May 15, 1972 - rically at 620 nm with a Bausch and Lomb Spectronic. 20. Cellmorphology was monitored by using phase microscopy. Viable cell counts were ...
JOURNAL OF BACTERIOLOGY, Oct. 1972, p. 170-175 Copyright 0 1972 American Society for Microbiology
Vol. 112, No. 1 Printed in U.SA.
Selective Inhibition of Deoxyribonucleic Acid Synthesis by 2-Deoxyadenosine in the BlueGreen Bacterium Agmenellum quadruplicatum The
L. 0. INGRAMI AND W. D. FISHER University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 Received for publication 15 May 1972
Concentrations of deoxyadenosine which have little effect on net ribonucleic acid (RNA) synthesis or on increase in cell mass selectively inhibit deoxyribonucleic acid (DNA) synthesis in Agmenellum quadruplicatum. Exogenously supplied deoxyadenosine, at concentrations above 10 ,ug/ml, stimulates DNA degradation. These results are correlated with a rapid loss in viability. Over a narrow concentration range (6-15 jig/ml), deoxyadenosine impairs the division process and induces the formation of elongated cells. Low exogenous concentrations of deoxyadenosine are readily incorporated into both DNA and RNA, with the major portion as DNA.
Deoxyribonucleic acid (DNA) synthesis appears to be intimately involved in the regulation of cell division (9). Thus, in our continuing study of the regulation of cell division in the blue-green bacterium Agmenellum quadruplicatum, we have examined deoxyadenosine as a potential, selective inhibitor of DNA synthesis. A number of agents have been characterized as potent, selective inhibitors of DNA synthesis in commonly used bacterial systems such as Escherichia coli (5, 7, 14, 20). However, no agents have been shown to selectively inhibit DNA synthesis in any of the blue-green bacteria. Wehr et al. (26) amply demonstrate the problems associated with applying inhibitors, with demonstrated selective properties in one system, to a new system. The blue-green bacterium Anacystis nidulans appears quite insensitive to high concentrations of two commonly employed inhibitors of DNA synthesis-nalidixic acid and hydroxyurea (17). Further,- studies involving DNA synthesis and its inhibition in the blue-green bacteria are complicated by the apparent failure of these bacteria to incorporate efficiently many exogenously supplied nucleic acid precursors such as thymidine (18). Deoxyadenosine has been shown to inhibit selectively DNA synthesis in plant cells (11), in animal cells (4), and in bacteria (13). In this paper, we describe the selective inhibition of I Present address: Department of Microbiology, McCarty Hall, Univ. of Florida, Gainesville, Fla. 32601.
DNA synthesis and stimulation of DNA degradation by deoxyadenosine. Further, we describe a method for labeling the DNA in the blue-green bacterium A. quadruplicatum by using lower concentrations of tritiated deoxyadenosine. MATERIALS AND METHODS Organism. A. quadruplicatum strain BG1 was originally isolated into axenic culture by Van Baalen (22); Nostoc sp. strain MAC was isolated by Bowyer and Skermann (1); and Anacystic nidulans strain TX20 was isolated by Kratz and Myers (12). Strain BG1 was grown autotrophically in liquid medium ASP2 plus B,, at 39 C with continuous gassing (1% CO,-enriched air) as described previously (10). For growth on petri plates, medium was solidified with 1% agar (Difco). Plates were sealed with Scotch tape (no. 801), inverted, and incubated beneath a bank of 25-w tungsten lamps. Both strains TX20 and MAC were grown in a similar manner by using CglO medium of Van Baalen (23). Both media ASP2 and CglO are composed of mineral salts with an organic buffer. Analyses. Growth was monitored turbidimetrically at 620 nm with a Bausch and Lomb Spectronic 20. Cell morphology was monitored by using phase microscopy. Viable cell counts were made by pourplating appropriate dilutions and incubating for 4 days. Nucleic acids were extracted by incubation of pellets in 10% perchloric acid for 30 min at 70 C. DNA was determined by the diphenylamine method as described by Giles and Myers (6), and ribonucleic acid (RNA) was determined by the orcinol method as described by Hatcher and Goldstein (8). Calf 170
VIOL. 1 12, 1972
DNA INHIBITION
thymus DNA (Worthington Biochemical Corporation, Freehold, N.J.) and yeast RNA (Schwarz BioResearch, Mount Vernon, N.Y.) served as standards. Linearity of reaction for each class of compounds was established by using various amounts of cell material and standards over the range of experimental variation. Auxanographic assay. An auxanographic assay was used to survey the toxicity of various purine and pyrimidine derivatives. Petri plates containing solidified medium were spread with 106 cells. One milligram of the compound to be tested was placed in the center. Plates were sealed and immediately incubated beneath a bank of tungsten lamps. Possible antagonisms of various related compounds were tested in a similar manner. Cells were spread on solidified medium containing 2'-deoxyadenosine (10 pg/ml). Various compounds to be tested were placed singly and in combination in the center of the petri plate, sealed, and incubated. Growth respones were recorded after 3 days of incubation. Labeling experiments. 2-Deoxyadenosine-8- 3H was obtained from Schwarz/Mann, Orangeburg, N.Y. Cells were routinely labeled by incubation under growth conditions with 2 or 4 pAg of deoxyadenosine per ml at a specific activity of 0.25 pCi/lg. For some experiments, higher concentrations at the same specific activity were used. Samples (0.1 ml) removed for measurement of radioactivity were applied to Whatman filter paper discs (3 mm by 2.5 cm diameter). These were immediately transferred to chilled 5% trichloroacetic acid. Fifteen minutes after the last sample was taken, the filter discs were washed through 10% trichloroacetic acid, 5% trichloroacetic acid, and three changes of 95% ethanol (15 min for each washing). Discs were dried at 60 C and counted in toluene containing 0.5% 2, 5-bis- [2-(5-tert-butylbenzoxazolyl) ]-thiophene (Packard Instrument Co.) by using a Packard scintillation counter. Chemicals. All purines and pyrimidines were obtained from Calbiochem (Los Angeles, Calif.). Orcinol and diphenylamine were obtained from Matheson, Coleman & Ball (Norwood, Ohio). Enzymes used in digestions were obtained from Worthington Biochemical Corporation (Freehold, N.J.).
RESULTS Auxanographic survey. Various purines and pyrimidines were tested for growth inhibition of A. quadruplicatum. In general, purines were more toxic than pyrimidines. No inhibition (no zone of clearing) was shown with thymine, thymidine, thymidine monophosphate, thymidine diphosphate, thymidine triphosphate, deoxyadenosine monophosphate, deoxyadenosine diphosphate, deoxyguanosine monophosphate, deoxyguanosine diphosphate, deoxyguanosine triphosphate, deoxycytosine diphosphate, deoxycytosine triphosphate, uracil, uridine, deoxyuridine, 5methyl-cytosine, and inosine.
171 Slight inhibition (1-cm zone) was shown with cytosine, cytidine, deoxycytidine, deoxycytidine monophosphate, and guanine. Moderate inhibition (3- to 4-cm zone) was shown with guanosine, deoxyguanosine, and adenine. Strong inhibition (6-cm zone) was shown with adenosine, deoxyadenosine, and deoxyadenosine triphosphate. On plates, just beyond the zone of complete growth inhibition by deoxyadenosine, cells of A. quadruplicatum formed serpentine filaments three to four cells in length. Of all the compounds tested, deoxyadenosine appeared to be the most potent inhibitor. The purines and pyrimidines, tested singly and in various combinations, failed to relieve the growth inhibition caused by deoxyadenosine (10 jig/ml). Two other blue-green bacteria were tested for their sensitivity to deoxyadenosine on seeded plates. Both Nostoc sp. MAC and A. nidulans TX20 appeared sensitive. MAC, in particular, displayed a very sharply delimited zone of inhibition. Incorporation of deoxyadenosine. During logarithmic growth, low concentrations of exogenous deoxyadenosine are incorporated into trichloroacetic acid-insoluble material by A. quadruplicatum (Fig. 1). Both the initial uptake and incorporation (after 15 min) (Fig. 2) and the continued incorporation of deoxyadenosine (Fig. 1) are strongly dependent upon the external concentration. Enzymatic digestion
E E
"IN
0
1.0 V 0 TIME (hr)
FIG. 1. Incorporation of deoxyadenosine by BG1. (0) 2 jg (0.5 uCi)/ml; (0) 4 pg (1.0 ACi)/ml.
172
J. BACTERIOL.
INGRAM AND FISHER
TABLE 1. Enzymatic digestionsa
14-
Counts remaining (%) A. quad-
Treatment
E. coli
ruplicatum
96 98 4 2 100 87 100
59 62 28 22 100 83 100
Ribonuclease A ............... Ribonuclease B ............... Deoxyribonuclease I (10 hr) ... Deoxyribonuclease I (24 hr) .... Pepsin ...................... Chymotrypsin ................ Control ......................
10-
-
CYO 6-
2-
0
10
20
30
40
50
DEOXYADENOSINE (Qg/ml)
FIG. 2. Concentration dependence of initial 2deoxyadenosine incorporation. A suspension of BGI was divided into five portions, incubated in the presence of various concentrations of 2-deoxyadenosine of the same specific activity (1 MiCi/4 pg), and sampled at the end of 15 min.
employed to determine the relative specificity of deoxyadenosine incorporation into DNA; E. coli K-12 labeled with tritiated thymidine was used as a standard (Table 1). From 60 to 75% of the trichloroacetic acid-precipitable deoxyadenosine was localized in the DNA of A. quadruplicatum, whereas virtually all of the tritiated thymidine was localized in the DNA of E. coli. Similar results with E. coli were reported previously by Van Tubergen and Setlow (24). Effects of deoxyadenosine on growth, survival, and macromolecular synthesis. Concentrations of deoxyadenosine above 10 ,ug/ml significantly inhibited the growth of A. quadruplicatum (Fig. 3). Higher concentrations were increasingly effective and lower concentrations had little effect during the first 12 hr. Short filaments averaging three to four times the length of normal cells were produced during this initial 12-hr incubation period (roughly 3.5 generation times) in the presence of low concentrations of deoxyadenosine (5-15 j.g/ml). At a concentration of 15 qg/ml, cell viability decreased sharply after an initial lag (Fig. 4). Since deoxyadenosine had previously been shown to inhibit DNA synthesis in quite di-
a Cells of Escherichia coli were grown in the presence of tritiated thymidine (5 Ag/ml) with adenosine (100 ug/ml) as recommended by Boyce and Setlow (2). Cells of A. quadruplicatum were grown in the presence of tritiated 2-deoxyadenosine. Samples (0.1-ml) of labeled cells were applied to filter paper discs and washed as described in Materials and Methods. The dried discs were moistened with 0.2 ml of enzyme solution (100 ttg/ml) and incubated at 37 C for 10 and 24 hr in a moist chamber. The discs were then washed a second time with trichloroacetic acid and ethanol, dried, and counted. Phosphate buffer (0.1 M, pH 7.0) served as a control. Results are expressed as the percentage of disintegrations remaining after treatment.
was
0
Dr
0
4
2
8
6
10
12
TIME (hr)
FIG. 3. Growth inhibition of BGJ1 by various concentrations of 2'-deoxyadenosine. (0) 0 pg/rql; (0) 5 ,ug/ml; (A) 10 Ag/ml; (A) 20 ug/ml; (0) 30 Ag/ml; 100 ug/ml. 50 Ag/mI; (-) 40 ug/ml;
(0)
(#)
verse systems, its effects on net DNA and RNA syntheses were examined in A. quadruplicatum. Initially these effects on RNA and DNA synthesis were examined by use of colorimetric assays. The results presented in Fig. 5 represent four experiments. The rate of growth is less than maximal because dense suspensions were needed for the colorimetric assay. The results were normalized to eliminate
173
DNA INHIBITION
VOL. 112, 1972
A
2.5- RNA
DNA
00
-0.8
A 260 0
z
-0.4 >l a
-0.2 i-
~1.0
4
.0< -0.1 z D
0 0 -i
r-0
4
6
2
4
6
0 TIME (hr)
2
4
6
FIG. 5. Effect of deoxyadenosine on RNA and DNA synthesis. Four cell suspensions were incubated under growth conditions in the presence of various levels of deoxyadenosine. Samples (10 ml) were withdrawn hourly, chilled, and centrifuged. Nucleic acids were extracted with 10% perchloric acid and both DNA and RNA and were estimated colorimetrically. All values were normalized by setting the initial level of RNA or DNA equal to 1, eliminating initial variations in culture density. Control, 0; 10 pg/mI, 0; 20 pg/ml, A; 50 pg/ml, A. 8"I
a6-
0-0-0-0 _---O--o ---* -&-,&
0
.I
aS
\A
E
0
2
4
\1A
6
TAME thr) FIG. 4. Effect of 2'-deoxyadenosine on growth and viability. BG1 was incubated in the presence and absence of 2-deoxyadenosine (15 gg/ml) under growth conditions. Turbidity and viable cell count were determined at intervals of 1 hr. Viability: 0 pg/ml, 0; 15 pg/ml, A. Turbidity: 0 pg/ml, 0; 15 pg/mi, A.
\--,A
0
-4-
3
0
2
4
6 TIME (hr)
8
A
24
FIG. 6. Effect of cold deoxyadenosine on the loss
of tritiated deoxyadenosine incorporated previously. A suspension of BGJ was grown for 24 hr in the presslight variations in initial titer. Concentrations ence of deoxyadenosine, 4 pg (1 1iCi)/ml. The susof deoxyadenosine which block the net in- pension was centrifuged, resuspended in fresh mecrease in DNA within 4 hr do not hinder the dium, and divided into four portions. Various connet synthesis of RNA. Furthermore, high con- centrations of cold deoxyadenosine were added (4 centrations of deoxyadenosine induce the deg- pg/ml, 0; 10 ug/ml, 0; 20 ig/ml, A; 50 ug/ml, A) radation of DNA after 4 hr of incubation. The and the suspensions incubated under growth condislight increase in the level of net RNA syn- tions. thesis in the presence of deoxyadenosine over that of the control is interpreted as being concentrations above 4 pg/ml. As shown in Fig. 7, the addition of 50 ,ug of tritiated deoxywithin the limits of experimental variation. The apparent decrease in net DNA induced adenosine per ml blocked its own net incorpoby high concentrations of deoxyadenosine was ration after 4 hr. The lack of subsequent loss of further examined by using cells prelabeled counts during continued incubation probably with tritiated deoxyadenosine. The addition of results from the compensation for DNA degra50 ,ug of cold deoxyadenosine per ml resulted dation by the incorporation of label into the in a decrease in trichloroacetic acid-precipi- RNA (Table 1). table counts within 5 hr (Fig. 6). Lower concenDISCUSSION trations were much less effective. However, In A. quadruplicatum, as in many other sysafter 24 hr, loss of counts occurred at all tested
174
INGRAM AND FISHER 0
*
0 c
E
Oeo--_O
.O-
10 o
0
5 0
2
4 6 8 TIME (hr) FIG. 7. Effect of deoxyadenosine corwcentration on its incorporation. Cells were prelabelted for 4.5 hr with 4 ug of deoxyadenosine per ml (1If1Ci/ml). The suspension was split into two parts, (and sufficient tritiated 2-deoxyadenosine (of the samie specific activity) was added to one part to raise tthe concentration of 50 ug/ml, whereas the other w(as maintained as a control (containing 5 pg/ml; 1 ACi I/mi). Control, 0; 50 pg/ml, 0.
aslmaintained
tems (4, 11, 13), deoxyadenosine cfan be used to
inhibit DNA synthesis with somie specificity. Deoxyadenosine triphosphate app)ears to be a key negative effector for the rediuctase which converts purine and pyrimidine ribosides to deoxyribosides (15, 16, 19), and t;his has been suggested as the probable cause ffor inhibition of DNA synthesis in some systenns. However, deoxyadenosine has also been sh own to exert regulatory activity on other enzyi mes involved in purine and pyrimidine metabo]lism (25, 27). The stimulation of DNA degradattion by deoxyadenosine suggests the possible caccurrence in A. quadruplicatum of specific nm icleases activated by deoxyadenosine or r elated compounds. Nucleases of this type htave been reported previously in a number c)f other bacterial systems (3, 21). The rapid loss oss of O viavlability caused by deoxyadenosine may result from an impairment of nucleic accid synthesis, caused by an imbalance in the prrecursor pool, coupled with DNA degradation. ACKNOWLEDGMENTS
L.O.I. is a postdoctoral investigator supp-orted by subcontract 3322 from the Biology Division, Oak Ridge National Laboratory. Oak Ridge National Laboratory is operatted by the Union
J. BACTERIOL.
Carbide Corporation for the United States Atomic Energy Commision.
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7. Gross, W. A., W. H. Deitz, and T. M. Cook. 1965. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 89:1068-1074. 8. Hatcher, D. W., and G. Goldstein. 1969. Improved methods for the determination of RNA and DNA. Anal. Y., Biochem. 31:42-50. 9. Hirota, A. Ryter, and F. Jacob. 1968. Thermosensi-
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1972. Effects of selected inhibitors on growth and cell division inAgmenellum. Arch. Mikrobiol. 81:1-12.
11. Kihlman, B. A., and G. Odmark. 1966. Effects of adenine nucleosides on chromosomes, cell division, and nucleic acid synthesis in Vicia faba. Hereditas 56:7182. 1. 12 Kratz, W. A., and J. Myers. 1955. Nutrition and growth of several blue-green algae. Amer. J. Bot. 42:282-287. 13. Lark, K. G. 1960. Studies on the mechanism regulating periodic DNA synthesis in synchronized cultures of
Alcaligenes fecalis. Biochim. Biophys. Acta 45:121132. 14. Lark, K. G., and C. Lark. 1966. Regulation of chromosome replication in Escherichia coli: a comparison of
the effects of phenethyl alcohol treatment with those
of amino acid starvation. J. Mol. Biol. 20:9-19. 15. Larsson, A., and P. Reichard. 1966. Enzymatic synthesis of deoxyribonucleotides. J. Biol. Chem. 241:2533-
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ficity of the enzyme system from Escherichia coli B.
J. Biol. Chem. 241:2540-2549. 17. Madan, V., and H. D. Kumar. 1971. Action of nalidixic acid and hydroxyurea on two blue-green algae. Z. Allg. Mikrobiol. 11:495-499. 18. Pigott, G. H., and N. G. Carr. 1971. The assimilation of nucleic acid precursors by intact cells and protoplasts of the blue-green alga, Anacystis nidulans. Arch. Mikrobiol. 79:1-6. 19. Reichard, P., Z. N. Canellakis, and E. S. Canellakis. 1961. Studies on a possible regulatory mechanism for
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DNA INHIBITION
the biosynthesis of DNA. J. Biol. Chem. 236:25142519.
20. Shiba, S., A. Terawaki, T. Taguchi, and J. Kawamata. 1959. Selective inhibition of formation of deoxyribonucleic acid in Escherichia coli by mitomycin C. Nature (London) 183:1056-1057. 21. Tsuda, Y., and B. S. Strauss. 1964. A deoxyribonuclease reaction requiring nucleoside di- or triphosphates. Biochemistry 3:1678. 22. Van Baalen, C. 1962. Studies on marine blue-green algae. Bot. Mar. 4:129-139. 23. Van Baalen, C. 1967. Further observations on the growth of single cells of coccoid blue-green algae. J. Phycol. 3:154-157. 24. Van Tubergen, R. P., and R. B. Setlow. 1961. Quantita-
175
tive radioautographic studies on exponentially growing cultures of Escherichia coli. The distribution of parental DNA, RNA, protein, and cell wall among progeny cells. Biophys. J. 1:589-625. 25. Vornovitskaja, G. I., V. S. Shapot, and T. I. Nicolskaja. 1968. Concerning the site of action of deoxyadenosine upon the synthesis of nucleic acids in the cancer cell. Biochim. Biophys. Acta 166:596-599. 26. Wehr, C. T., R. D. Kudma, and L. W. Parks. 1970. Effect of putative deoxyribonucleic acid inhibitors on macromolecular synthesis in Saccharomyces cerevisiae. J. Bacteriol. 102:636-641. 27. Yagil, E., and A. Rosner. 1970. Effect of adenosine and deoxyadenosine on the incorporation and breakdown of thymidine in Escherichia coli K-12. 103:417-421.
