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Effect of deoxyribonucleosides on deoxyribonucleic acid synthesis in vitamin B12-deficient Lactobacillus leichmannii
222
SHORT COMMUNICATIONS
Effect of deoxyribonucleosides on deoxyribonucleic acid synthesis in vitamin B1~leficient lactobacillus leichmannii The first metabolic function proposed for vitamin B12 concerned its role in the biosynthesis of thymidine 1 which was traced to the formation of the deoxyribosyl moiety in certain lactobacilli t. Subsequently, the biosynthesis of deoxyribosyl derivatives was shown to occur in rats from ribosyl derivatives without cleavage of the base and carbohydrate moieties 8. In cultures of Lactobacillus l e i c h m a n n i i 313 the DNA concentration per unit dry cell mass has been reported by WACKER et al L to be 5-fold greater in cells grown in vitamin B12-supplemented medium than in cells grown in deoxycytidine-supplemented medium. DINNING et al. 5 demonstrated a role of vitamin Bt2 in increasing the incorporation of formate into the DNA thymine of L. l e i c h m a n n i i 313, and in the conversion of ribose to DNA deoxyribose6in this organism. In a recent communication, BIRNIE AND CROSBIE 7 considered the possibility that alterations in the DNA concentration per unit dry cell mass might affect the conclusions of DINNING et al. 5 On reinvestigation of the DNA content of cultures of L . l e i c h m a n n i i 313, these authors found that under their experimental conditions there is initially a greater increase in the rate of DNA synthesis of cultures grown on deoxycytidinesupplemented medium, but after 8 h growth there is no significant difference in the amount of DNA concentration/mg of cells grown on the deoxycytidine-supplemented or vitamin Bx2-supplemented media. Since differences of the type reported by WACKER et al. 4 had been observed in this laboratory, the discrepancies in the relative DNA content of cultures of L. leichmannii 313 grown under different conditions merited further study. These data are also important in studies concerning the conversion of ribonucleosides to deoxyribonucleosides because vitamin Bl~-deficient cell preparations with diminished DNA content might give results concerning the synthesis of de0xyribonucleosides which could be easily misinterpreted. Cells of L. leichmannii used for the inoculation of the growth flasks were grown on lO ml of a previously described medium s supplemented with O.Ol 9g vitamin B12 ml. The cells were harvested after 20-24 h, washed twice with sterile saline, and resuspended in lO ml of sterile saline, and 0.2 ml of the cell suspension was used to inoculate 25 ml of growth medium. The growth medium was modified to contain o.2# mole/ml of cytidine, and vitamin Bx~ and deoxynucleosides were added as indicated. When deoxynucleosides were used in place of vitamin B12, 0.025 units of apoerythein 9 (kindly supplied by Prof. R. E. EAKIN) Was added without heating to each 25 ml of sterile medium. This amount of apoerythein will bind o.o25 ~g of vitamin BI~ and thereby removes trace amounts of vitamin B12 which might be present in the medium. The growth flasks were incubated for 16 h at 37 °. The cells were then precipitated, washed and extracted with HCI04 as previously described ~, and the DNA content of the extracts was determined by the method of CERIOTT119.The amount of growth was determined turbidimetrically, and the mg dry weight of cells was calculated by comparing the turbidimetric readings to a standard curve of mg dry weight of cells plotted versus turbidity readings. Cell morphology was observed using a phasecontrast microscope, and average cell length given in Table I is an average of 80 to lOO individual cell-length measurements. The experiments of BIRNIE AND CROSBIE were repeated and essentially the same Biochim. Biophys. Acta, 55 (1962) 222-224
SHORT COMMUNICATIONS
223
results were obtained. However, under their experimental conditions, the inoculurn cells for both the vitamin B u and deoxycytidine-supplemented media were grown in a medium supplemented with vitamin Blz, and the inocula were very large so that only approximately one cell generation occurred before DNA measurements were made. Thus, under these conditions, it is unlikely that a vitamin Bu deficiency was obtained in the deoxycytidine-supplemented medium. Using a small inoculum of washed cells with apoerythein added to react with any residual vitamin Bls and a longer incubation time, differences in the amount of DNA per dry cell weight were observed (Table I). TABLE I EFFECTS OF VITAMIN B I | AND DEOXYEIBONUCLEOSIDESUPON THE DNA CONTENT AND CELL LENGTH OF Lactobacillus leichmannii 313
Supplements per (ml) Expt. I
D¢ozycytidine (l~moles) 0.002
G r o w t h (rag cells/ml) D N A (/~g/mg cells) Average cell length (p)
0.36 3 12.6
O.OO5
O.OI
0.05
0.52 5 7.5
0.62 7 4.3
0.64 14 3-3
0.2
0.53 2i 3.z
0.54 17 3.2
0. 5 0.54 9 4.x
Deozyfibonud¢osid~s* (pmoles)
Expt. II O.OO2 G r o w t h (rag cells/ml)~, D N A (/~.g/mg cells) Average cell length (/U)
O.I
o.53 4 II. 4
O.OO5
O.OX
0.05
O.I
0.2
0. 5
o.82 6 7.1
z.o 3 8 4.5
z.io x4 3-3
z.z 4 20 2-9
1.26 25 2.8
z.3o 37 2.6
Vitamin Bta (o.oz pg) 0.94 28 2.7
Vitamin Bzz (o.oz pg) 1.22 36 2. 4
" Total. c o n c e n t r a t i o n of deoxyribonucleosides composed of one-fourth the indicated concent r a t i o n s of deoxycytidine, thymidine, deoxyadenosine, and deoxyguanosine.
It has previously been observedn,xa that certain vitamin B12-requiring lactobacilli grown in the absence of vitamin B12 but in the presence of low concentrations of deoxsn:ibonucleosides were elongated and filamentous rather than in the normal short-rod condition when adequate vitamin B12 was present. This observation could account for the apparent difference in DNA per unit dry cell mass if comparisons were made between cells grown with sufficient vitamin Bz2 but with insufficient amounts of deox3n'ibonucleosides. When observations of cell morphology were made using a phase-contrast microscope, it was found that at low concentrations of deoxycytidine the average cell length was from 2-5 times longer than the length of cells grown in vitamin Bz2-supplemented medium. Accordingly, cell morphology and DNA content per unit dry cell mass were studied over a wide range of deoxycytidine concentrations and compared with the corresponding results from cells grown in adequate amounts of vitamin Bz2. The results are tabulated in Table I. With increasing concentrations of deoxycyt:idineup to o.z pmole/ml, the cell length decreases and the DNA content (on a dry weight basis) increases, but neither of these values quite attain that of cells grown in the presence Of vitamin BI~. Further increases in the deoxycytidine concentration have an adverse effect and apparently interfere with DNA synthesis, since a combination of deoxycytidine, thymidine, deoxyadenosine, and deoxyguanosine in total concentration of o.I pmole/ml (Table I) approximates the effect of deoxycytidine alone, but at higher concentrations continues to decrease cell length and increase Biochim. Biophys. Acta, 55 (I962) 222-224
224
SHORT COMMUNICATIONS
DNA content per unit dry weight of cells. In contrast to deoxycytidine alone, appropriately high concentrations of the mixture give cells which in morphology and DNA content/unit mass of cells do not signifiCantly differ from vitamin Bx2-grown cells. Thus, a mixture of deoxyribosyl derivatives, but not deoxycytidine alone, can replace vitamin B12 not only in promoting growth but also in replacing its role in DNA synthesis. Therefore, investigations involving comparative studies between deoxyribonucleosides and vitamin Bt2 as growth supplements for L. ldchmannii should be considered and evaluated in light of the above findings.
Clayton Foundation Biochemical Institute and the Department o/ Chemistry, The University o/ Texas Austin, Texas (U.S.A.)
S. J. NORTON" J. M. RAVEL W.
SHIVE
1 W . SHIVE, J. M. RAVEL AND R. E. EAKI~, J. Am. Chem. Soc., 7 ° (1948) 2614. I W . SHIVE, J. M. RAVEL AND W . M. HARDING, J. Biol. Chem., 176 (1948) 991 8 I. A. ROSE AND B. S. SCHWEIGERT, f. Biol. Chem., 202 (I953) 635. 4 A. WACKER, D. PFAHL AND I. SCHRODER, Z. Natur/orsch., 126 (1957) 5 lo5 j . S. DINNING, B. K. ALLEN. R. S. YOUNG AND P. L. DAY, J. Biol. Chem., 233 (I958) 674' W . H. SPELL, JR. AND J. S. DINNING, J. Am. Chem. Soc., 81 (I959) 3804 • T G. D. BIRNIE AND G. W . CROSBIE, Biochim. Biophys. Acta, 46 (I96I) 397. s U. S. Pharmacopeia, Vol. 16, M a c k P u b l i s h i n g Co., E a s t o n , Pa., p. 888. g J. L. TERNBERG AND R. E. EAKIN, J. Am. Chem. Soc., 71 (1949) 3858. 10 G. CERIOTTI, J. Biol. Chem., 198 (1952) 297. ax H. JEENER ANn R. JEENER, Exptl. Cell Research, 3 (I95 z) 675. xz W. S. BECK, B. HURLOCK AND M. LEVm, Federation Proc., I9 (196o) 417 .
Received July 24th, 1961 " P r e d o e t o r ~ l Fellow ( C F - I o 779) N a t i o n a l Cancer I n s t i t u t e , U n i t e d S t a t e s Public H e a l t h Service.
Biochim. Biophys. Acta, 55 (1962) 2 2 2 - 2 2 4
SHORT COMMUNICATIONS
Effect of deoxyribonucleosides on deoxyribonucleic acid synthesis in vitamin B1~leficient lactobacillus leichmannii The first metabolic function proposed for vitamin B12 concerned its role in the biosynthesis of thymidine 1 which was traced to the formation of the deoxyribosyl moiety in certain lactobacilli t. Subsequently, the biosynthesis of deoxyribosyl derivatives was shown to occur in rats from ribosyl derivatives without cleavage of the base and carbohydrate moieties 8. In cultures of Lactobacillus l e i c h m a n n i i 313 the DNA concentration per unit dry cell mass has been reported by WACKER et al L to be 5-fold greater in cells grown in vitamin B12-supplemented medium than in cells grown in deoxycytidine-supplemented medium. DINNING et al. 5 demonstrated a role of vitamin Bt2 in increasing the incorporation of formate into the DNA thymine of L. l e i c h m a n n i i 313, and in the conversion of ribose to DNA deoxyribose6in this organism. In a recent communication, BIRNIE AND CROSBIE 7 considered the possibility that alterations in the DNA concentration per unit dry cell mass might affect the conclusions of DINNING et al. 5 On reinvestigation of the DNA content of cultures of L . l e i c h m a n n i i 313, these authors found that under their experimental conditions there is initially a greater increase in the rate of DNA synthesis of cultures grown on deoxycytidinesupplemented medium, but after 8 h growth there is no significant difference in the amount of DNA concentration/mg of cells grown on the deoxycytidine-supplemented or vitamin Bx2-supplemented media. Since differences of the type reported by WACKER et al. 4 had been observed in this laboratory, the discrepancies in the relative DNA content of cultures of L. leichmannii 313 grown under different conditions merited further study. These data are also important in studies concerning the conversion of ribonucleosides to deoxyribonucleosides because vitamin Bl~-deficient cell preparations with diminished DNA content might give results concerning the synthesis of de0xyribonucleosides which could be easily misinterpreted. Cells of L. leichmannii used for the inoculation of the growth flasks were grown on lO ml of a previously described medium s supplemented with O.Ol 9g vitamin B12 ml. The cells were harvested after 20-24 h, washed twice with sterile saline, and resuspended in lO ml of sterile saline, and 0.2 ml of the cell suspension was used to inoculate 25 ml of growth medium. The growth medium was modified to contain o.2# mole/ml of cytidine, and vitamin Bx~ and deoxynucleosides were added as indicated. When deoxynucleosides were used in place of vitamin B12, 0.025 units of apoerythein 9 (kindly supplied by Prof. R. E. EAKIN) Was added without heating to each 25 ml of sterile medium. This amount of apoerythein will bind o.o25 ~g of vitamin BI~ and thereby removes trace amounts of vitamin B12 which might be present in the medium. The growth flasks were incubated for 16 h at 37 °. The cells were then precipitated, washed and extracted with HCI04 as previously described ~, and the DNA content of the extracts was determined by the method of CERIOTT119.The amount of growth was determined turbidimetrically, and the mg dry weight of cells was calculated by comparing the turbidimetric readings to a standard curve of mg dry weight of cells plotted versus turbidity readings. Cell morphology was observed using a phasecontrast microscope, and average cell length given in Table I is an average of 80 to lOO individual cell-length measurements. The experiments of BIRNIE AND CROSBIE were repeated and essentially the same Biochim. Biophys. Acta, 55 (1962) 222-224
SHORT COMMUNICATIONS
223
results were obtained. However, under their experimental conditions, the inoculurn cells for both the vitamin B u and deoxycytidine-supplemented media were grown in a medium supplemented with vitamin Blz, and the inocula were very large so that only approximately one cell generation occurred before DNA measurements were made. Thus, under these conditions, it is unlikely that a vitamin Bu deficiency was obtained in the deoxycytidine-supplemented medium. Using a small inoculum of washed cells with apoerythein added to react with any residual vitamin Bls and a longer incubation time, differences in the amount of DNA per dry cell weight were observed (Table I). TABLE I EFFECTS OF VITAMIN B I | AND DEOXYEIBONUCLEOSIDESUPON THE DNA CONTENT AND CELL LENGTH OF Lactobacillus leichmannii 313
Supplements per (ml) Expt. I
D¢ozycytidine (l~moles) 0.002
G r o w t h (rag cells/ml) D N A (/~g/mg cells) Average cell length (p)
0.36 3 12.6
O.OO5
O.OI
0.05
0.52 5 7.5
0.62 7 4.3
0.64 14 3-3
0.2
0.53 2i 3.z
0.54 17 3.2
0. 5 0.54 9 4.x
Deozyfibonud¢osid~s* (pmoles)
Expt. II O.OO2 G r o w t h (rag cells/ml)~, D N A (/~.g/mg cells) Average cell length (/U)
O.I
o.53 4 II. 4
O.OO5
O.OX
0.05
O.I
0.2
0. 5
o.82 6 7.1
z.o 3 8 4.5
z.io x4 3-3
z.z 4 20 2-9
1.26 25 2.8
z.3o 37 2.6
Vitamin Bta (o.oz pg) 0.94 28 2.7
Vitamin Bzz (o.oz pg) 1.22 36 2. 4
" Total. c o n c e n t r a t i o n of deoxyribonucleosides composed of one-fourth the indicated concent r a t i o n s of deoxycytidine, thymidine, deoxyadenosine, and deoxyguanosine.
It has previously been observedn,xa that certain vitamin B12-requiring lactobacilli grown in the absence of vitamin B12 but in the presence of low concentrations of deoxsn:ibonucleosides were elongated and filamentous rather than in the normal short-rod condition when adequate vitamin B12 was present. This observation could account for the apparent difference in DNA per unit dry cell mass if comparisons were made between cells grown with sufficient vitamin Bz2 but with insufficient amounts of deox3n'ibonucleosides. When observations of cell morphology were made using a phase-contrast microscope, it was found that at low concentrations of deoxycytidine the average cell length was from 2-5 times longer than the length of cells grown in vitamin Bz2-supplemented medium. Accordingly, cell morphology and DNA content per unit dry cell mass were studied over a wide range of deoxycytidine concentrations and compared with the corresponding results from cells grown in adequate amounts of vitamin Bz2. The results are tabulated in Table I. With increasing concentrations of deoxycyt:idineup to o.z pmole/ml, the cell length decreases and the DNA content (on a dry weight basis) increases, but neither of these values quite attain that of cells grown in the presence Of vitamin BI~. Further increases in the deoxycytidine concentration have an adverse effect and apparently interfere with DNA synthesis, since a combination of deoxycytidine, thymidine, deoxyadenosine, and deoxyguanosine in total concentration of o.I pmole/ml (Table I) approximates the effect of deoxycytidine alone, but at higher concentrations continues to decrease cell length and increase Biochim. Biophys. Acta, 55 (I962) 222-224
224
SHORT COMMUNICATIONS
DNA content per unit dry weight of cells. In contrast to deoxycytidine alone, appropriately high concentrations of the mixture give cells which in morphology and DNA content/unit mass of cells do not signifiCantly differ from vitamin Bx2-grown cells. Thus, a mixture of deoxyribosyl derivatives, but not deoxycytidine alone, can replace vitamin B12 not only in promoting growth but also in replacing its role in DNA synthesis. Therefore, investigations involving comparative studies between deoxyribonucleosides and vitamin Bt2 as growth supplements for L. ldchmannii should be considered and evaluated in light of the above findings.
Clayton Foundation Biochemical Institute and the Department o/ Chemistry, The University o/ Texas Austin, Texas (U.S.A.)
S. J. NORTON" J. M. RAVEL W.
SHIVE
1 W . SHIVE, J. M. RAVEL AND R. E. EAKI~, J. Am. Chem. Soc., 7 ° (1948) 2614. I W . SHIVE, J. M. RAVEL AND W . M. HARDING, J. Biol. Chem., 176 (1948) 991 8 I. A. ROSE AND B. S. SCHWEIGERT, f. Biol. Chem., 202 (I953) 635. 4 A. WACKER, D. PFAHL AND I. SCHRODER, Z. Natur/orsch., 126 (1957) 5 lo5 j . S. DINNING, B. K. ALLEN. R. S. YOUNG AND P. L. DAY, J. Biol. Chem., 233 (I958) 674' W . H. SPELL, JR. AND J. S. DINNING, J. Am. Chem. Soc., 81 (I959) 3804 • T G. D. BIRNIE AND G. W . CROSBIE, Biochim. Biophys. Acta, 46 (I96I) 397. s U. S. Pharmacopeia, Vol. 16, M a c k P u b l i s h i n g Co., E a s t o n , Pa., p. 888. g J. L. TERNBERG AND R. E. EAKIN, J. Am. Chem. Soc., 71 (1949) 3858. 10 G. CERIOTTI, J. Biol. Chem., 198 (1952) 297. ax H. JEENER ANn R. JEENER, Exptl. Cell Research, 3 (I95 z) 675. xz W. S. BECK, B. HURLOCK AND M. LEVm, Federation Proc., I9 (196o) 417 .
Received July 24th, 1961 " P r e d o e t o r ~ l Fellow ( C F - I o 779) N a t i o n a l Cancer I n s t i t u t e , U n i t e d S t a t e s Public H e a l t h Service.
Biochim. Biophys. Acta, 55 (1962) 2 2 2 - 2 2 4