepare the cells for autoradiography. By measuring the size of each individual cell as well as the number of grains, the rate of DNA synthesis as a function of cell size was determined. These experiments showed: (a) the presence of a DNA cycle, i.e. cells do not synthesize any DNA for 20% of the generation time when growing exponentially on the defined medium; (b) that those cells that are synthesizing DNA are 609

610

D.

ZUSMAN

AND

E. ROSENBERG

doing so at a constant rate, independent of cell size or cell age; and (c) the position of the DNA cycle within the cell division cycle. Chemical analysis of the average DNA content of M. xanthus growing in the defined medium, indicated an average of one replicating chromosome per cell.

2. Materials and Methods (a) Bacteria The

organism used was M. zaathus strain FBmp. This strain was derived from M. xanthus FB by repeated culturing on defined media. Strain FB was obtained from Martin Dworkin and has been described previously (Dworkin & Volez, 1962). Strain FBmp has an absolute requirement for glycine, isoleucine, leucine and valine; it is stimulated by

alanine,

asparagine,

Witkin,

personal

histidine,

lysine,

phenylalanine,

proline,

serine and tbreonine

(S.

communication). (b) Medium

The medium contained/l.: 2 g MgSO,*7H,O, 0.2 g NaCl, 0.136 g KHePO,, O-002 g 0.1 g arginine, 0.5 g ssparagine, O-1 g cystine FeC1s*6Hz0, 0.002 g CaCla, 1 g alanine, 0.1 g glycine, 0.1 g histidine, 1 g isoleucine, 2 g leucine, 0.5 g lysine, 0.5 g methionine, 1 g phenylalanine, 1 g proline, 0.2 g serine, 0.1 g threonine, 0.4 g tyrosine, 0.2 g valine (all ammo acids used were the L isomer and were obtained from Nutritional Biochemical Corp.) and 10 ml. of 1 M-Tris buffer; final pH is 7.2.

(c) Growth conditions The organism was grown at 30°C under conditions of vigorous gyratory shaking. For each experiment 60 ml. of defined medium were inoculated with bacteria and aerated by vigorous gyratory shaking for 48 hr at 30°C. At this time the cells were in exponential growth with a generation time of about 6.5 hr. The medium was then supplemented with 10 cc@;thymidine/ml. 4 hr before the labeling period. (d) [3H]Thymidine

puke

An exponential phase culture of M. zanthzLs was pulsed for 5 or 10% of the generation time by incubating 2 ml. aerated culture with 0.2 mo of [naeth&3H]thymidine (3.0 c/mmole, Schwarz Bio Research, Inc.) for 20 and 40 min, respectively. The incubation was terminated by the addition of 0.5 mg thymidine/ml. and placing the cultures on ice. The bacteria were collected by centrifugation at 6000 g for 7 mm at 4°C. The pellet was then washed with 0.01 M-Tris buffer, pH 7.4, containing 0.001 M-magnesium acetate and 0.6 mg thymidine/ml. The washed cells were then resuspended in 0.4 ml. 0.01 M-Tris, pH 7.4, and spread on cleaned microscope slides, and air dried. The bacteria were fixed with 2% formaldehyde for 30 set and washed by dipping successively in 4 beakers containing cold 5% trichloroacetic acid and 0.5% sodium pyrophosphate. The slides were then rinsed with distilled water and air dried.

(e) Autoradiography Ilford Nuclear Research emulsion L4 (Ilford, Ltd.) was prepared as described (Caro, van Tubergen & Kolb, 1962) for quantitative autoradiography. 15 g of emulsion were added (in the dark) to 10 ml. water and gently stirred at 45°C with a glass rod for 16 mm. The slides were dipped rapidly in the emulsion. After the emulsion was dry, the slides were stored in light-proof Bakelite boxes containing a tube of anhydrous calcium chloride. Exposure times were adjusted so that the mean number of grains/cell was between 2 and 3. The slides were developed for 4 min at 22°C in Kodak D19 developer, rinsed rapidly with distilled water, and fixed for 4 min in Kodak F24 fixer. The slides were then washed with water and dipped for 30 set in a 0.02 o/0 solution of methylene blue in water; excess stain was removed by soaking the slides in water for 10 min. After the slides were dry, the bacteria were examined under 1875 times magnification with a Wild M20 microscope equipped with a screw micrometer eyepiece calibrated with a stage micrometer (Wild Heerbrugg, Ltd.). The standard deviation for length measurements was 0.13 CL.

DNA

CYCLE

OF

MYXOCOCCUS

611

XANTHUS

The slides were examined by placing immersion oil directly on the slide without a coverslip. Cells were selected randomly for analysis: gram counts and length measurements. The bacteria were plainly visible and well dispersed. Those bacteria that were clearly dividing but not yet physically separated were scored as dividing cells and listed separately. Background grain counts were less than O.l/cell. Control slides containing cells labeled with radioactive valine showed that over 99% of the bacteria were metabolically active. (f) Analysis

for DNA

per cell

DNA was estimated chemically after fractionation by a modification of the SchmidtThannhauser procedure (Leslie, 1955). M. zunthus FBmp was grown in defined medium to a cell concentration of 1.24 x 10s cells/ml. Four 20-ml. samples were removed and collected by centrifugation at 6000 g for 7 mm at 4°C. The pellets were washed with cold 0.01 M-Tris buffer, pH 7.4, containing 0.001 M-magnesium acetate. The washed cells were then resuspended in iced 10% perchloric acid. After 1 hr, the precipitates were collected by centrifugation at 18,000 g for 20 min at 4’C, washed with iced 5% perchloric acid, resuspended in 0.5 M-NaOH, and incubated at 37°C for 20 hr. The alkali hydrolysates were chilled to 0°C and the DNA reprecipitated with 5% perchloric acid. The pellets were collected by centrifugation at 18,000 g for 20 min at 4”C, and resuspended in 0.3 ml. water. Diphenylamine reagent (0.6 ml.) was added to each sample and the amount of DNA estimated calorimetrically (Dische, 1955). Cell counts were performed on samples fixed in 2% formaldehyde by use of a Petroff-Hausser counting chamber.

3. Results (a) Rate of thymidine

incorporation

during

the division

cycle

The rate of thymidine incorporation into cells of different sizes in populations of was determined by pulse-labeling an exponential phase culture with [3H]thymidine, removing acid-soluble radioactivity, and then preparing the cells for autoradiography. Each cell was scored both for length and number of grains. Tables 1 and 2 show the autoradiographic data for cells pulse-labeled for 10% of the doubling time; Tables 3 and 4 present similar data for a 5% pulse. The Tables are arranged into 20 arbitrary size groups, each of which contains an equal number of bacteria. The distribution of grains observed and the mean for each size group is presented in these Tables. The mean number of silver grains in each size group yields a direct measure of the rate of DNA synthesis. Direct observation of the methylene blue-stained bacteria revealed that approximately 10% of the cells were in the act of division. These dividing cells would be scored as one large cell by standard Coulter Counter methods, but physiologically might very well behave as individuals. The dividing cells were therefore scored either as one large “mother” cell (Tables 2 and 4) or two small “sister” cells (Tables 1 and 3). By presenting the data in both of these ways, additional information is obtained relating the rate of DNA synthesis to the cell cycle. The total grain distributions in Tables 1 to 4 indicate that in each experiment the total zero class is much higher than would be expected for a Poisson distribution of grains for the observed mean. In Table 1 the predicted zero class for a Poisson distribution of mean 1.66 is 0.19; the observed zero class is 0.35. In Table 3 the predicted zero class for the mean of 1.83 is 0.16; the observed zero class is 0.32. This indicates that in a population of exponential phase M. xanthus growing in the defined medium, a class of cells exists which is not synthesizing DNA at any given time. M. xanthus

612

D. ZUSMAN

AND

E. ROSENBERG

TABLE 1 Autoradiographic data as a function of cell size for M. xanthus pulse-labeled for 10% of a generati4m-dividin.g cells scored as two cells Size groupst length (4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

1.88 2.40 2.64 2.76 2.88 3.00 3.08 3.16 3.28 3.36 3.48 3-60 3-72 3.84 3.96 4.12 4.28 4-48 4.72 5.12 Total

to to to to to to to to to to to to to to to to to to to to

2.40 2.64 2.76 2.88 2.96 3.08 3.16 3.28 3.36 3.48 3.60 3.72 3.84 3.96 4.12 4.28 4.48 4,72 5.12 7.00

0 36 32 23 20 10 15 7 9 7 8 6 5 2 2 10 13 11 19 24 22 281

1

2

3 7 6 7 9 9 10 9 6 10 5 11 12 6 3 8 11 13 9 11 165

0 1 5 4 10 6 7 7 8 9 10 6 11 19 13 5 6 1 0 3 131

t The 800 baoterie scored in this experiment of which contains 40 cells.

Grains per cell (n) 3 4 5 0 0 3 6 4 5 5 6 10 6 6 7 6 5 6 7 7 2 3 2 94

1 0 1 1 3 4 5 3 5 1 6 7 6 3 7 4 1 3 1 2 64

are divided

0 0 2 3 1 1 5 4 3 4 5 4 3 1 0 1 2 1 1 0 41

6

7

8

Mean

0 0 0 0 2 0 1 1 0 1 1 0 0 2 1 1 1 1 2 0 14

0 0 0 0 1 0 0 1 1 1 1 0 0 2 0 1 1 0 0 0 9

0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

0.18 0.23 0.98 1.23 1.93 1.43 2.25 2.16 2.35 2.08 2.75 2.30 2.28 2.55 2.03 1.83 1.78 1.10 0.98 0.78 1.66

into 20 arbitrary

size groups, each

TABLE 2 Autoradiographic data as a function of cell size for M. xanthus pulse for 10% of a generation-+Jividing cells scored as one cell Size groupst length (14 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

1.88 2.72 2-88 3.00 3.12 3.20 3.32 3.44 3.52 3.60 3.72 3.84 3.96 4.12 4.24 4.40 4.60 4.88 5.12 5.56 Total

to to to to to to to to to to to to to to to to to to to to

2-72 2.88 2.96 3.12 3.20 3.32 3.44 3.52 3.60 3.72 3.84 3.96 4.12 4.24 4.40 4.56 4.84 5.12 5.56 7.00

0 19 12 9 7 3 4 5 7 6 3 2 2 11 10 13 17 22 26 24 2.5 227

1

2

10 7 8 10 9 10 8 7 6 10 12 5 2 7 11 9 9 8 9 6 163

4 6 10 8 7 8 6 10 8 6 10 18 11 8 5 1 1 0 1 3 131

t The 744 bacteria scored in this experiment which contains 37 or 38 oells.

Grains per cell (n) 4 5 3 1 7 4 6 6 6 9 6 5 7 5 5 5 6 5 5 2 2 0

1 2 3 5 4 4 5 1 5 8 5 2 7 4 1 1 2 0 3

2 3 1 1 7 3 2 4 6 3 3 I 0 1 1 2 0 1 0

labeled

6

7

8

Mean

0 0 2 0 1 1 1 0 1 0 0 2 1 1 1 1 1 1 0

0 0 1 0 0 1 1 1 I 0 0 2 0 1 0 1 0 0 0

0 0 0 0 0 0 0 :,

0.95 1.70 2.00 1.86 2.65 2.38 2.43 2.27 2.63 2.43 2.22 2.57 1.97 1.97 1.38 143 0.84 0.66 0.62 1j.65 1.78

0 0 0 0 0 0 0 0 0 0

2

0

0

1

0

0

94

63

41

15

9

1

are divided

into 20 erbitrary

size groups, each of

DNA

CYCLE

OF M YXOCOOCUS

data as a function

of cell size for

for 5% of generation---dividing Size groups? length (1.4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

1.88 to 2.48 to 2.68 to 2.80 to 2.88 to 3.04 to 3.12 to 3.24 to 3.36 to 3.48 to 3.60 to 3.72 to 3.80 to 3.92 to 4.08 to 4.20 to 4.36 to 4.62 to 4.76 to 5.12 to Total

2.48 2.68 2.80 2-88 3.04 3.12 3.24 3.36 3.48 3.60 3.72 3.80 3.92 4.08 4.20 4.32 4.52 4.76 5.08 7.00

M. xanthus

1

2

3

66 67 47 45 39 33 22 15 5 14 9 6 5 5 8 13 19 25 38 39 519

13 8 20 18 15 10 14 17 13 11 12 15 11 21 20 12 16 15 15 20 296

1 3 8 6 8 16 14 16 21 14 16 22 24 14 13 23 18 13 13 7 267

1 2 5 6 8 13 16 13 15 14 15 15 11 15 14 14 16 8 8 5 214

0 0 0 3 6 4 6 9 14 15 13 7 17 11 14 7 4 12 .5 7 154

t The 1600 bacteria scored in this experiment of which contains 80 cells.

pulse labeled

cells scored as two cells

Grains per cell (n) 4 5 6

0

613

TH US

3

TABLE

Autoradiographic

XAN

0 0 0 1 4 3 5 4 6 8 12 8 8 5 7 7 5 6 1 2 92

are divided

0 0 0 1 0 2 3 4 4 3 3 3 3 6 3 2 2 0 0 0 39

7

8

9

0 0 0 0 0 0 0 1 1 0 1 2 1 3 1 2 0 I 0 0 13

0 0 0 0 0 0 0 1 1 1 0 2 0 0 0 0 0 0 0 0 5

0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1

into 20 arbitrary

Mean 0.23 0.25 0.64 0.89 1.24 1.53 1.96 2.38 2.82 2.69 2.85 2.75 2.82 2.75 2.55 2.36 l-91 1.87 1.12 1.09 1.83

size groups, each

TABLE 4

data as a function of cell size for IK xanthus pulse labeled for 5% of generation4ividing celb scored as one cell

Autoradiographic

Size groupst length (4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 16. 16. 17. 18. 19. 20.

1.88 to 2.88 to 3.12 to 3.20 to 3.32 to 3.44 to 3.56 to 3.68 to 3.76 to 3.88 to 4.00 to 4.12 to 4.24 to 4.36 to 4.62 to 4.72 to 4.92 to 5.16 to 5.48 to 5.92 to Total

2.84 3.08 3.20 3-32 3.44 3.56 3.68 3.76 3.88 4.00 4.12 4.24 4.36 4.52 4.72 4.92 6.16 6.48 6.92 7.00

Grains per cell (n) 4 5 6

0

1

2

3

28 9 11 5 6 7 7 6 3 5 4 10 12 18 24 34 38 39 41 42 349

23 13 13 13 14 9 6 12 11 10 27 12 10 13 15 13 13 20 17 14 278

7 16 16 18 13 16 19 15 18 19 9 17 19 16 10 10 10 6 4 7 264

10 15 16 15 14 11 14 12 15 12 11 13 12 14 9 5 5 3 4 4 214

t The 1412 baoteria soared in this experiment of which contains 70 or 71 oella. 40

2 9 7 7 14 16 11 10 11 11 IO 12 6 4 7 7 3 2 3 3 155

0 7 4 6 3 9 11 9 8 5 5 4 8 4 5 1 2 1 1 1 94

are divided

1 1 4 4 4 2 2 3 3 5 4 3 1 2 0 0 0 0 0 0 39

7

8

9

0 0 0 1 1 0 1 1 2 3 1

0 0 0 1 1 1 0 2 0 0 0

0 0 0 0 1 0 0 0 0 0

0

0

0

2 0 1 0 0 0 0 0 13

0 0 0 0 0 0 0 0 5

0 0 0 0 0 0 0 0 1

into 20 arbitrary

0

Mean 1.14 2.39 2.33 2.73 2.83 2.87 2.87 2.91 2.93 2.92 2.46 2.41 2.23 1.87 1.72 1.16 0.98 0.76 0.77 0.80 2.06

size groups, eaoh

614

D.

ZUSMAN

AND

E. ROSENBERG

FIG. 1. Frequency distribution of cell size for M. zatathzla. ilI. zanthwr FBmp w8a grown in defined medium, pulse-lsbeled with [3H]thymidine, and prepared for 8utOr8diOgr8phy 88 described in Makwi81s and Methods. The bacteria were examined under 1876 times magnification with a Wild M20 microsoope equipped with 8 screw miorometer eyepiece. Cells were selected randomly for analysis: gr8in counts and length measurements. The frequency distributions of cell size for 811 the 2400 bacteria scored 8re shown. Dividing cells were scored as two smati cells (8) or 88 one large cell (b). The frequency distributions for dividing cells is also presented. -a-m-, Frequency distribution for all cells ; -O-O-, frequency distribution for dividing cells.

The frequency distribution of cell size for all the 2400 bacteris scared is presented in Figure 1 (closed circles). Length alone wss considered a measure of cell size inasmuch as cell diameter, O-89 CL,was never found to vary in any single population over the cell division cycle. This is in agreement with the findings of Schaechter, Williamson, Hood & Koch (1962) for Escherichia coli and Xalmonella typhimurium and with those of Marr, Harvey & Trentini (1966) for E. coli. The mean length observed in Figure 1(a) is 3.62 f O-84 p; in Figure 1(b) the mean length is 4-01 f @93 I”. These lengths correspond to volumes of 2.26 $ and 2.51 p3, respectively. The shape of these frequency distribution curves indicates that the cells chosen for analysis were, indeed, chosen at random and that the grain distributions observed in Tables 1 to 4 represent the entire population. Figure l(b) is similar in shape to that observed by Harvey, Marr & Painter (1967) for E. coli and Azotobacter agilis from electronic measurement of volumes of cells. The frequency distribution of dividing cells is also presented in Figure 1 (open circles). The mean length observed for “sister” cells (Fig. l(a)) is 2.75 f 0.36 p; the mean length of “mother” cells (Fig. l(b)) is 5.50 & O-65CL.These lengths correspond

DNA

CYCLE

OF MYXOCOCCUS

616

XANTHUS

to volumes of 1.72 p3 and 344 p3, respectively. The different shapes of the curves in Figure l(a) and (b) (closed circles) is due to the method of scoring dividing cells. Figure 2 presents graphically the mean number of grains per cell listed in Tables 3 and 4 as a function of cell size. These data were chosen for analysis because of the greater resolution offorded by the shorter pulse. The abscissa contains the 20 size groups as an exponential function (2O to 2’). This representation places the rate of DNA synthesis on a linear time and age scale. The data in Figure 2(a) show : (a) that for the first 8% of the division cycle the cells synthesize little DNA ; this is followed by (b) a linear increase in grains per cell, (c) a plateau region with a mean of 2.8 grains per cell, and (d) a linear decrease in the rate of DNA synthesis. Representing the dividing cells as one cell, yields a similar curve displaced in the cell cycle (Fig. 2(b)). This large displacement is due to the low rate of DNA synthesis observed for dividing cells. While the mean for all cells scored in Table 3 is 1.83 grains per cell, “sister” cells have a mean of only O-17. I , ,

,

I

I

I

I

I

30-

IIIIIII

I

. 2,5-

l+*-*l

z

“v 2-o-

.

2 .cs

l

I

‘1,

*

.

I

b6 1.5L-T i-o-

I

\ 0.

lD

i

I

*..-.*.-&.

\g

/ /

l

t

l

\

.

1 %-.-.

i

0.5~/ M 0 I 0

lll1II 2 4 6 I

0.2

8

IO I2 I

I 14 I

0.4

0x5 (a)

I 16

I I8 I

00

I IIIilII 20 0 2 4 6 Size groups I I I

I.0

0

0.2

8

IO 12

I

I 14

I

0.4

O-6

If 16

I8 I

08

I 20 I

I.0

Age groups (b)

FIQ. 2. Mean grsins per cell es 8 function of cell size groups. M. m&us FBmp ~8s grown in defined medium, pulse-lebeled for 20 min (6% of generation time), 8nd prepared for 8utomdiogr8phy 8s described in Materials 8nd Methods. Cells were selected randomly for 8n8lysis : gr8in counts and length measurements. The 1600 bacterie scored in this experiment 8re divided into 20 arbitrary size groups, eech of which contains an equal number of bacterie. The means for eech size group (Tebles 3 and 4) are plotted as a function of cell size groups. The 8bscies8 also contains the 20 size groups 88 an exponenti function. This representation places the rate of DNA synthesis on a linear time and age scale. Dividing cells were scored 8s two smell cells (8) or as one large cell (b). The data shown in Figure:2 present only the means of the different size groups. These data are, therefore, subject to at least two possible interpretations: (1) the means reflect the rate of DNA synthesis of the entire population of any given age group, i.e. cells synthesize DNA at different rates as a function of cell age, and (2) all cells that are synthesizing DNA are doing so at an identical rate, independent of cell age; observed changes in means are caused by corresponding changes in the fraction of cells that are not synthesizing any DNA. These two models are distinguished

616

D.

ZUSMAN

AND

E. ROSENBERG

by analysis of the distributions of grains observed in Tables 3 and 4. For a simple Poisson distribution, a straight line is obtained when the log [Pc,,a!] is plotted against n. As presented in Figure 3(b), the distribution of grains obtained at representative points on the plateau region (Fig. 2(a)) closely fit a straight line. This indicates that each cell incorporated the same amount of radioactivity during the pulse period. Therefore, the cells at the plateau region are all synthesizing DNA; the rate of synthesis is constant and independent of cell size. I

I

I

I

I

I

I

I

IOC

IC -. E: : 2

,.c

I

01

i. .LllLL-I 01234567 (a)

I

I

I

I

I

01234567

I

I

I

I

I

I

I

I

I

I

I

I

01234567 " (b)

Cc)

FIG. 3. Distribution of rates of DNA synthesis within size groups. The distribution of silver greins for each of the 20 size groups (Table 3) were analyzed. The frequency function, log [Pc,,-~!], is plotted as the ordinate. The abscissa indicstes the number of greins per cell. Points on the positive slope (a), pkh8U (b), and negative slope (c) from Fig. 2(e) are represented. The values for the zero class of eaoh distribution are corrected as described in the text (Results); the observed zero olaeses are enclosed in boxes. (8) (0) Size group 6; (0) size group 6; (A) size group 7; (X) size group 8. (b) (a) Size group 9; (0) size group 12; (A) size group 14. (c) ( l ) Size group 15; (0) size group 10; (A) size group 17, (X) size group 18.

Figure 3(a) and (c) present the distribution of grains obtained at representative points on the positive slope and negative slope of Figure 2(a). The uncorreoted distributions clearly do not fit the Poisson equation since the zero classes (enclosed in boxes) are much higher than would be predicted. However, if the zero classes are arbitrarily fixed at the level found at the plateau region and the new distributions plotted, straight lines, i.e. Poisson distributions, are obtained (Fig. 3(a) and (c)). The slopes of these lines are identical to that found on the plateau. This indicates that two classes of cells contribute to the means in these regions: (a) a zero class that is not synthesizing any DNA, and (b) cells that are synthesizing DNA at the same rate aa those at the plateau. Therefore, it follows that those cells that are synthesizing DNA are doing so at a constant rate, independent of cell size or age.

DNA CYCLE OF MYXOCOCCUS (b) Chromosome replication

01;

XASTHCS

during the division

cycle

Previously we have shown that M. xanthu.s in its dormant state, the microcyst, contains completed chromosomes (Rosenberg et al., 1967). The number of chromosomes per microcyst was determined by labeling exponentially growing cells with radioactive thymidine, microcysting the cells, and then germinating the microoysts in non-radioactive medium. The average number of grains per labeled cell decreased from an initial level of 32.4 to a limit of 4.9, indicating that the microcyst contained 6.6 conserved units, or three to four chromosomes per microcyst (Zusman & Rosenberg, 1968). Correlation of the DNA content and chromosome number yielded a molecular weight for the non-replicating chromosome, 4.9 x log daltons. Chemical analysis of the DNA content of vegetative cells of M. xanthu.s growing in the defined medium was performed by extracting DNA from a known number of bacteria with a modified Schmidt-Thannhauser procedure, and then determining the amount of DNA by the Dische reaction. The results yielded 14.0 f 1.1 x 10Wgpg DNA per cell. Since the non-replicating chromosome of M. xanthus weighs 8.2 x lo-” pg (Zusman & Rosenberg, 1968) cells growing on the defined medium contain an average of l-7 & O-2 chromosomes per cell. The predicted value for an exponential population in which 80% of the cells contain one replicating chromosome and the remaining 20% contain two completed chromosomes, is 1.53 chromosomes per cell. Therefore, it follows that the rates of DNA synthesis reported here correspond to that of one replicating chromosome per cell. In contrast, M. xanthus growing in a complex medium contains two to four chromosomes per cell: the two chromosomes appear to be replicated sequentially during 8Oo/o of the division cycle (Rosenberg et al., 1967). In the complex medium, cells contain 22.4 x 10eg pg DNA per cell, corresponding to 2.73 chromosomes per cell (Zusman & Rosenberg, 1968).

4. Discussion (a) Rate of DNA

synthesis

Data have been presented (Tables 3 and 4; Fig. 2) which relate the rate of DNA synthesis to cell size. These data clearly demonstrate that all cells which are synthesizing DNA are doing so at a constant rate, independent of cell size or age. The variations in observed mean grain counts per cell are due to the presence of cells that are not synthesizing any DNA (Fig. 3). Since the single chromosome of ill. xanthus (mol. wt. 4.9 x log) was completed in 312 minutes (80% of the doubling time), the rate of DNA synthesis at 30°C in the defined medium is 2.4~ lo4 base pairs per minute. The assumption of a constant rate of DNA synthesis per replication point (Maalee & Kjeldgaard, 1966) is supported by autoradiographic data of individual replication points in E. coli (Cairns, 1963) and the increased rate of DNA synthesis observed with the appearance of multiple replication points (Clark & Maalee, 1967 ; Helmstetter, 1967). This assumption is basic to an understanding of the DNA cycle and its relation to the division cycle. The assumption of a constant rate of DNA synthesis per replication point can be considered in two parts : Constant rate during the division cycle and constant rate in different media. It should be emphasized that the present study only provides evidence for a constant rate of DNA synthesis during the division cycle.

618

D. ZUSMAN

(b) Relationship

AND

of DNA

E. ROSENBERG

cycle to the division

cycle

The existence of a DNA cycle in M. xatihus growing in a defined medium is indicated in Figure 2. Since the standard deviation for length measurements was 0.13 p, the error in placing individual cells within a particular size group was one to two size groups; the observed slopes were more gradual than this. Therefore, the incline of the slopes are it measure of the broad range of sizes in which DNA synthesis is initiated and terminated. These data do not support any model which predicts that DNA synthesis is initiated at a particular cell mass or mass per DNA ratio. The position of the DNA cycle within the division cycle (Fig. 4) was established by analysis of the zero classes of the distributions listed in Tables 3 and 4. Since

Ia)obiz

090 IO DNA

DNA synthesis starts

(b) :m DNA starts

DNA

synthesis

Physical (cl

DNA

Septum formotlo”

synthesis 0.81 stops

syntheses stops

Septum formatan

IO Physical separation

syntheses stops

,seporation 0 0 02 -

DNA

synthesis

starts

O90&

DNA synthesis ot constant rate

FIQ. 4. DNA cycle es a function of the cell division cyole. The position of the DNA cycle within the division cycle is illustrated when dividing aella are scored as two smell cells (a) or ae one large cell (b). Combining these Figures (c) yields the DNA and physical separation of sister cells. cycle as a function of both septum fOITII8tiOn

the rate of DNA synthesis for those cells engaged in DNA synthesis is con&ad, the fractions of cells containing anomalous zero grains (those in excess to that prescribed for Poisson distributions) reflect the fractions of cells not engaged in DNA synthesis. In Table 3, for example, the anomalous zero class in size groups one to eight is 293 out of the 1600 cells scored. Therefore, the fraction of cells which have not initiated DNA synthesis is 0.183. For an exponential population, this fraction of cells corresponds to an age of 0.12 generation. In a similar manner, the fraction of anomalous zero cells in size groups 15 to 20 can be used to calculate the average age (0.90 generation) of chromosome completion. Combining the data for dividing cells scored as two small cells (Fig. 4(a)) or as one large cell (Fig. 4(b)), yields the DNA cycle as a function of both septum formation and physical separation of sister cells (Fig. 4(c)). DNA synthesis extends from 0.02 to 0.81 generation, septum formation occurs at O-90, and physical separation of sister cells at I.0 generation. The experimental procedure used yields detailed information on the DNA cycle not generally available by other methods. The direct microscopic observations relate the rate of DNA synthesis to physiological division as indicated by septum formation.

DNA

CYCLE

OF MYXOCOCCUS

XANTHUS

G19

The method yields direct values for the time for a round of chromosome replication, and the time between the end of a round of replication and the following cell division. It is not dependent on cell synchrony and can be used with bacilli that normally form chains. We are indebted to Dr F. Eiserling for criticism of the manuscript and to Dr P. Gottlieb for assistance in relating size groups to age groups. This investigation was supported by National Science Foundation grant GB-8708. One of us (D.Z.) is a recipient of a National Institutes of Health predoctoral traineeship (5-TOI-GM 01297-05). REFERENCES Bacon, K. & Rosenberg, E. (1967). J. Bad. 94, 1883. Cairns, J. (1963). J. Mol. Biol. 6, 208. Caro, L. G., van Tubergen, R. P. & Kolb, J. A. (1962). J. Cell BioZ. 15, 173. Clark, D. J. & Maalee, 0. (1967). J. Mol. BioZ. 23, 99. Cooper, S. & Helmstetter, C. (1968). J. Mol. BioZ. 31, 519. Dische, Z. (1955). In The NucZeic Acids, ed. by E. Chargaff & J. N. Davidson, vol. 1, p. 287. New York: Academic Press. Dworkin, M. & Voles, E. (1962). J. f&n. Microbial. 28, 81. Harvey, R., Marr, A. & Painter, P. (1967). J. Bact. 93, 606. Helm&et&r, C. E. (1967). J. Mol. BioZ. 24, 417. Helmstetter, C. E. (1969a). In The Cell Cycle, ed. by G. M. Padilla, G. L. Whitson, & I. L. Cameron, p. 15. New York: Academic Press. Helm&et&, C. E. (19696). Ann. Rev. Microbial. 23, 223. Helmstetter, C. E., Cooper, S., Pierucci, 0. & Revelas, E. (1968). Cold Spr. Harb. Symp. &ant. BioZ. 33, 809. Lark, K. G. (1969). Anrt. Rev. Biochem. 38, 569. Leslie, I. (1955). In The Nucleic Am&, ed. by E. Chargaff & J. N. Davidson, vol. 2, p. 2. New York: Academic Press. Maalee, 0. & Kjeldgaard, N. 0. (1966). Control of MacromoZecular Synthesis. New York: W. A. Benjamin. Marr, A., Harvey, R. & Trentini, W. (1966). J. Ract. 91, 2388. Rosenberg, E., Katarski, M. & Gottlieb, P. (1967). J. Bact. 93, 1402. Schaechter, M., Williamson, J., Hood, J. R. & Koch, A. L. (1962). J. Geen. Microbial. 29, 421. Zusman, D. R. & Rosenberg, E. (1968). J. Bact. 96, 981.