JOURNAL OF BACTERIOLOGY, Oct. 1979, p. 65-72 0021-9193/79/10-0065/08$02.00/0
Vol. 140, No. 1
Macromolecular Synthesis in Synchronized Cultures of Anacystis nidulans YUKIO ASATO Department of Biology, Southeastern Massachusetts University, North Dartmouth, Massachusetts 02747 Received for publication 18 July 1979
Synchronous culture of Anacystis nidulans has been induced by the lightdark-light regimen. At various time intervals during synchronous growth, samples were pulsed with radioactive labels to determine phospholipid, protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) syntheses within the cell division cycle. A temporal order of protein, RNA, and DNA syntheses occurred within the cell division cycle, whereas phospholipid was characteristically synthesized during midcycle (during cell enlargement) and during the time of cell division. Chemically determined protein, RNA, and DNA syntheses were found to support the schedule of these macromolecules in cultures growing at an 8-h doubling time. The study of macromolecular synthesis in the unicellular cyanobacterium Anacystis nidulans should simplify to a great extent our attempts in determining the major molecular events that occur within the cell division cycle of these organisms. Previous reports (2, 7, 12) have clearly established that the DNA synthesis occurred in the late stages of the cell cycle of Anacystis. An initiator protein has been implicated in the initiation of DNA synthesis in Anacystis (11). On the other hand, bulk RNA synthesis was initiated about the time when cells divided and terminated just before the subsequent cell division period (12). Other macromolecules could very well be synthesized at specific times within the cell cycle of Anacystis, although very few details are available (23) or known. To describe further details of the macromolecular synthesis patterns of A. nidulans, the synthesis of DNA, RNA, protein, and phospholipid was investigated by applying pulse-labeling techniques to a dark-induced synchronous culture. This report presents the results of the experiments, which show that increased rates of bulk protein, RNA, DNA, and phospholipid syntheses occurred at specific time frames within the cell division cycle of A. nidulans.
10. The flasks were placed on a shaker (shaking rate, 280 rpm) and incubated in an environmental chamber (Conviron, model PGW 36; Controlled Environments, Inc., Pembina, N.Dak.). A bank of four cool white fluorescent bulbs (Sylvania, F96 Tll CW) provided light intensity of 200 footcandles (ca. 2.1 klx). To induce cell synchrony, exponential cultures were placed in the dark for 12 to 24 h. When these cultures were exposed to light, synchronized growths occurred
(2). Total cell count. Cells were counted on a PetroffHausser bacterial cell counter. Pulse-labeling of macromolecules. In labeling nucleic acids and phospholipid, 5-ml samples of synchronized cultures were distributed into 50-ml flasks.
H332P04 (Schwarz/Mann, Orangeburg, N.Y.) was added to a final concentration of 0.1 Ci/ml per flask containing HEPES (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid)-buffered Dm medium (2) at selected time intervals for 30 or 60 min. The methods of Roodyn and Mandel (20) were followed to determine 32p incorporation in DNA, RNA, and phospholipid fractions. To determine DNA synthesis, a set of duplicate 0.5-ml samples was placed in test tubes. To the samples 0.5-ml amounts of 1.1 N NaOH were added, and the mixtures were incubated in a 37°C water bath for 2 h. After this incubation period, the samples were placed in ice for 15 min. Volumes of 0.1 ml of 6 N HCI were added, and the mixtures were chilled for 15 min. The DNA fractions were precipitated with cold perchloric acid (PCA) (final concentration, 0.5 N), collected, and washed with 0.5 N PCA on membrane filters. In the determination of RNA synthesis, a second set of duplicate 0.5-ml samples was treated with 2 volumes of 95% ethyl alcohol. The alcohol-treated samples were incubated in a 70°C water bath for 30 min and then placed in an ice bath. The chilled samples were filtered on membrane filters and washed with cold 0.5 N PCA. This fraction contained both DNA and RNA. The radioactivity from
MATERIALS AND METHODS Organisms and growth media. A. nidulans IU625 was originally obtained from the Algal Culture Collection, Indiana University (Bloomington, Ind.). Dm broth and agar were described by Van Baalen
(22). Growth condition and synchronization of culture. Cultures of A. nidulans were grown in Erlenmeyer flasks with a medium-flask ratio (vol/vol) of 1: 65
66
ASATO
J. BACTERIOL.
the alcohol-treated sample was subtracted from that from the alkali-treated sample to yield the RNA fraction. A third set of duplicate 0.5-ml samples was treated with cold 0.5 N (final concentration) PCA. I'he samples were collected on membrane filters and washed with cold 0.5 N PCA. These samples contained labeled phospholipid, RNA, and DNA. The difference in radioactivity between the cold I'CA fraction and the alcohol-treated sample gave the phospholipid value. RNA was alternatively pulse-labeled with [rH]uracil (specific activity, 25 mCi/mmol; New England Nuclear Corp., Boston, Mass.) at a final concentration of 1 to 10 [iCi/ml. Protein was pulse-labeled with [14C]valine (specific activity, 30 mCi/mmol; New England Nuclear) for 30 to 60 min at a final concentration of 0.1 ,iCi/ml. All labeled samples collected on membrane filters were air dried and placed in a toluene-based scintillation fluid. The radioactivity was measured with a Packard scintillation counter. Colorimetric determinations of DNA, RNA, and protein. DNA was determined by the indole method (13, 21). RNA was analyzed by the orcinol method (3). The standard used for DNA determination was fish roe DNA (Calbiochem, La Jolla, Calif.), and for RNA determination yeast RNA (Calbiochem) was the standard. The method of Lowry et al. (18) was used to determine the protein content. Bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) served as the standard.
RESULTS Macromolecular synthesis in synchronized culture of A. nidulans growing at 8-h doubling time. When exponential cultures are incubated in the dark for 12 to 24 h and subsequently exposed to light, synchronously dividing cultures are obtained (2). A typical synchronized culture (Fig. 1) at an incubation temperature of 32°C demonstrated abrupt increases in cell number at h 4 and h 12. Interdivision time, i.e., the time between the long vertical lines drawn through the midpoints of increases in cell number during the first and second division periods, was 8 h. Cell division periods, taking approximately 50%/c of the cell cycle, indicated a fairly broad distribution of interdivision time. Nevertheless, partial synchronous growth patterns have been maintained through two successive cell division periods without severe deterioration in the slope of the second cell division period. In any event, synchronized cultures have been shown to be experimentally useful. For example, temporal genetic maps of Anacystis were constructed from synchronized culture (2, 7, 12). The construction of temporal genetic maps is based on the premises that genome replication in synchronized cultures occurs in a specific, ordered manner and that the induced mutation frequency of a specific gene increases when that gene replicates (2).
Another possible use of synchronized culture of Anacystis is to determine whether an ordering of macromolecular synthesis occurs during the synchronous cell cycle. To test this possibility, determinations of DNA, RNA, protein, and phospholipid syntheses were made from synchronized cultures. The results are shown in Fig. 1 and 2. In the pulse-labeling experiments, macromolecular synthesis curves exhibited characteristic peaks. Such peaked curves indicate that there is a wide distribution of initiation times of respective macromolecular syntheses. After the peaks, the amounts of radioactive labels incorporated in DNA (Fig. 1) and RNA (Fig. 2) decreased to lower levels than expected. On the other hand, the amounts of ['4C]valine incorporated in protein decreased to expected levels after attaining the peak incorporation rate. Balanced growth apparently was not obtained in the synchronized cultures from which DNA and IRNA syntheses were determined. Although there are a number of factors that may cause unbalanced growth, changes in cell size as cell densities increase could be a significant one. tUpon microscopic observations, cell lengths were found to decrease as the cell densities increased beyond approximately 5 x 10' cells per ml (probably resulting from a shading effect). For example, the cell densities of a typical synchronized culture were 4 x 10' cells per ml at h 8 and 8 x 10' cells per ml at h 16. The average cell lengths at h 8 and h 16 were 6.5 and 5.4 ftm, respectively. DNA (Fig. 1) and RNA (Fig. 2) syntheses were determined for synchronized cultures growing at cell densities of about 8 x 10 cells per ml, whereas protein synthesis (Fig. 2) was determined at cell densities of 4 x 10' cells per ml. A more detailed study must be made to prove the possible correspondence of synthesis rates and cell size changes as observed in the present study. Nevertheless, active syntheses of DNA, RNA, protein, and phospholipid could be shown to occur at different times in the cell cycle. Vertical lines in Fig. 1 mark the midpoints of increase and decrease in the DNA synthesis rate curve. This period bounded by the midpoints signifies the time frame of DNA synthesis. The 32p incorporation during the time devoid of DNA synthesis in synchronized cells was most likely attributable to a fraction of unsynchronized cells. In the chemically determined DNA synthesis curve, a plateau preceded the period of increase, which in turn was followed by a plateau. The plateau periods indicated the periods devoid of DNA synthesis in synchronized cells. There was a doubling of DNA content at the end of the synthesis period. Exact correspondence in the time frames of chemically de-
MACROMOLECULAR SYNTHESIS IN ANACYSTIS
VOL. 140, 1979
10 8 E 6 a 0)
o
4
67
10 8 6 4
.E WI
2 3
10 a E
1
6
V.0
10 8
4 3
2
1
TIME, HOURS FIG. 1. DNA and phospholipid syntheses of synchronized cultures growing at an 8-h doubling time. (First curve [counting from the top]) DNA synthesis determined by the indole method; (second curve) rate of 32p incorporation in the DNA fraction; (third curve) cell number; (fourth curve) rate of 32P incorporation in phospholipid. The pulse time of Hi 32UP04 label in the rate curves was 1 h. Vertical lines through the midpoints of cell division periods indicate the times when 50%o of the population have divided. Shorter vertical lines are drawn through the midpoints in the increases and decreases of the rate curves. C and PL denote DNA and phospholipid synthesis periods.
termined and pulse-labeled DNA synthesis curves was not obtained. An exact correspondence time schedule between any two experiments was difficult to obtain. The time frames of active phospholipid, RNA, and protein syntheses are demarcated by vertical lines through the midpoints of increases and decreases in the synthesis rate curves in Fig. 1 and 2. RNA and protein syntheses were also
determined chemically, and the contents of RNA and protein doubled within the respective synthesis periods. The overall patterns of the chemically determined RNA and protein syntheses were consistent with the synthesis patterns obtained by pulse-labeling experiments. The molecular species of bulk RNA and protein were not identified. The relatively long pulses of radioactive labels suggest that only stable RNA
68
TJ BACTF.RIOL.
ASAT'O
3 10
10 8
0
8 to 6 3
6 RNA
3H]URACIL
E
i 20.P
6
~~~~~~10 10 -~~~~~~~
60 BER t: 2
40 X
1
A
3~~~~~~~~~~~~~~~~~~ 3 2~~~~
8 4 [14C]VALINE
1~~~~~~~~~~~~~~~~~0
10
2~~~~~~~~~~~~~
8k
2
TIME, HOURS
FIG. 2. RNA and protein synthese.s of .synchronized cultulres grow!ing at aIn 8-h dloubling time. (Fir.st curiwe [ counting from the top],) RNA synthe.si.s determined by the orcinol method; (.second c urvte) rate o)f [fsH/uracil incorporation (pul.se time o)f label, 30 mmn); (third curve,)1)rotein .synthesi.s dletermined1 by thc mnethodl of Lowir et al. (18); (fourth curve) raste of f'40/valine incorpovration ( pulse-labeling time, .30 mnin); (fifth cu1rvle) cell number. Verticall line.s througJh the m7idpoint.s oJf ('dl1 divi.sion period.s indlicatc the timcs o'hcnl 5r0% at the po)pulaltion havle (livzide(I. Shortevr vnertical line.s arZe drawln through the midpo0intrs in the) icrcas>lse.s and decreasxes of the roltc curves. ft an(l P dlenote RN1A alnd protcin1 sytnthe.si.s periodls.
and protein molecular species would have been detectable. Unstable R{NA and protein molecXules and others made in small quantities could not be differentiated with the techniques used. These molecules couldl be made continuously throughout the cell ( yele or induc ed at specific
times;. The
radioactive j)uls
Vol. 140, No. 1
Macromolecular Synthesis in Synchronized Cultures of Anacystis nidulans YUKIO ASATO Department of Biology, Southeastern Massachusetts University, North Dartmouth, Massachusetts 02747 Received for publication 18 July 1979
Synchronous culture of Anacystis nidulans has been induced by the lightdark-light regimen. At various time intervals during synchronous growth, samples were pulsed with radioactive labels to determine phospholipid, protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) syntheses within the cell division cycle. A temporal order of protein, RNA, and DNA syntheses occurred within the cell division cycle, whereas phospholipid was characteristically synthesized during midcycle (during cell enlargement) and during the time of cell division. Chemically determined protein, RNA, and DNA syntheses were found to support the schedule of these macromolecules in cultures growing at an 8-h doubling time. The study of macromolecular synthesis in the unicellular cyanobacterium Anacystis nidulans should simplify to a great extent our attempts in determining the major molecular events that occur within the cell division cycle of these organisms. Previous reports (2, 7, 12) have clearly established that the DNA synthesis occurred in the late stages of the cell cycle of Anacystis. An initiator protein has been implicated in the initiation of DNA synthesis in Anacystis (11). On the other hand, bulk RNA synthesis was initiated about the time when cells divided and terminated just before the subsequent cell division period (12). Other macromolecules could very well be synthesized at specific times within the cell cycle of Anacystis, although very few details are available (23) or known. To describe further details of the macromolecular synthesis patterns of A. nidulans, the synthesis of DNA, RNA, protein, and phospholipid was investigated by applying pulse-labeling techniques to a dark-induced synchronous culture. This report presents the results of the experiments, which show that increased rates of bulk protein, RNA, DNA, and phospholipid syntheses occurred at specific time frames within the cell division cycle of A. nidulans.
10. The flasks were placed on a shaker (shaking rate, 280 rpm) and incubated in an environmental chamber (Conviron, model PGW 36; Controlled Environments, Inc., Pembina, N.Dak.). A bank of four cool white fluorescent bulbs (Sylvania, F96 Tll CW) provided light intensity of 200 footcandles (ca. 2.1 klx). To induce cell synchrony, exponential cultures were placed in the dark for 12 to 24 h. When these cultures were exposed to light, synchronized growths occurred
(2). Total cell count. Cells were counted on a PetroffHausser bacterial cell counter. Pulse-labeling of macromolecules. In labeling nucleic acids and phospholipid, 5-ml samples of synchronized cultures were distributed into 50-ml flasks.
H332P04 (Schwarz/Mann, Orangeburg, N.Y.) was added to a final concentration of 0.1 Ci/ml per flask containing HEPES (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid)-buffered Dm medium (2) at selected time intervals for 30 or 60 min. The methods of Roodyn and Mandel (20) were followed to determine 32p incorporation in DNA, RNA, and phospholipid fractions. To determine DNA synthesis, a set of duplicate 0.5-ml samples was placed in test tubes. To the samples 0.5-ml amounts of 1.1 N NaOH were added, and the mixtures were incubated in a 37°C water bath for 2 h. After this incubation period, the samples were placed in ice for 15 min. Volumes of 0.1 ml of 6 N HCI were added, and the mixtures were chilled for 15 min. The DNA fractions were precipitated with cold perchloric acid (PCA) (final concentration, 0.5 N), collected, and washed with 0.5 N PCA on membrane filters. In the determination of RNA synthesis, a second set of duplicate 0.5-ml samples was treated with 2 volumes of 95% ethyl alcohol. The alcohol-treated samples were incubated in a 70°C water bath for 30 min and then placed in an ice bath. The chilled samples were filtered on membrane filters and washed with cold 0.5 N PCA. This fraction contained both DNA and RNA. The radioactivity from
MATERIALS AND METHODS Organisms and growth media. A. nidulans IU625 was originally obtained from the Algal Culture Collection, Indiana University (Bloomington, Ind.). Dm broth and agar were described by Van Baalen
(22). Growth condition and synchronization of culture. Cultures of A. nidulans were grown in Erlenmeyer flasks with a medium-flask ratio (vol/vol) of 1: 65
66
ASATO
J. BACTERIOL.
the alcohol-treated sample was subtracted from that from the alkali-treated sample to yield the RNA fraction. A third set of duplicate 0.5-ml samples was treated with cold 0.5 N (final concentration) PCA. I'he samples were collected on membrane filters and washed with cold 0.5 N PCA. These samples contained labeled phospholipid, RNA, and DNA. The difference in radioactivity between the cold I'CA fraction and the alcohol-treated sample gave the phospholipid value. RNA was alternatively pulse-labeled with [rH]uracil (specific activity, 25 mCi/mmol; New England Nuclear Corp., Boston, Mass.) at a final concentration of 1 to 10 [iCi/ml. Protein was pulse-labeled with [14C]valine (specific activity, 30 mCi/mmol; New England Nuclear) for 30 to 60 min at a final concentration of 0.1 ,iCi/ml. All labeled samples collected on membrane filters were air dried and placed in a toluene-based scintillation fluid. The radioactivity was measured with a Packard scintillation counter. Colorimetric determinations of DNA, RNA, and protein. DNA was determined by the indole method (13, 21). RNA was analyzed by the orcinol method (3). The standard used for DNA determination was fish roe DNA (Calbiochem, La Jolla, Calif.), and for RNA determination yeast RNA (Calbiochem) was the standard. The method of Lowry et al. (18) was used to determine the protein content. Bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) served as the standard.
RESULTS Macromolecular synthesis in synchronized culture of A. nidulans growing at 8-h doubling time. When exponential cultures are incubated in the dark for 12 to 24 h and subsequently exposed to light, synchronously dividing cultures are obtained (2). A typical synchronized culture (Fig. 1) at an incubation temperature of 32°C demonstrated abrupt increases in cell number at h 4 and h 12. Interdivision time, i.e., the time between the long vertical lines drawn through the midpoints of increases in cell number during the first and second division periods, was 8 h. Cell division periods, taking approximately 50%/c of the cell cycle, indicated a fairly broad distribution of interdivision time. Nevertheless, partial synchronous growth patterns have been maintained through two successive cell division periods without severe deterioration in the slope of the second cell division period. In any event, synchronized cultures have been shown to be experimentally useful. For example, temporal genetic maps of Anacystis were constructed from synchronized culture (2, 7, 12). The construction of temporal genetic maps is based on the premises that genome replication in synchronized cultures occurs in a specific, ordered manner and that the induced mutation frequency of a specific gene increases when that gene replicates (2).
Another possible use of synchronized culture of Anacystis is to determine whether an ordering of macromolecular synthesis occurs during the synchronous cell cycle. To test this possibility, determinations of DNA, RNA, protein, and phospholipid syntheses were made from synchronized cultures. The results are shown in Fig. 1 and 2. In the pulse-labeling experiments, macromolecular synthesis curves exhibited characteristic peaks. Such peaked curves indicate that there is a wide distribution of initiation times of respective macromolecular syntheses. After the peaks, the amounts of radioactive labels incorporated in DNA (Fig. 1) and RNA (Fig. 2) decreased to lower levels than expected. On the other hand, the amounts of ['4C]valine incorporated in protein decreased to expected levels after attaining the peak incorporation rate. Balanced growth apparently was not obtained in the synchronized cultures from which DNA and IRNA syntheses were determined. Although there are a number of factors that may cause unbalanced growth, changes in cell size as cell densities increase could be a significant one. tUpon microscopic observations, cell lengths were found to decrease as the cell densities increased beyond approximately 5 x 10' cells per ml (probably resulting from a shading effect). For example, the cell densities of a typical synchronized culture were 4 x 10' cells per ml at h 8 and 8 x 10' cells per ml at h 16. The average cell lengths at h 8 and h 16 were 6.5 and 5.4 ftm, respectively. DNA (Fig. 1) and RNA (Fig. 2) syntheses were determined for synchronized cultures growing at cell densities of about 8 x 10 cells per ml, whereas protein synthesis (Fig. 2) was determined at cell densities of 4 x 10' cells per ml. A more detailed study must be made to prove the possible correspondence of synthesis rates and cell size changes as observed in the present study. Nevertheless, active syntheses of DNA, RNA, protein, and phospholipid could be shown to occur at different times in the cell cycle. Vertical lines in Fig. 1 mark the midpoints of increase and decrease in the DNA synthesis rate curve. This period bounded by the midpoints signifies the time frame of DNA synthesis. The 32p incorporation during the time devoid of DNA synthesis in synchronized cells was most likely attributable to a fraction of unsynchronized cells. In the chemically determined DNA synthesis curve, a plateau preceded the period of increase, which in turn was followed by a plateau. The plateau periods indicated the periods devoid of DNA synthesis in synchronized cells. There was a doubling of DNA content at the end of the synthesis period. Exact correspondence in the time frames of chemically de-
MACROMOLECULAR SYNTHESIS IN ANACYSTIS
VOL. 140, 1979
10 8 E 6 a 0)
o
4
67
10 8 6 4
.E WI
2 3
10 a E
1
6
V.0
10 8
4 3
2
1
TIME, HOURS FIG. 1. DNA and phospholipid syntheses of synchronized cultures growing at an 8-h doubling time. (First curve [counting from the top]) DNA synthesis determined by the indole method; (second curve) rate of 32p incorporation in the DNA fraction; (third curve) cell number; (fourth curve) rate of 32P incorporation in phospholipid. The pulse time of Hi 32UP04 label in the rate curves was 1 h. Vertical lines through the midpoints of cell division periods indicate the times when 50%o of the population have divided. Shorter vertical lines are drawn through the midpoints in the increases and decreases of the rate curves. C and PL denote DNA and phospholipid synthesis periods.
termined and pulse-labeled DNA synthesis curves was not obtained. An exact correspondence time schedule between any two experiments was difficult to obtain. The time frames of active phospholipid, RNA, and protein syntheses are demarcated by vertical lines through the midpoints of increases and decreases in the synthesis rate curves in Fig. 1 and 2. RNA and protein syntheses were also
determined chemically, and the contents of RNA and protein doubled within the respective synthesis periods. The overall patterns of the chemically determined RNA and protein syntheses were consistent with the synthesis patterns obtained by pulse-labeling experiments. The molecular species of bulk RNA and protein were not identified. The relatively long pulses of radioactive labels suggest that only stable RNA
68
TJ BACTF.RIOL.
ASAT'O
3 10
10 8
0
8 to 6 3
6 RNA
3H]URACIL
E
i 20.P
6
~~~~~~10 10 -~~~~~~~
60 BER t: 2
40 X
1
A
3~~~~~~~~~~~~~~~~~~ 3 2~~~~
8 4 [14C]VALINE
1~~~~~~~~~~~~~~~~~0
10
2~~~~~~~~~~~~~
8k
2
TIME, HOURS
FIG. 2. RNA and protein synthese.s of .synchronized cultulres grow!ing at aIn 8-h dloubling time. (Fir.st curiwe [ counting from the top],) RNA synthe.si.s determined by the orcinol method; (.second c urvte) rate o)f [fsH/uracil incorporation (pul.se time o)f label, 30 mmn); (third curve,)1)rotein .synthesi.s dletermined1 by thc mnethodl of Lowir et al. (18); (fourth curve) raste of f'40/valine incorpovration ( pulse-labeling time, .30 mnin); (fifth cu1rvle) cell number. Verticall line.s througJh the m7idpoint.s oJf ('dl1 divi.sion period.s indlicatc the timcs o'hcnl 5r0% at the po)pulaltion havle (livzide(I. Shortevr vnertical line.s arZe drawln through the midpo0intrs in the) icrcas>lse.s and decreasxes of the roltc curves. ft an(l P dlenote RN1A alnd protcin1 sytnthe.si.s periodls.
and protein molecular species would have been detectable. Unstable R{NA and protein molecXules and others made in small quantities could not be differentiated with the techniques used. These molecules couldl be made continuously throughout the cell ( yele or induc ed at specific
times;. The
radioactive j)uls
