JOURNAL OF BACTERIOLOGY, Aug. 1979, p. 694-696 0021-9193/79/08-0694/03$02.00/0

Vol. 139, No. 2

Increased Synthesis of Ribonucleotide Reductase After Deoxyribonucleic Acid Inhibition in Various Species of Bacteria DAVID FILPULA AND JAMES A. FUCHS* College of Biological Sciences, University of Minnesota, St. Paul, Minnesota Department of Biochemistry, 55108

Received for publication 18 April 1979

The specific activity of ribonucleotide reductase was found to increase significantly after deoxyribonucleic acid inhibition in seven species of bacteria investigated. This group of bacteria includes species with B12-dependent ribonucleotide reductase as well as some with an Escherichia coli-type ribonucleotide reductase.

The enzyme ribonucleotide reductase catalyzes the reduction of ribonucleotides to deoxyribonucleotides and thus constitutes the first reaction unique to the pathway of DNA synthesis. Two very different types of ribonucleotide reductases have been purified and characterized from bacteria. The enzyme purified from Escherichia coli is composed of two subunits (3), uses ribonucleotide diphosphates as substrates, and has an organic radical that can be destroyed by hydroxyurea at the active site (4). The enzyme purified from Lactobacillus leichmannii consists of a single polypeptide chain, requires B12 coenzyme for activity, and uses ribonucleoside triphosphate as substrate (14). A ribonucleotide reductase that requires B12 coenzyme has been reported for Bacillus megaterium, Rhizobium meliloti, Corynebacterium nephridii, and several Lactobacillus species (5, 12, 15). Bacillus subtilis appears to have a ribonucleotide reductase that does not require B12 coenzyme and is inhibited by hydroxyurea (1). The control of the synthesis of ribonucleotide reductase is not well understood in any organism. Recently, it was found that in E. coli DNA inhibition resulting from either addition of chemical inhibitors or use of mutants defective in DNA initiation or elongation led to greatly increased synthesis of ribonucleotide reductase (8). These results necessitated a reinterpretation of experiments which showed that thymine deprivation in E. coli resulted in increased ribonucleotide reductase synthesis (2) and were interpreted as suggesting that a thymidine nucleotide served as a corepressor for synthesis of ribonucleotide reductase. The control of synthesis of ribonucleotide reductase in L. leichmannii and R. meliloti has been investigated. Increasing the concentrations of vitamin B12 or deoxyribonucleosides in the growth medium of L. leichmannii

represses ribonucleotide reductase synthesis (11). R. meliloti cells grown in a medium deficient in cobalt, which is a constituent of B12 compounds, have 5- to 10-fold-increased specific activity of ribonucleotide reductase (6). Since all of these growth conditions would appear to reduce the intracellular concentration of deoxyribonucleotides as well as derepress ribonucleotide reductase synthesis, it has been suggested that deoxynucleotides in general or a thymine nucleotide in particular acts as a corepressor in the control of reductase synthesis in these bacteria (6, 11). Since these conditions also cause DNA inhibition due to deoxyribonucleotide depletion, it would not be unreasonable to assume that ribonucleotide reductase synthesis in these organisms is controlled in the same manner as in E. coli. However, the B-12 type ribonucleotide reductase is so different from that of E. coli that it seems possible that organisms may have evolved different control for the synthesis of this enzyme. We therefore wished to analyze ribonucleotide reductase in a number of species of bacteria to see whether activity would increase after DNA inhibition. The cultures of bacteria were obtained from the following sources at the University of Minnesota: B. subtilis WT 6051 and B. megaterium NCTC 9848 from P. Chapman, Biochemistry; Streptococcus faecalis and Klebsiella strain A422 (species not identified) from J. Anderson, Biochemistry; Lactobacillus caseii from L. McKay, Food Science and Nutrition; C. nephridii from H. Hogenkamp, Biochemistry; and R. meliloti strain 2 from C. Vance, Agronomy and Plant Genetics. R. meliloti was grown in a mannitol medium previously described (5). L. caseii was grown in ADP medium which contained, per liter: tryptone, 10 g; yeast extract, 5 g; K2HPO4, 5 g; NaCl, 5 g; sodium citrate, 5 g;

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glucose, 10 g; Tween 80, 1 ml; MgSO4. 7H20, 0.8 g; FeSO4.7H20, 8 mg; MnCl2 4H20, 140 mg. Other bacteria were grown in L broth which was prepared as previously described (13). All media were

supplemented with vitamin

shown). Five hours was required for R. meliloti to stop DNA synthesis after addition of nalidixic acid. C. nephridii did not take up [3H]thymidine in labeling experiments, although nalidixic acid B12 (0.5 jg/ stopped growth 4 h after its addition, which

liter) plus CoCl2.6H20 (200 pg/liter) plus thymidine (20 ug/ml) to insure that these compounds were not limiting. Ribonucleotide reductase activity was measured in ether-permeabilized cells prepared as previously described for E. coli (9). In a standard

assay, the reaction mixture contained 0.8 umol of MgCl2, 2 jzmol of N-2-hydroxyethyl piperazine-N'-2'-ethanesulfonic acid (HEPES) buffer (pH 8.3), 2 ,umol of dithiothreitol, 50 nmol of NADPH, 60 nmol of ATP, 50 nmol of EDTA, 5 nmol of coenzyme B12, 25 nmol of [3H]CDP (3.4 x 10' cpm/nmol), and ether-treated cells in a fmal volume of 50 ,ul. After 0, 10, or 20 min of incubation at 370C, the assay mixture was boiled at 1000C for 2 min to stop the reaction. After the addition of 5 pl of dCMP (50 mM), the mixture was then treated with 5,ul of apyrase (12 mg/ml) for 30 min at 370C. The mixture was again heated to 1000C for 2 min and then centrifuged for 2 min in an Eppendorf microcentrifuge, and 10 Al of the supernatant was spotted on polyethyleneimine-cellulose plates and chromatographed and processed as described previously (9).

Two antibiotics, nalidixic acid and mitomycin C, which have been established as preferential inhibitors of DNA synthesis in a wide variety of bacteria (7, 10), were used in this study. Rapid inhibition of DNA synthesis with the concentration of antibiotic used was verified for B. subtilis, B. megaterium, L. caseii, Klebsiella sp., and S. faecalis by measuring incorporation of [3H]thymidine into acid-insoluble material (data not

suggests that DNA synthesis was inhibited. Exponentially growing cultures of the various organisms were divided into three cultures; one received DNA synthesis inhibitor, one received DNA synthesis inhibitor as well as chloramphenicol, and one remained untreated. After 2 h of treatment (9 h for R. meliloti), the cultures were chilled, harvested, and permeabilized by ether treatment and assayed for ribonucleotide reductase. Table 1 reports the results of these experimnents. In all cases, ribonucleotide reductase specific activity was significantly increased. The prevention of these increased activities by chloramphenicol suggests that de novo enzyme synthesis is required. It is interesting to note that in C. nephridii and R. meliloti chloramphenicol reduces ribonucleotide reductase specific activity six- to sevenfold, suggesting that the enzyme is being rapidly synthesized and degraded in these organisms. These are the two organisms that also show the least increase in ribonucleotide reductase specific activity. Since optimal conditions for assays of the enzyme from various bacteria were not determined, the differences in specific activity among organisms may not be meaningful. To determine the type of enzyme present in species we utilized that had not previously been investigated, we tested ether-permeabilized cells for sensitivity to hydroxyurea. The B. subtilis and Klebsiella sp. enzyme was inhibited 90% by incubation at 00C for 30 min in the presence of 10 mM hydroxyurea before enzyme assay. The enzyme from S. faecalis was inhibited 60%. The ribonucleotide

TABLE 1. Ribonucleotide reductase specific activity in several unrelated bacteria after DNA synthesis

inhibition Bacterium Bacterium

~Doublingb DNA inhibitor usedb time (min)a

Sp act (pmol of dCDP/min per mg of protein) +DNA inUntreated +DNA + hibitorin- hibitor CMC

B. subtilis 24 20 ug of NAL per ml, 2 h 16 B. megaterium 22 10 lAg of NAL per ml, 2 h 17 L. caseii 100 51 perml, 2 h 5jugofMITO S. faecalis 69 5 ug of MITO per ml, 2 h 22 Klebsiella sp. 25 40 ug of NAL per ml, 2 h 5 C. nephridii 70 of NAL per ml, 2 h 45 20jug R. meliloti 170 40 ug of NAL per ml, 9 h 120 a Cells were grown as described in the text at 300C for Lactobacillus, Streptococcus, 370C for others. b NAL, Nalidixic acid; MITO, mitomycin C. c CM, Chloramphenicol (100 ytg/ml).

98 13 60 10 290 94 170 15 52 4 110 6 270 21 and Rhizobium and

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reductase in these organisms is presumably similar to the E. coli type enzyme. The other organisms studied had previously been shown to have a B12 coenzyme-requiring enzyme which is insensitive to hydroxyurea (5, 12, 15). It is interesting that although procaryotic organisms have evolved two very different types of enzymes to reduce ribonucleotides to deoxyribonucleotide, they have evolved a similar means of controlling the synthesis of these enzymes. This work was supported by Public Health Service grant GM20884 from the National Institute of General Medical Science to J.A.F. and Public Health Service training grant GM07094 from the National Institute of General Medical Science to D.F. LITERATURE CITED 1. Bazill, G. W., and D. Karamata. 1972. Temperaturesensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis. Mol. Gen. Genet. 117:19-29. 2. Biswas, C., J. Hardy, and W. S. Beck. 1965. Release of repressor control of ribonucleotide reductase by thymine starvation. J. Biol. Chem. 240:3631-3640. 3. Brown, N. C., Z. N. Canellakis, B. Lundin, P. Reichard, and L. Thelander. 1969. Ribonucleoside diphosphate reductase. Purification of the two subunits, proteins B1 and B2. Eur. J. Biochem. 9:561-573. 4. Brown, N. C., R. Eliasson, P. Reichard, and L Thelander. 1969. Spectrum and iron content of protein B2 from ribonucleoside diphosphate reductase. Eur. J. Biochem. 9:512-518. 5. Cowles, J. R., and H. J. Evans. 1968. Some properties of the ribonucleotide reductase from Rhizobium meliloti. Arch. Biochem. Biophys. 127:770-778.

J. BACTERIOL. 6. Cowles, J. R., H. J. Evans, and S. A. Russell. 1969. B,2 coenzyme-dependent ribonucleotide reductase in Rhizobium species and the effects of cobalt deficiency on the activity of the enzyme. J. Bacteriol. 97:1460-1465. 7. Cozzarelli, N. R. 1977. The mechanism of action of inhibitors of DNA synthesis. Annu. Rev. Biochem. 46: 641-668. 8. Filpula, D., and J. A. Fuchs. 1977. Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 130:107-113. 9. Fuchs, J. A., and H. R. Warner. 1975. Isolation of an Escherichia coli mutant deficient in glutathione synthesis. J. Bacteriol. 124:140-148. 10. Gale, E. F., E. Cundliffe, P. E. Reynolds, M. H. Richmond, and M. J. Waring. 1972. The molecular basis of antibiotic action. Wiley, London. 11. Ghambeer, R. K., and R. L. Blakeley. 1966. Cobamide and ribonucleotide reduction. III. Factors influencing the level of cobamide-dependent ribonucleoside triphosphate reductase in Lactobacillus leichmannii. J. Biol. Chem. 241:4710-4716. 12. Gleason, F. J., and H. P. Hogenkamp. 1972. 5'-Deoxyadenosylcobalamin-dependent ribonucleotide reductase: a survey of its distribution. Biochim. Biophys. Acta 227:446-470. 13. Luria, S. E., and J. W. Burrous. 1957. Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74: 461476. 14. Panagou, D., M. D. Orr, J. D. Dunstone, and R. Blakeley. 1972. A monomeric, allosteric enzyme with a single polypeptide chain. Ribonucleotide reductase of Lactobacillus leichmannii. Biochemistry 11:23782388. 15. Yau, S., and J. T. Wachsman. 1973. The Bacillus megaterium ribonucleotide reductase: evidence for a B12 coenzyme requirement. Mol. Cell. Biochem. 1:101105.

Increased synthesis of ribonucleotide reductase after deoxyribonucleic acid inhibition in various species of bacteria.

JOURNAL OF BACTERIOLOGY, Aug. 1979, p. 694-696 0021-9193/79/08-0694/03$02.00/0 Vol. 139, No. 2 Increased Synthesis of Ribonucleotide Reductase After...
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