Vol. 126, No. 2 Printed in U.SA.
JOURNAL OF BACTERIOLOGY, May 1976, p. 630-633 Copyright X) 1976 American Society for Microbiology
Formation, in the Dark, of Virus-Induced Deoxyribonuclease Activity in Anacystis nidulans, an Obligate Photoautotroph J. UDVARDY, B. SIVOK, G. BORBgLY, AND G. L. FARKAS* Institute ofPlant Physiology, Hungarian Academy of Sciences, 6701 Szeged, Hungary Received for publication 11 February 1976
In Anacystis nidulans, upon infection with cyanophage AS-1, after a lag period of 1 h the level of deoxyribonuclease (DNase) activity increased rapidly, up to 15- to 20-fold in 4 to 5 h in the light. In contrast, the ribonuclease and phosphomonoesterase activities increased significantly only 4 to 5 h after infection, i.e., as late as 1 h prior to lysis. In complete darkness, the nuclease levels remained unaltered. However, when the infected cells were exposed to light for 1 or 2 h after infection, the DNase level increased essentially to the same extent in the dark as in continuous light, although the complete replication cycle of the virus was impaired in the dark and cells lysed only in the continuously illuminated cultures. Inhibition of photosystem II with 3-(3,4-dichlorophenyl)-1,1dimethylurea during the early illumination period strongly decreased the subsequent, infection-dependent increase in DNase activity in the dark. The virusinduced increase in DNase activity was also inhibited by chloramphenicol. The data suggest that, in spite of the obligate photoautotrophic nature of A. nidulans, dark metabolism is able to support fully the formation of some specific proteins if the triggering of their synthesis takes place in light. For a number of blue-green bacteria (obligate photoautotrophs) light appears to be the only acceptable energy source (1). This may be true for the majority of synthetic processes leading to cell growth and division. Indeed, light has been shown to stimulate overall protein synthesis in Anacystis nidulans by an order of magnitude (13). Therefore, the observation that in Anacystis some proteins are preferentially synthesized in the dark is of great interest (13). These proteins, e.g., glycogen phosphorylase (EC 2.4.1.1) and glucose-6-phosphate dehydrogenase -(EC 1.1.1.49), are supposed to be essential for dark metabolism by maintaining viability (but not allowing multiplication) in the dark (3, 10). We have wondered whether only those proteins that are needed for survival in darkness can be synthesized extensively in the dark in the cells of an "obligate photoautotroph." To answer this question we have chosen virus-infected bluegreen bacterial cells as a model system. In such cells extensive synthesis of enzymes that are not "necessary" for the bacterium (and are even deleterious) is expected to occur. The virusinduced nucleases are examples of such enzymes. It was expected that blue-green bacteria would respond with an increase in nuclease level to phage infection, just as a number of heterotrophic bacteria do (8). Indeed, we have 630
found that in A. nidulans the very low nuclease levels (9) do increase dramatically after infection with the cyanophage AS-1 described by Safferman et al. (12). It will be shown that the virus-induced increase in deoxyribonuclease (DNase) level, which is clearly not needed for "normal" metabolism and/or survival in the dark, can be supported by dark metabolism if the dark period is preceded by a short exposure of the infected cells to light. MATERIALS AND METHODS Organism and growth conditions. A wild-type strain ofA. nidulans was maintained on agar slants enriched with modified Chu no. 10 medium (5). From the agar slants A. nidulans cells were transferred, under sterile conditions, to liquid medium "C" of Kratz and Myers (6). The cultures were grown in 6-liter glass vessels illuminated with cool-white fluorescent light (10,000 lux). Aeration of the cultures, kept at 37 C, was achieved by bubbling sterile air containing 5% CO2. Cells were harvested by centrifugation in the late log phase of growth (approximately 108 cells/ml), washed with Kratz-Myer's medium, and used for the experiments. Virus, virus assay, and infection. AS-1 cyanophage was obtained for the experiments by infecting batch cultures of A. nidulans (3 x 108 cells/ml) with the phage at a multiplicity of infection of 0.1. After lysis, the lysates were centrifuged to remove the cell debris in a Sorvall RC-2 centrifuge for 10 min at 11,000 x g or in a continuous flow rotor of SzentGyorgyi and Blum at 27,000 x g at a flow rate of 30
VOL. 126, 1976
VIRUS-INDUCED DNase ACTIVITY IN A. NIDULANS
ml/min. The virus concentration was determined throughout the procedure by using the plaque assay of Safferman et al. (12). For enzyme studies Anacystis cultures in the late log phase were adjusted to a density of 7 x 107 cells/ml and infected with a multiplicity of infection of 3. The cultures were kept under the conditions described above. Under these conditions lysis started approximately 5 h after infection. This time period was established by counting the cell number in a Burker chamber (see Fig. 1) as well as in one-step growth experiments, as described by Safferman et al. (12). Assay of enzyme activities. Crude extracts were prepared from A. nidulans cells washed with distilled water and adjusted to a concentration of 5 x 109 cells/ml before sonic oscillation. Each sample was treated in the cold in an MSE ultrasonic disintegrator for a total of 10 min in 15-s intervals, followed by 15-s cooling periods in ice. The 10,000 x g supernatant fluid served as the enzyme source. The enzyme assays were carried out as described earlier (14-16). Both native and denatured deoxyribonucleic acids (DNAs) were used in the assays of DNase activity. The results obtained with the two substrates were essentially the same; only the results obtained with denatured DNA will be presented. Denatured DNA was prepared by heating doublestranded DNA from chicken erythrocytes in 0.001 M sodium citrate containing 0.015 M NaCl for 15 min, followed by rapid cooling in ice. The results of the assays are expressed as change in absorbancy at 260 nm (nucleases) and change in absorbancy at 400 nm (phosphomonoesterase) (14-16). 1
107
D2.4
142
9 x?
2.0
o 1.O
x
0 7x107
RESULTS Anacystis cultures infected with AS-1 phage as described above were illuminated for 2 h. Then, the cultures were divided into two parts; one of them was further illuminated throughout the experiment (Fig. 1A) and the other was kept in darkness for the rest of the time under otherwise identical conditions (Fig. 1B). It may be seen from Fig. 1A that in the permanently illuminated sample, prior to and/or during lysis (5 to 6 h after infection), all enzyme activities measured were substantially higher than in the zero-time control. The time course of the increase in enzyme activities was, however, different. Ribonuclease (RNase) activity remained essentially constant for about 5 h after infection and increased only in the late phase of the infection cycle. Phosphomonoesterase activity decreased significantly during the first 2 h after infection and started to increase rapidly at the same time as RNase activity. In contrast, the DNase level increased dramatically immediately after infection and continued to rise steadily until complete lysis occurred. Complete adsorption of the phage took place in about 1 h (cf. also Safferman et al. [12]). As shown by the decrease in cell number, excessive lysis started around h 5 after infection. In cultures infected and kept in the dark
A ,0
-*
631
0, ,0
0/6
4>> W51 1.2&
0~~~~ .
8 0 2 4 6 6 8 AFTER INFECTION HOURS FIG. 1. Nuclease activities in A. nidulans cells after infection with cyanophage AS-1. (A) Continuously illuminated cultures; (B) cultures illuminated for2 h and kept in darkness afterwards. Symbols: cell number per milliliter (0); DNase activity (0); RNase activity (A); phosphomonoesterase activity (A). All data on enzyme activities are calculated for identical cell numbers: nucleases, 101 cells; phosphomonoesterase, 108 0
cells.
2
4
632
J. BACTERIOL.
UDVARDY ET AL.
throughout the experiment, there was no change in cell number (i.e., no lysis took place), and no change was detected in enzyme activities relative to the zero-time values (data not shown). In the cells illuminated for 2 h and kept in darkness for the rest of the experiment (Fig. 1B) the trend of changes in enzyme activities differed from those of the permanently illuminated ones (Fig. 1A). DNase activity reached essentially the same level in both cultures by h 7 after inoculation, although only the illuminated cultures lysed (cf. cell number). In some experiments, but not in all, we experienced some delay (approximately 1 h) in the rapid increase in DNase activity in the darkened cultures as compared to the continuously illuminated ones. In sharp contrast to DNase, the increase in RNase and phosphomonoesterase activities was considerably less in the cultures darkened after 2 h of illumination than in the continuously illuminated ones (Fig. 1A and B). Clearly, light applied in the early phase of the infection had created conditions that could drive, in the dark, the virus-induced increase in DNase activity but not the increase in RNase (or to a much lesser extent). To provide further evidence for the role of illumination in the early period of infection in the virus-induced formation of DNase later in the dark, 10- M 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor that interferes with the activity of photosystem II, was added to the cultures simultaneously with the inoculum. DCMU greatly inhibited the virusinduced increase in DNase activity (Fig. 2A). This observation suggests that photosynthesis provides, in stored form, the driving force for the dark synthesis of DNase. Since the level of phosphomonoesterase also exhibited an early change (a transitory decrease in contrast to the increase of DNase activity) in the infected cells (Fig. 1A), the effect of DCMU on the virus-induced decrease in phosphomonoesterase activity was also tested. DCMU retarded considerably the virusinduced decrease in phosphomonoesterase level (Fig. 2B). Evidence that the virus-induced increase in enzyme activities is due to new protein synthesis rather than enzyme activation was obtained in experiments in which, 2 h after infection, 3 x 10-4 M chloramphenicol was added to the cultures. This treatment completely inhibited the further rise in DNase activity, as well as the rise in RNase and phosphodiesterase activity that coincides with the lytic process (data not shown).
DISCUSSION A number of blue-green bacteria are typically obligate photoautotrophs. Since they are prokaryotes but probably phylogenetically related to the chloroplasts of photosynthetic eukaryotes, much work, and even more speculation, was done on the regulatory mechanisms that distinguish these prokaryotes from the heterotrophic bacteria. It has been questioned whether, in general, these blue-green bacteria are able to regulate their metabolism at the level of gene expression (1, 2, 7). The shift of their energy metabolism in favor of the oxidative pentose phosphate cycle in the dark (3, 10) has been attributed to light inactivation of one of the key enzymes of the oxidative pathway (glucose-6-phosphate dehydrogenase) and light activation of a reductive pentose phosphate cycle enzyme, ribulose-5-phosphate kinase (4). Perhaps more important is the recent evidence (13) for the preferential "dark induction" (de novo synthesis) of two enzymes specifically required for dark endogenous energy metabolism (glycogen phosphorylase, glucose-6-phosphate dehydrogenase). The above observations suggest that some synthetic processes of the obligate photoauto~~~~0
1. r *
A
E 8
10
J," *'/
B
O/ E
/
I
>0 t
C
o
I -
0-~~
I;,,"
C1
r
V)
c
z
D
04
I,.
k
"aa
0 °
Q2
\ k
\A
\
i.
0
2
4
a__I
4 2 6 0 HOURS AFTER INFECTION
6
FIG. 2. Effect of DCMU on the changes in DNase (A) and phosphomonoesterase (B) activities induced by the infection ofA. nidukans cells with cyanophage AS-I. Symbols: DNase activity in cells illuminated for 2 h after infection and kept in darkness afterwards (-); DNase activity in cells illuminated for 2 h in the presence of 1O-5 M DCMU and kept in darkness afterwards (0); DNase activity in cells illuminated throughout the experiment in the presence of iO-5 M DCMU (A); phosphomonoesterase activity in cells illuminated for 2 h and kept in darkness afterwards (A); phosphomonoesterase activity in cells illuminated for 2 h in the presence of 1O-5 M DCMU and kept in darkness afterwards (U); phosphomonoesterase activity in cells illuminated throughout the experiment in the presence of O-5 M DCMU (0).
VOL. 126, 1976
VIRUS-INDUCED DNase ACTIVITY IN A. NIDULANS
trophic blue-green bacteria may be quite extensive in the dark. The results presented in this paper extend this concept to another group ofenzymes: virusinduced nuclease(s). It was shown that the dramatic, virus-induced increase in DNase level, once it has been initiated in light, can take place in complete darkness. This system seems to be one that deserves further study regarding the synthetic capacities and mechanisms of biosyntheses in obligate photoautotrophs in the dark. In addition, it seems worthwhile to stress that in higher plant cells virus infection per se, as studied in detail in our laboratory, does not induce major changes in the nuclease levels (17). Nothing similar to the dramatic increase in DNase activity in virus-infected A. nidulans cells was found in the early phase of virus replication in virus-infected, photosynthesizing tissues ofhigher plants. In its response, with an early increase in DNase level, to virus infection, A. nidulans resembles much more the heterotrophic bacteria (Escherichia coli, etc.) than a photosynthetic eukaryote cell. However, in the late stage of infection, when cell damage is already excessive, the RNase level has been found to increase dramatically both in A. nidulans and in virus-infected higher plant cells (17). Thus, it is tempting to speculate that the increase in RNase activity in A. nidulans late during infection, when lytic processes are already in progress, may be similar in nature to the increase in RNase level of green, photosynthetic cells of eukaryote plants.
2.
3.
4. 5.
6. 7.
8.
9. 10. 11. 12.
13. 14.
ACKNOWLEDGMENT A sample of cyanophage AS-1 was kindly provided by R. S. Safferman, Office of Research and Monitoring, Environmental Protection Agency, Cincinnati, Ohio.
15.
LITERATURE CITED 1. Carr, N. G. 1973. Metabolic control and autotrophic physiology, p. 39-65. In N. G. Carr and B. A. Whitton
16.
633
(ed.), The biology of blue-green algae. Blackwell Scientific Publishers, Oxford. Delaney, S. F., A. Dickson, and N. G. Carr. 1973. Control of homoserine-O-transsuccinylase in a methionine-requiring mutant of the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 79:89-94. Doolittle, F. W., and R. A. Singer. 1974. Mutational analysis of dark endogenous metabolism in the bluegreen bacterium Anacystis nidulans. J. Bacteriol. 119:677-683. Duggan, J. K., and L. E. Anderson. 1975. Light-regulation of enzyme activity in Anacystis nidulans (Richt.). Planta 122:293-297. Gerloff, G. C., G. P. Fitzgerald, and G. F. Skoog. 1950. The isolation, purification and culture of blue-green algae. Am. J. Bot. 37:216-218. Kratz, W. A., and J. Myers. 1955. Nutrition of several blue-green algae. Am. J. Bot. 42:282-287. Mann, N., and N. G. Carr. 1974. Control of macromolecular composition and cell division in the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 83:399405. Matthews, C. K. 1971. Bacteriophage biochemistry. Nostrand Reinhold Co., New York. Norton, J., and J. S. Roth. 1967. Some aspects of nuclease activity in Anacystis nidulans and other algae. Comp. Biochem. Physiol. 23:361-371. Peiroy, R. A., R. Rippka, and R. Y. Stanier. 1972. Metabolisn of glucose by unicellular blue-green algae. Arch. Mikrobiol. 87:303-322. Safferman, R. S. 1973. Phycoviruses, p. 214-237. In N. G. Carr and B. A. Whitton (ed.), The biology of bluegreen algae. Blackwell Scientific Publishers, Oxford. Safferman, R. S., T. 0. Diener, P. R. Desjardins, and M. E. Morris. 1972. Isolation and characterisation of AS-1, a phycovirus infecting the blue-green algaAnacystis nidulans and Synechococcus cedrorum. Virology 47:105-113. Singer, R. A., and W. F. Doolittle. 1975. Control of gene expression in blue-green algae. Nature (London) 253:650-651. Udvardy, J., E. Marre, and G. L. Farkas. 1970. Purification and properties of a phosphodiesterase from Avena leaf tissues. Biochim. Biophys. Acta 206:392403. Wyen, N. V., S. Erdei, and G. L. Farkas. 1971. Isolation from Avena leaf tissues of a nuclease with the same type of specificity towards RNA and DNA. Accumulation of the enzyme during leaf senescence. Biochim. Biophys. Acta 232:472-483. Wyen, N. V., J. Udvardy, S. Erdei, and G. L. Farkas. 1972. The level of a relatively purine-specific ribonuclease increases in virus-infected tobacco leaves. Virology 48:337-341.
JOURNAL OF BACTERIOLOGY, May 1976, p. 630-633 Copyright X) 1976 American Society for Microbiology
Formation, in the Dark, of Virus-Induced Deoxyribonuclease Activity in Anacystis nidulans, an Obligate Photoautotroph J. UDVARDY, B. SIVOK, G. BORBgLY, AND G. L. FARKAS* Institute ofPlant Physiology, Hungarian Academy of Sciences, 6701 Szeged, Hungary Received for publication 11 February 1976
In Anacystis nidulans, upon infection with cyanophage AS-1, after a lag period of 1 h the level of deoxyribonuclease (DNase) activity increased rapidly, up to 15- to 20-fold in 4 to 5 h in the light. In contrast, the ribonuclease and phosphomonoesterase activities increased significantly only 4 to 5 h after infection, i.e., as late as 1 h prior to lysis. In complete darkness, the nuclease levels remained unaltered. However, when the infected cells were exposed to light for 1 or 2 h after infection, the DNase level increased essentially to the same extent in the dark as in continuous light, although the complete replication cycle of the virus was impaired in the dark and cells lysed only in the continuously illuminated cultures. Inhibition of photosystem II with 3-(3,4-dichlorophenyl)-1,1dimethylurea during the early illumination period strongly decreased the subsequent, infection-dependent increase in DNase activity in the dark. The virusinduced increase in DNase activity was also inhibited by chloramphenicol. The data suggest that, in spite of the obligate photoautotrophic nature of A. nidulans, dark metabolism is able to support fully the formation of some specific proteins if the triggering of their synthesis takes place in light. For a number of blue-green bacteria (obligate photoautotrophs) light appears to be the only acceptable energy source (1). This may be true for the majority of synthetic processes leading to cell growth and division. Indeed, light has been shown to stimulate overall protein synthesis in Anacystis nidulans by an order of magnitude (13). Therefore, the observation that in Anacystis some proteins are preferentially synthesized in the dark is of great interest (13). These proteins, e.g., glycogen phosphorylase (EC 2.4.1.1) and glucose-6-phosphate dehydrogenase -(EC 1.1.1.49), are supposed to be essential for dark metabolism by maintaining viability (but not allowing multiplication) in the dark (3, 10). We have wondered whether only those proteins that are needed for survival in darkness can be synthesized extensively in the dark in the cells of an "obligate photoautotroph." To answer this question we have chosen virus-infected bluegreen bacterial cells as a model system. In such cells extensive synthesis of enzymes that are not "necessary" for the bacterium (and are even deleterious) is expected to occur. The virusinduced nucleases are examples of such enzymes. It was expected that blue-green bacteria would respond with an increase in nuclease level to phage infection, just as a number of heterotrophic bacteria do (8). Indeed, we have 630
found that in A. nidulans the very low nuclease levels (9) do increase dramatically after infection with the cyanophage AS-1 described by Safferman et al. (12). It will be shown that the virus-induced increase in deoxyribonuclease (DNase) level, which is clearly not needed for "normal" metabolism and/or survival in the dark, can be supported by dark metabolism if the dark period is preceded by a short exposure of the infected cells to light. MATERIALS AND METHODS Organism and growth conditions. A wild-type strain ofA. nidulans was maintained on agar slants enriched with modified Chu no. 10 medium (5). From the agar slants A. nidulans cells were transferred, under sterile conditions, to liquid medium "C" of Kratz and Myers (6). The cultures were grown in 6-liter glass vessels illuminated with cool-white fluorescent light (10,000 lux). Aeration of the cultures, kept at 37 C, was achieved by bubbling sterile air containing 5% CO2. Cells were harvested by centrifugation in the late log phase of growth (approximately 108 cells/ml), washed with Kratz-Myer's medium, and used for the experiments. Virus, virus assay, and infection. AS-1 cyanophage was obtained for the experiments by infecting batch cultures of A. nidulans (3 x 108 cells/ml) with the phage at a multiplicity of infection of 0.1. After lysis, the lysates were centrifuged to remove the cell debris in a Sorvall RC-2 centrifuge for 10 min at 11,000 x g or in a continuous flow rotor of SzentGyorgyi and Blum at 27,000 x g at a flow rate of 30
VOL. 126, 1976
VIRUS-INDUCED DNase ACTIVITY IN A. NIDULANS
ml/min. The virus concentration was determined throughout the procedure by using the plaque assay of Safferman et al. (12). For enzyme studies Anacystis cultures in the late log phase were adjusted to a density of 7 x 107 cells/ml and infected with a multiplicity of infection of 3. The cultures were kept under the conditions described above. Under these conditions lysis started approximately 5 h after infection. This time period was established by counting the cell number in a Burker chamber (see Fig. 1) as well as in one-step growth experiments, as described by Safferman et al. (12). Assay of enzyme activities. Crude extracts were prepared from A. nidulans cells washed with distilled water and adjusted to a concentration of 5 x 109 cells/ml before sonic oscillation. Each sample was treated in the cold in an MSE ultrasonic disintegrator for a total of 10 min in 15-s intervals, followed by 15-s cooling periods in ice. The 10,000 x g supernatant fluid served as the enzyme source. The enzyme assays were carried out as described earlier (14-16). Both native and denatured deoxyribonucleic acids (DNAs) were used in the assays of DNase activity. The results obtained with the two substrates were essentially the same; only the results obtained with denatured DNA will be presented. Denatured DNA was prepared by heating doublestranded DNA from chicken erythrocytes in 0.001 M sodium citrate containing 0.015 M NaCl for 15 min, followed by rapid cooling in ice. The results of the assays are expressed as change in absorbancy at 260 nm (nucleases) and change in absorbancy at 400 nm (phosphomonoesterase) (14-16). 1
107
D2.4
142
9 x?
2.0
o 1.O
x
0 7x107
RESULTS Anacystis cultures infected with AS-1 phage as described above were illuminated for 2 h. Then, the cultures were divided into two parts; one of them was further illuminated throughout the experiment (Fig. 1A) and the other was kept in darkness for the rest of the time under otherwise identical conditions (Fig. 1B). It may be seen from Fig. 1A that in the permanently illuminated sample, prior to and/or during lysis (5 to 6 h after infection), all enzyme activities measured were substantially higher than in the zero-time control. The time course of the increase in enzyme activities was, however, different. Ribonuclease (RNase) activity remained essentially constant for about 5 h after infection and increased only in the late phase of the infection cycle. Phosphomonoesterase activity decreased significantly during the first 2 h after infection and started to increase rapidly at the same time as RNase activity. In contrast, the DNase level increased dramatically immediately after infection and continued to rise steadily until complete lysis occurred. Complete adsorption of the phage took place in about 1 h (cf. also Safferman et al. [12]). As shown by the decrease in cell number, excessive lysis started around h 5 after infection. In cultures infected and kept in the dark
A ,0
-*
631
0, ,0
0/6
4>> W51 1.2&
0~~~~ .
8 0 2 4 6 6 8 AFTER INFECTION HOURS FIG. 1. Nuclease activities in A. nidulans cells after infection with cyanophage AS-1. (A) Continuously illuminated cultures; (B) cultures illuminated for2 h and kept in darkness afterwards. Symbols: cell number per milliliter (0); DNase activity (0); RNase activity (A); phosphomonoesterase activity (A). All data on enzyme activities are calculated for identical cell numbers: nucleases, 101 cells; phosphomonoesterase, 108 0
cells.
2
4
632
J. BACTERIOL.
UDVARDY ET AL.
throughout the experiment, there was no change in cell number (i.e., no lysis took place), and no change was detected in enzyme activities relative to the zero-time values (data not shown). In the cells illuminated for 2 h and kept in darkness for the rest of the experiment (Fig. 1B) the trend of changes in enzyme activities differed from those of the permanently illuminated ones (Fig. 1A). DNase activity reached essentially the same level in both cultures by h 7 after inoculation, although only the illuminated cultures lysed (cf. cell number). In some experiments, but not in all, we experienced some delay (approximately 1 h) in the rapid increase in DNase activity in the darkened cultures as compared to the continuously illuminated ones. In sharp contrast to DNase, the increase in RNase and phosphomonoesterase activities was considerably less in the cultures darkened after 2 h of illumination than in the continuously illuminated ones (Fig. 1A and B). Clearly, light applied in the early phase of the infection had created conditions that could drive, in the dark, the virus-induced increase in DNase activity but not the increase in RNase (or to a much lesser extent). To provide further evidence for the role of illumination in the early period of infection in the virus-induced formation of DNase later in the dark, 10- M 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor that interferes with the activity of photosystem II, was added to the cultures simultaneously with the inoculum. DCMU greatly inhibited the virusinduced increase in DNase activity (Fig. 2A). This observation suggests that photosynthesis provides, in stored form, the driving force for the dark synthesis of DNase. Since the level of phosphomonoesterase also exhibited an early change (a transitory decrease in contrast to the increase of DNase activity) in the infected cells (Fig. 1A), the effect of DCMU on the virus-induced decrease in phosphomonoesterase activity was also tested. DCMU retarded considerably the virusinduced decrease in phosphomonoesterase level (Fig. 2B). Evidence that the virus-induced increase in enzyme activities is due to new protein synthesis rather than enzyme activation was obtained in experiments in which, 2 h after infection, 3 x 10-4 M chloramphenicol was added to the cultures. This treatment completely inhibited the further rise in DNase activity, as well as the rise in RNase and phosphodiesterase activity that coincides with the lytic process (data not shown).
DISCUSSION A number of blue-green bacteria are typically obligate photoautotrophs. Since they are prokaryotes but probably phylogenetically related to the chloroplasts of photosynthetic eukaryotes, much work, and even more speculation, was done on the regulatory mechanisms that distinguish these prokaryotes from the heterotrophic bacteria. It has been questioned whether, in general, these blue-green bacteria are able to regulate their metabolism at the level of gene expression (1, 2, 7). The shift of their energy metabolism in favor of the oxidative pentose phosphate cycle in the dark (3, 10) has been attributed to light inactivation of one of the key enzymes of the oxidative pathway (glucose-6-phosphate dehydrogenase) and light activation of a reductive pentose phosphate cycle enzyme, ribulose-5-phosphate kinase (4). Perhaps more important is the recent evidence (13) for the preferential "dark induction" (de novo synthesis) of two enzymes specifically required for dark endogenous energy metabolism (glycogen phosphorylase, glucose-6-phosphate dehydrogenase). The above observations suggest that some synthetic processes of the obligate photoauto~~~~0
1. r *
A
E 8
10
J," *'/
B
O/ E
/
I
>0 t
C
o
I -
0-~~
I;,,"
C1
r
V)
c
z
D
04
I,.
k
"aa
0 °
Q2
\ k
\A
\
i.
0
2
4
a__I
4 2 6 0 HOURS AFTER INFECTION
6
FIG. 2. Effect of DCMU on the changes in DNase (A) and phosphomonoesterase (B) activities induced by the infection ofA. nidukans cells with cyanophage AS-I. Symbols: DNase activity in cells illuminated for 2 h after infection and kept in darkness afterwards (-); DNase activity in cells illuminated for 2 h in the presence of 1O-5 M DCMU and kept in darkness afterwards (0); DNase activity in cells illuminated throughout the experiment in the presence of iO-5 M DCMU (A); phosphomonoesterase activity in cells illuminated for 2 h and kept in darkness afterwards (A); phosphomonoesterase activity in cells illuminated for 2 h in the presence of 1O-5 M DCMU and kept in darkness afterwards (U); phosphomonoesterase activity in cells illuminated throughout the experiment in the presence of O-5 M DCMU (0).
VOL. 126, 1976
VIRUS-INDUCED DNase ACTIVITY IN A. NIDULANS
trophic blue-green bacteria may be quite extensive in the dark. The results presented in this paper extend this concept to another group ofenzymes: virusinduced nuclease(s). It was shown that the dramatic, virus-induced increase in DNase level, once it has been initiated in light, can take place in complete darkness. This system seems to be one that deserves further study regarding the synthetic capacities and mechanisms of biosyntheses in obligate photoautotrophs in the dark. In addition, it seems worthwhile to stress that in higher plant cells virus infection per se, as studied in detail in our laboratory, does not induce major changes in the nuclease levels (17). Nothing similar to the dramatic increase in DNase activity in virus-infected A. nidulans cells was found in the early phase of virus replication in virus-infected, photosynthesizing tissues ofhigher plants. In its response, with an early increase in DNase level, to virus infection, A. nidulans resembles much more the heterotrophic bacteria (Escherichia coli, etc.) than a photosynthetic eukaryote cell. However, in the late stage of infection, when cell damage is already excessive, the RNase level has been found to increase dramatically both in A. nidulans and in virus-infected higher plant cells (17). Thus, it is tempting to speculate that the increase in RNase activity in A. nidulans late during infection, when lytic processes are already in progress, may be similar in nature to the increase in RNase level of green, photosynthetic cells of eukaryote plants.
2.
3.
4. 5.
6. 7.
8.
9. 10. 11. 12.
13. 14.
ACKNOWLEDGMENT A sample of cyanophage AS-1 was kindly provided by R. S. Safferman, Office of Research and Monitoring, Environmental Protection Agency, Cincinnati, Ohio.
15.
LITERATURE CITED 1. Carr, N. G. 1973. Metabolic control and autotrophic physiology, p. 39-65. In N. G. Carr and B. A. Whitton
16.
633
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