JOURNAL
OF
Vol. 130, No. 1 Printed in U.S.A.
BACTriuOWOGY, Apr. 1977, p. 118-127
Copyright C) 1977 American Society for Microbiology
Medium-Dependent Variation of Deoxyribonucleic Acid Segregation in Escherichia coli STEPHEN COOPER* AND MARTIN WEINBERGER' Department of Microbiology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109
Received for publication 4 November 1976
The degree to which deoxyribonucleic acid segregates nonrandomly has been investigated for Escherichia coli B/r growing in different media. The degree of nonrandom segregation observed is dependent on the medium, with segregation becoming less random as the growth rate decreases. This indicates that there must be some varying probabilistic component to the segregation process. A probabilisitic modification of the Pierucci-Zuchowski model is proposed as well as a probabilistic model, in which it is proposed that deoxyribonucleic acid strands segregate, with a probability greater than 0.5, in the same direction (toward the same pole) as at the previous cell division.
It is not yet known how a dividing procaryotic cell apportions its deoxyribonucleic acid (DNA) into two daughter cells. It is possible that an understanding of this process may be gained by seeing whether the segregation of DNA is random or nonrandom. Segregation may be considered random if the direction in which a DNA strand segregates in one division bears no relationship to the direction in which it segregated at the previous cell division. Segregation is nonrandom if such a relationship exists. Pierucci and Zuchowski (7) studied the segregation of DNA in Escherichia coli B/r and concluded that segregation was nonrandom. They grew thymidine-labeled cells in a viscous growth medium so that the progeny cells from a number of divisions were preserved in a chain that reflected the order of cell division. After autoradiography to determine which cells were labeled, they compared their results with the predictions of a number of deterministic models for nonrandom segregation. They concluded that one strand (-) segregates randomly and the complementary DNA strand (+) segregates nonrandomly as though the strand permanently associates with one pole of the bacterial cell, associating or attaching as soon as it has first been used as a template. We have performed a number of experiments that indicate that the degree of nonrandom segregation observed is dependent on the medium, with segregation being more nonrandom as the growth rate decreases. This observation shows that there must be some probabilistic component in the segregation process, and so we have
reanalyzed the data of Pierucci and Zuchowski (7) and our own data to determine what kinds of probabilisitic models may explain the data. One of these is a modification of the Pierucci and Zuchowski model, and the other is a model that does not postulate any differences in segregation behavior between the different strands of DNA.
MATERIALS AND METHODS E. coli B/rA (2) was grown in MOPS medium (5) containing glycerol, glucose, or glucose and Casamino Acids, all at final concentrations of 0.2%. Bacteria growing exponentially at 37°C were labeled with tritiated thymidine (55 Ci/mmol; 40 ,Ci/ml) for approximately 10% of a doubling time, centrifuged, washed, and suspended in the same medium without thymidine. Tritiated thymidine was obtained from the General Dynamics Corp., stored in a freezer, and generally used within 1 month to minimize any destruction of the thymidine by decay processes. Growth was then continued for two, three, and four doubling times (more in the case of rapidly growing cells); at each time cells were removed, centrifuged, and concentrated to an optical density at 450 nm of 10.0 to 20.0, and 4 to 7 drops of the cells were added to a vial (approximately 2 ml) of Methocel medium. Methocel medium was prepared by adding Methocel (hydroxypropylmethylcellulose, 90HG standard; 400 cP/s) to warm MOPS medium (with all additions) to a final concentration of 3.5%. After 2 h of stirring with a magnetic stirrer, the medium was heated for 20 min in an 800C water bath, stirred in the cold room overnight, and then distributed to small capped vials. The vials with the Methocel medium were sterilized at 80°C for 20 min. Methocel was a gift of the Dow Chemical Corp., Midland, Mich. The cells were stirred into the Methocel with a small stirring rod, and then the mixture was 1 Present address: Department of Experimental Biology, placed on a number of labeled glass slides. The Roswell Park Memorial Institute, Buffalo, NY 14203. Methocel medium with the cells was then spread 118
VOL. 130, 1977 into a thin film with an aluminum foil-covered slide, as described by Lin et al. (4), and the slide was inverted over a concave depression slide, which was coated with sterile Methocel medium. The depression slide seals off an area of the thin Methocel film and prevents the drying of the Methocel. The slides were incubated at 370C for various periods of time to form chains of 4, 8, or 16 cells. A number of slides were taken at each time to follow the growth of the cells on the slides. The cells are observed without staining by using phase-contrastI microscopy. Growth on the slides was stopped by opening up the slides and exposing the thin film of Methocel to the air for drying. After the Methocel film was dry (2 to 3 days), it was made insoluble by treatment with 10% p-toluenesulfonylchloride in pyridine (4). After another 1 to 3 days of drying, the slides were coated with Kodak LTB-2 liquid emulsion (diluted with an equal volume of distilled water) and stored in slide boxes in the cold with Drierite. After 4 days to a week, the slides were developed with D-19 developer (Eastman Kodak, Rochester, N.Y.) for 2.0 min, dipped in 2% acetic acid for 10 s, and fixed for 5 min in Fixol. The developed slides were rinsed for 30 min in running tap water and then dipped, for 2 min, in a solution of methylene blue (200 mg/liter) to stain the cells. The chains and the labeled cells were counted using a Leitz microscope. Counting was performed by scanning fields, counting all chains that had 4, 8, or 16 cells with only one labeled cell, and noting the position of the labeled cell. Different individuals counting the same slide arrived at essentially the same results. Figure 1A is a representative sample of some of the cell chains with labeled cells.
RESULTS Experimental rationale. As will be seen in more detail below, the Pierucci-Zuchowski model predicts that if one subsequently grows thymidine-pulse-labeled cells for a number of divisions in non-radioactive medium, a steadystate situation will be produced in which all cells with label will contain only one labeled strand, with half containing a + (nonrandom) strand and half a - (random) strand. The nonrandom (+) strand segregates as though bound permanently to one pole of the cell. If four cell chains are then made in viscous medium from these cells, then it will be expected according to the Pierucci-Zuchowski model (see Discussion) that the ratio of chains labeled in a pole position (outer position) to a chain labeled in a nonpole position (inner cells of four cell chains) will be 3.0. Random segregation would yield a ratio of 1.0. This experimental approach (Fig. 1B) allows one to look at a simpler indicator of segregation and thus to examine a number of different conditions with regard to the degree of nonrandom segregation. In addition, this approach allows the analysis of the segregation pattern from cells containing chromosome con-
DNA SEGREGATION IN E. COLI
119
figurations that would otherwise be too complex to analyze by the formation of chains immediately after labeling. It will be assumed that such a steady state is achieved after extensive growth, and the experimental evidence (see below) supports this assumption. Analysis of DNA segregation in different media. Cells growing in an exponential culture in different media were labeled for approximately 10% of a generation with tritiated thymidine, centrifuged and washed, and suspended in fresh growth medium. The labeled cells were then allowed to grow for two to five generations in unlabeled medium to allow the segregation of the labeled strands. The cells, now labeled in only one strand, along with a varying number of unlabeled cells, were concentrated by centrifugation, mixed with Methocel medium, spread on glass slides, and incubated to form chains of 4, 8, or 16 cell lengths. The position of radioactive cells in these chains was determined by autoradiography. The general experimental procedure is illustrated in Fig. 1B, typical fields of labeled chains are shown in Fig. 1A, and the description and results of a number of experiments are presented in Table 1. The data are presented for three different steady-state cultures (the same medium for growth, labeling, segregation, and chain formation) and for experiments in which changes of medium were involved. These medium shifts were performed immediately after labeling so that segregation and chain formation occurred in the second medium. In each experiment there were a number of different generations of segregation allowed prior to spreading the cells on slides for chain fornation. There was no significant difference between the different generations. For example, the results after three generations of segregation in glucose were the same as those after four generations of segregation, and therefore the results were pooled and not treated independently. Only slides with a large fraction of unlabeled chains were counted. The results for a number of independent experiments are presented in Table 1. The results of any experiment can be summarized in terms of an "R" value. The R value is the ratio of the number of chains labeled in one position(s) to the number labeled in another position(s). A subscript indicates the positions comprising the ratio, such that R112 means that the ratio is calculated as the number of chains labeled in position 1 divided by the number of chains labeled in position 2. (The chain numbering system of Pierucci and Zuchowski [7] was followed. The numbering of chains starts from a pole
120
J. BACTERIOL.
COOPER AND WEINBERGER w
A. Dv
B ..
Ar
,. . . . . JIIYJL AND -, .'-
A
G R4;
9'
iTIIIZLKJEJZETT 7iTZiF
FIG. 1. (A) Typical field observed after growth of cells in Methocel and autoradiography. (B) Outline of the basic segregation experiment. Four parts of the basic experiment are outlined. First, cells are grown exponentially in batch culture, and then they are labeled for a short period of time. Assuming that the cell to be labeled had one replicating chromosome, this leaves two labeled strands in the cell. The labeling period is followed by a period of growth in batch culture to allow segregation of the labeled strands. Three generations are illustrated here, resulting in eight cells, two of which contain labeled strands. These cells are then spread on a slide in Methocel medium, and chains are allowed to form. It would be expected from this experiment that one-quarter of the chains would have label. The chains illustrated are labeled in position 2 of the 4-cell chain and in position 7 (not 10) of the 16-cell chain. The 8-cell chain has no observable autoradiographic grains.
nearest a radioactive cell. Therefore, in a fourcell chain with one labeled cell, only positions 1 and 2 have to be considered, as these positions are equivalent to positions 4 and 3, respectively.) A superscript is placed above the R value to indicate the number of cells in a chain that give the ratio, so that RI,3 4 is the ratio of the sum of cells labeled in positions 1 and 2 divided by the number of labeled cells in positions 3 and 4 from chains that contain eight
cells. For a given sample of labeled cells one would expect that R12/,3,4 is the same as R1,2, because cells 1 and 2 in the eight-cell chain must have been derived from cell 1 in the fourcell chain, and cells 3 and 4 similarly, must have come from cell 2 of the four-cell chain. This ability to "condense" data from longer chains into equilavence with shorter chains allows the pooling of data, and therefore a superscript P (as in R'2) indicates that pooled data
DNA SEGREGATION IN E. COLI
VOL. 130, 1977
&o
,
OF
Vol. 130, No. 1 Printed in U.S.A.
BACTriuOWOGY, Apr. 1977, p. 118-127
Copyright C) 1977 American Society for Microbiology
Medium-Dependent Variation of Deoxyribonucleic Acid Segregation in Escherichia coli STEPHEN COOPER* AND MARTIN WEINBERGER' Department of Microbiology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109
Received for publication 4 November 1976
The degree to which deoxyribonucleic acid segregates nonrandomly has been investigated for Escherichia coli B/r growing in different media. The degree of nonrandom segregation observed is dependent on the medium, with segregation becoming less random as the growth rate decreases. This indicates that there must be some varying probabilistic component to the segregation process. A probabilisitic modification of the Pierucci-Zuchowski model is proposed as well as a probabilistic model, in which it is proposed that deoxyribonucleic acid strands segregate, with a probability greater than 0.5, in the same direction (toward the same pole) as at the previous cell division.
It is not yet known how a dividing procaryotic cell apportions its deoxyribonucleic acid (DNA) into two daughter cells. It is possible that an understanding of this process may be gained by seeing whether the segregation of DNA is random or nonrandom. Segregation may be considered random if the direction in which a DNA strand segregates in one division bears no relationship to the direction in which it segregated at the previous cell division. Segregation is nonrandom if such a relationship exists. Pierucci and Zuchowski (7) studied the segregation of DNA in Escherichia coli B/r and concluded that segregation was nonrandom. They grew thymidine-labeled cells in a viscous growth medium so that the progeny cells from a number of divisions were preserved in a chain that reflected the order of cell division. After autoradiography to determine which cells were labeled, they compared their results with the predictions of a number of deterministic models for nonrandom segregation. They concluded that one strand (-) segregates randomly and the complementary DNA strand (+) segregates nonrandomly as though the strand permanently associates with one pole of the bacterial cell, associating or attaching as soon as it has first been used as a template. We have performed a number of experiments that indicate that the degree of nonrandom segregation observed is dependent on the medium, with segregation being more nonrandom as the growth rate decreases. This observation shows that there must be some probabilistic component in the segregation process, and so we have
reanalyzed the data of Pierucci and Zuchowski (7) and our own data to determine what kinds of probabilisitic models may explain the data. One of these is a modification of the Pierucci and Zuchowski model, and the other is a model that does not postulate any differences in segregation behavior between the different strands of DNA.
MATERIALS AND METHODS E. coli B/rA (2) was grown in MOPS medium (5) containing glycerol, glucose, or glucose and Casamino Acids, all at final concentrations of 0.2%. Bacteria growing exponentially at 37°C were labeled with tritiated thymidine (55 Ci/mmol; 40 ,Ci/ml) for approximately 10% of a doubling time, centrifuged, washed, and suspended in the same medium without thymidine. Tritiated thymidine was obtained from the General Dynamics Corp., stored in a freezer, and generally used within 1 month to minimize any destruction of the thymidine by decay processes. Growth was then continued for two, three, and four doubling times (more in the case of rapidly growing cells); at each time cells were removed, centrifuged, and concentrated to an optical density at 450 nm of 10.0 to 20.0, and 4 to 7 drops of the cells were added to a vial (approximately 2 ml) of Methocel medium. Methocel medium was prepared by adding Methocel (hydroxypropylmethylcellulose, 90HG standard; 400 cP/s) to warm MOPS medium (with all additions) to a final concentration of 3.5%. After 2 h of stirring with a magnetic stirrer, the medium was heated for 20 min in an 800C water bath, stirred in the cold room overnight, and then distributed to small capped vials. The vials with the Methocel medium were sterilized at 80°C for 20 min. Methocel was a gift of the Dow Chemical Corp., Midland, Mich. The cells were stirred into the Methocel with a small stirring rod, and then the mixture was 1 Present address: Department of Experimental Biology, placed on a number of labeled glass slides. The Roswell Park Memorial Institute, Buffalo, NY 14203. Methocel medium with the cells was then spread 118
VOL. 130, 1977 into a thin film with an aluminum foil-covered slide, as described by Lin et al. (4), and the slide was inverted over a concave depression slide, which was coated with sterile Methocel medium. The depression slide seals off an area of the thin Methocel film and prevents the drying of the Methocel. The slides were incubated at 370C for various periods of time to form chains of 4, 8, or 16 cells. A number of slides were taken at each time to follow the growth of the cells on the slides. The cells are observed without staining by using phase-contrastI microscopy. Growth on the slides was stopped by opening up the slides and exposing the thin film of Methocel to the air for drying. After the Methocel film was dry (2 to 3 days), it was made insoluble by treatment with 10% p-toluenesulfonylchloride in pyridine (4). After another 1 to 3 days of drying, the slides were coated with Kodak LTB-2 liquid emulsion (diluted with an equal volume of distilled water) and stored in slide boxes in the cold with Drierite. After 4 days to a week, the slides were developed with D-19 developer (Eastman Kodak, Rochester, N.Y.) for 2.0 min, dipped in 2% acetic acid for 10 s, and fixed for 5 min in Fixol. The developed slides were rinsed for 30 min in running tap water and then dipped, for 2 min, in a solution of methylene blue (200 mg/liter) to stain the cells. The chains and the labeled cells were counted using a Leitz microscope. Counting was performed by scanning fields, counting all chains that had 4, 8, or 16 cells with only one labeled cell, and noting the position of the labeled cell. Different individuals counting the same slide arrived at essentially the same results. Figure 1A is a representative sample of some of the cell chains with labeled cells.
RESULTS Experimental rationale. As will be seen in more detail below, the Pierucci-Zuchowski model predicts that if one subsequently grows thymidine-pulse-labeled cells for a number of divisions in non-radioactive medium, a steadystate situation will be produced in which all cells with label will contain only one labeled strand, with half containing a + (nonrandom) strand and half a - (random) strand. The nonrandom (+) strand segregates as though bound permanently to one pole of the cell. If four cell chains are then made in viscous medium from these cells, then it will be expected according to the Pierucci-Zuchowski model (see Discussion) that the ratio of chains labeled in a pole position (outer position) to a chain labeled in a nonpole position (inner cells of four cell chains) will be 3.0. Random segregation would yield a ratio of 1.0. This experimental approach (Fig. 1B) allows one to look at a simpler indicator of segregation and thus to examine a number of different conditions with regard to the degree of nonrandom segregation. In addition, this approach allows the analysis of the segregation pattern from cells containing chromosome con-
DNA SEGREGATION IN E. COLI
119
figurations that would otherwise be too complex to analyze by the formation of chains immediately after labeling. It will be assumed that such a steady state is achieved after extensive growth, and the experimental evidence (see below) supports this assumption. Analysis of DNA segregation in different media. Cells growing in an exponential culture in different media were labeled for approximately 10% of a generation with tritiated thymidine, centrifuged and washed, and suspended in fresh growth medium. The labeled cells were then allowed to grow for two to five generations in unlabeled medium to allow the segregation of the labeled strands. The cells, now labeled in only one strand, along with a varying number of unlabeled cells, were concentrated by centrifugation, mixed with Methocel medium, spread on glass slides, and incubated to form chains of 4, 8, or 16 cell lengths. The position of radioactive cells in these chains was determined by autoradiography. The general experimental procedure is illustrated in Fig. 1B, typical fields of labeled chains are shown in Fig. 1A, and the description and results of a number of experiments are presented in Table 1. The data are presented for three different steady-state cultures (the same medium for growth, labeling, segregation, and chain formation) and for experiments in which changes of medium were involved. These medium shifts were performed immediately after labeling so that segregation and chain formation occurred in the second medium. In each experiment there were a number of different generations of segregation allowed prior to spreading the cells on slides for chain fornation. There was no significant difference between the different generations. For example, the results after three generations of segregation in glucose were the same as those after four generations of segregation, and therefore the results were pooled and not treated independently. Only slides with a large fraction of unlabeled chains were counted. The results for a number of independent experiments are presented in Table 1. The results of any experiment can be summarized in terms of an "R" value. The R value is the ratio of the number of chains labeled in one position(s) to the number labeled in another position(s). A subscript indicates the positions comprising the ratio, such that R112 means that the ratio is calculated as the number of chains labeled in position 1 divided by the number of chains labeled in position 2. (The chain numbering system of Pierucci and Zuchowski [7] was followed. The numbering of chains starts from a pole
120
J. BACTERIOL.
COOPER AND WEINBERGER w
A. Dv
B ..
Ar
,. . . . . JIIYJL AND -, .'-
A
G R4;
9'
iTIIIZLKJEJZETT 7iTZiF
FIG. 1. (A) Typical field observed after growth of cells in Methocel and autoradiography. (B) Outline of the basic segregation experiment. Four parts of the basic experiment are outlined. First, cells are grown exponentially in batch culture, and then they are labeled for a short period of time. Assuming that the cell to be labeled had one replicating chromosome, this leaves two labeled strands in the cell. The labeling period is followed by a period of growth in batch culture to allow segregation of the labeled strands. Three generations are illustrated here, resulting in eight cells, two of which contain labeled strands. These cells are then spread on a slide in Methocel medium, and chains are allowed to form. It would be expected from this experiment that one-quarter of the chains would have label. The chains illustrated are labeled in position 2 of the 4-cell chain and in position 7 (not 10) of the 16-cell chain. The 8-cell chain has no observable autoradiographic grains.
nearest a radioactive cell. Therefore, in a fourcell chain with one labeled cell, only positions 1 and 2 have to be considered, as these positions are equivalent to positions 4 and 3, respectively.) A superscript is placed above the R value to indicate the number of cells in a chain that give the ratio, so that RI,3 4 is the ratio of the sum of cells labeled in positions 1 and 2 divided by the number of labeled cells in positions 3 and 4 from chains that contain eight
cells. For a given sample of labeled cells one would expect that R12/,3,4 is the same as R1,2, because cells 1 and 2 in the eight-cell chain must have been derived from cell 1 in the fourcell chain, and cells 3 and 4 similarly, must have come from cell 2 of the four-cell chain. This ability to "condense" data from longer chains into equilavence with shorter chains allows the pooling of data, and therefore a superscript P (as in R'2) indicates that pooled data
DNA SEGREGATION IN E. COLI
VOL. 130, 1977
&o
,
