Incorporation of Deoxycytidine Into Deoxyribonucleic Acid Deoxycytidylate in Lactobacillus Acidophilus R-26 MINH-TAM B

JouRNAL OF BACTERIOLOGY, June 1976, p. 1136-1140 Vol. 126, No. 3 Copyright © 1976 American Society for Microbiology Printed in U.S.A. Incorporation of Deoxycytidine into Deoxyribonucleic Acid Deoxycytidylate in Lactobacillus acidophilus R-26 MINH-TAM B. DAVIS AND DAVID H. IVES* Department ofBiochemistry, College ofBiological Sciences, The Ohio State University, Columbus, Ohio 43210 Received for publication 14 January 1976

Lactobacillus acidophilus R-26, a strain deficient in ribonucleotide reductase, was grown with [G-'4C]deoxycytidine as the only source of deoxyribose in the medium. Of the radioactivity incorporated into deoxyribonucleic acid, a fifth moved directly into deoxyribonucleic acid deoxycytidylate, without deamina- tion. Furthermore, deoxycytidine and thymidine nucleotides had similar sugar/ base ratios, suggesting a direct conversion of deoxycytidine nucleotides to thy- midine nucleotides through deamination, without further dilution by glycosyl transfer. Although radioactivity was incorporated into both the sugar and base moieties of deoxyribonucleic acid pyrimidine deoxyribonucleotides, only the sugar moiety of purine deoxyribonucleotides was labeled. Purine deoxyribonu- cleotides probably were synthesized by glycosyl transfer from [G- '4C]deoxycytidine to unlabeled purines, followed by phosphorylation of the deoxynucleotides. A number of bacteria are incapable of incor- American Type Culture Collection (ATCC 11506). porating deoxycytidine (dCyd) directly into de- The bacteris were grown overnight at 37 C in B,2 oxyribonucleic acid (DNA) deoxycytidylate assay medium USP (Difco) containing a minimum without prior deamination or of the amount of unlabeled dCyd necessary for normal cleavage growth (3.52 uM dCyd). The cells were then col- glycosidic bond or both. Examples reported in- lected, washed in 0.85% NaCl, and resuspended in clude Escherichia coli, Salmonelkl typhimu- 37 C in 10 ml offresh B,2 assay medium containing 5 rium (8), and pneumococci (1). This deficiency ,uCi (142 mCi/mmol) of [G-'4CJdCyd and 2.5 mg of is believed to be due to the lack of dCyd kinase adenosine. The presence of excess adenosine in the in these organisms. However, Lactobacillus medium increased the cellular uptake of ['4C]dCyd acidophilus, strain R-26, which is incapable of about fourfold. The bacteria then grew logarithmic- forming deoxynucleotides by direct reduction of ally for 3 h and 10 min, during which time the ribonucleotides, has kinase activities toward optical density at 540 nm increased from 0.15 to 0.81. all four as described in an No growth lag was observed after the addition ofthe deoxynucleosides, radioactive medium. All absorbances were read earlier report from this laboratory (2). The with a Gilford spectrophotometer. The cells were growth requirements of this strain can be met harvested by centrifugation at 3,000 x g for 5 min at by any single deoxynucleoside, and it has been 5 C. used for microbiological assays ofDNA or deox- The cell-free medium was chromatographed in yribose-containing compounds (4). Neverthe- solvent system III (below). Fresh [3HldCyd was co- less, it is not clear how deoxyribonucleosides chromatographed with the medium to allow un- are incorporated and distributed in the DNA of changed ['4C]dCyd to be distinguished from its lactobacilli. breakdown products since materials in the medium We the of interfered with ultraviolet detection ofan unlabeled studied incorporation [G-'4C]dCyd marker. into L. acidophilus R-26 under conditions in Distribution of '4C radioactivity in DNA nucleo- which the labeled compound was the only tides and other cellular fractions. The cell pellet source of deoxyribose. The distribution of the was quickly washed twice with cold 0.85% NaCl and base and sugar moieties was analyzed, and the then extracted with cold 0.4 N perchloric acid for 1 h. predominant pathways responsible for the syn- The acid-soluble fraction was neutralized to pH 7 thesis of purine and pyrimidine deoxyribonu- with KOH, the KCl04 precipitate was removed by cleotides in this organism were defined. centrifugation, and the supernatant was analyzed by paper electrophoresis. The lipid fraction was collected by washing the MATERIALS AND METHODS acid precipitate with 100% ethanol and then 100% Bacterial growth and incorporation procedures. ethanol-ether (3:1, vol/vol), dried, and counted. L. acidophilus R-26 stock was obtained from the The pellet containing DNA, ribonucleic acid 1136 VOL. 126, 1976 INCORPORATION OF dCyd INTO BACTERIAL DNA 1137 (RNA), and protein was treated overnight with 1 N and then counted in modified Triton-toluene solvent KOH at 37 C. After neutralization with HCl, tri- (7). To count the radioactivity of whole cells, 1 ml of chloroacetic acid was added to give a final concen- NCS solubilizer (Amersham/Searle) was added to tration of 10%. The resulting precipitate containing the cell pellet and incubated overnight at room tem- DNA and protein was treated with 50 ug of deoxyri- perature. In this case the counting solution con- bonuclease in 25 ,lA of 4 mM MgSO4 and 0.02 M ace- sisted only of 4 g of PPO (2,5-diphenyloxazole) and tate buffer, pH 5, for 6 to 7 h at room temperature. 100 mg of POPOP[1,4-bis-(2)-(5-phenyloxazolyl)- The solution was then brought to pH 8 with 0.1 M benzene] per liter of toluene. tris(hydroxymethyl)aminomethane (Tris), pH 9.622 Radioactivity was also detected with a radiochro- (subscript indicates the temperature, degrees Cel- matogram scanner (Packard model 7201). sius, at which the pH was measured), and 5 ,g of Nucleosidase preparation. L. acidophilus R-26 phosphodiesterase was added and incubated over- cells, grown in modified rich broth (2), were washed night at room temperature. The reaction was with 0.85% NaCl and then suspended in 50 mM Tris- stopped by heating in boiling water for 1 to 2 min hydrochloride, pH 8.022. The cell suspension was and then centrifuged. The resulting nucleoside-5'- passed through a French press at 10,000 lb/in2 and phosphates were separated by paper electrophoresis. centrifuged at 27,000 x g for 1 h. The resulting The region containing each nucleotide was cut out supernatant was treated with 2.5% final concentra- and eluted with water, and the eluates were concen- tion of streptomycin sulfate and then precipitated trated under a stream of nitrogen. The nucleotides with 60% ammonium sulfate. The ammonium sul- were then hydrolyzed to nucleosides by treating fate pellet was dissolved in 20 mM Tris-hydrochlo- with 6.5 gg of alkaline phosphatase in 20 g.l of 0.5 ride, pH 7.422, dialyzed overnight against 10 mM mM Tris, pH 822, for 4 to 5 h at room temperature. Tris-succinate-0.1 mM magnesium acetate (pH The reaction was stopped by immersing tubes in 7.422), and stored at -20 C. The nucleosidase activ- boiling water for 1 to 2 min. The solutions were ity was found to be more active at pH 4.5 to 5 than at then adjusted to pH 4.9 with 5 ,ul of maleic acid and pH 7.4. treated overnight at room temperature with 224 ug Chemicals and enzymes. Unlabeled nucleosides of bacterial nucleosidase (described below). The and nucleotides were purchased from P-L Biochemi- reaction was stopped by heating in boiling water cals, Calbiochem, and Sigma Chemical Co. [G- for 1 to 2 min, followed by centrifugation. Compo- '4C]dCyd was obtained from Amersham/Searle. The nents of the nucleotide hydrolysates were separated purity of the [14C]dCyd was checked by paper chro- by paper chromatography. matography and found to be greater than 98%. De- Paper chromatography and electrophoresis. De- oxyribonuclease (type I from bovine pancreas) and scending paper chromatography was carried out on alkaline phosphatase (type III from E. coli) were Schleicher and Schuell 589 orange ribbon. The paper purchased from Sigma Chemical Co. Phosphodies- was prewashed with 0.5 N formic acid and then terase (Crotalis terrificus terrificus venom) was ob- washed with water. The following solvents were tained from Boehringer Mannheim. used: solvent system I, isobutyric acid-water-0. 1 M sodium ethylenediaminetetraacetate-concentrated RESULTS ammonium hydroxide-toluene (160:22:3:2:20, by vol- Incorporation of [G-14C]dCyd into different ume) (12); solvent system II, tert-butanol-methyl ethyl ketone-water-concentrated ammonium hy- cell fractions. L. acidophilus R-26 was grown droxide (40:30:20:10, by volume) (3); solvent system with [G-'4C]dCyd as the only source of deoxyri- III, isopropanol-water-concentrated HCl (65:18.4: bose, and the incorporation ofradioactivity into 16.6, by volume) (3). different cell fractions was deternined. Of the Bases, nucleosides, and nucleotides were located total radioactivity initially available in the me- with a 254 nM Mineralite. Deoxyribose was detected dium, 41% was taken up by the cells (Fig. 1). by spraying with 3% p-anisidine-HCl in n-butanol- This relatively efficient incorporation was due ethanol-water containing a trace of stannous chlo- to the presence of excess adenosine; in its ab- ride (4:1:1, by volume) only to the papers that were sence 10% to be analyzed by chromatographic scanning rather only of the isotope was retained by than by counting. the cells. About half of the labeled dCyd in the Paper electrophoresis was carried out on pre- medium was undegraded at the end of the washed sheets of Whatman 3MM paper. Electropho- growth period, and chromatographic analysis retic separation of the deoxynucleotides from DNA in solvent system III revealed that cytosine was hydrolysates was performed in 0.05 M ammonium the primary degradation product (data not formate, pH 3.92, at 3,200 V (80 mA) for 65 min. The shown). cellular acid-soluble fraction was analyzed by elec- The cellular acid soluble fraction constituted trophoresis in 0.05 M sodium acetate buffer, pH 5.2, only 4.5% ofthe radioactivity found in the cells. at 1,300 V (50 to 70 mA) for 85 to 105 min. Most of this fraction (74%) gave To aid in identifying metabolic products of dCyd electrophoretic in the above separations, unlabeled standards were mobilities in the nucleoside polyphosphate re- generally mixed with cellular extracts before sub- gion, 19% was nucleoside monophosphate, and jecting them to chromatography or electrophoresis. only 7% was nucleoside (not shown). Thus, Radioactivity analysis. Paper chromatograms most of the incorporated dCyd was phosphoryl- and electrophorograms were cut into 1-cm sections ated. A very small portion of the total radioac- and eluted with 1 ml of 0.2 M KCl-0.1 M HCl for 1 h tivity of the cells, 0.3%, appeared in the lipid 1138 DAVIS AND IVES J. BACTERIOL. fraction and 11% was found in RNA. The com- cells was incorporated into DNA (Fig. 1). The bined action of nucleosidase and transglycosi- DNA was enzymatically hydrolyzed, and the dation between [14C]dCyd and another base four purine or pyrimidine nucleoside-5'-phos- would probably provide labeled free pyrimidine phates were separated by electrophoresis (data base to the RNA precursor pool. not shown). The DNA hydrolysis was complete Most of the radioactivity taken up by the since no significant radioactivity was found in any regions other than those occupied by the Loi.1io5 MEDIUM nucleoside monophosphates. All four were la- beled and, ofthe total DNA label, deoxycytidyl- ate constituted about 21%, thymidylate 36%, 60.OX105 SUPERNATE 45.io CELLS deoxyadenylate 28%, and deoxyguanylate 15%. Distribution of 14C radioactivity in DNA I I I 1 base and sugar moieties. The isolated mononu- 40.7-10' 5.2X15- 10l1051 p2.3x105 were DNA RNA LIPID ACID- cleotides of DNA each eluted from the SOLUBLE paper electrophorogram and analyzed for the isotope content of the base and sugar. An ap- r 610 I } X1SI I 1O I I4 10 parent nucleosidase activity found in extracts dCMP dTMP dAMP dGMP ofL. acidophilus R-26 proved to be very useful in relatively complete hydrolysis of FIG. 1. Distribution of radioactivity from gener- obtaining ally labeled dCyd in medium and intracellular frac- all four deoxynucleosides, without damaging tions of L. acidophilus. Growth conditions were as the deoxyribose moiety. Figure 2 shows the described in the text. Radioactivities are expressed in chromatographic separation of hydrolysates of disintegrations per minute. the four deoxynucleotides into free sugars and

dR 6( 30 dR 54

44 20 0- , 0 o 21 0 / ~~~~~~~~~~~~~T 10

II 00 dTMP 10dTh2d j\

0 10 20 30 40 0 10 20 30 40 DISTANCE, cm DISTANCE, cm u dAMP 20 30 +dR dR

20

Q 14 0 c

o

A Do.+dAdo\ dGMP G d a 0,4444 *-*4444-*44-/4**4,_44_444*-A 10 20 30 40 1l 20 30 40 DISTANCE, cm DISTANCE, cm FIG. 2. Chromatograms ofhydrolysates of(A) dCMP, (B) dTMP, and (D) dAMP in solvent system I, and (C) dGMP in solvent system II (see text). (?) = unidentified peak. dR, Deoxyribose; C, cytosine; dAdo, deoxyadenosine; dThd, deoxythymidine; T, thymine; G, guanine; dGuo, deoxyguanosine. VOL. 126, 1976 INCORPORATION OF dCyd INTO BACTERIAL DNA 1139 bases. Except for the deoxyadenosine 5'-mono- suggests that most of the thymidylate was syn- phosphate (dAMP) hydrolysate, the free deoxy- thesized directly from dCMP by deamination ribose and the bases, as well as any unhydro- and methylation at the nucleotide level, with- lyzed nucleoside or nucleotide, were well sepa- out breaking the glycosidic bond. This orga- rated. Chromatography of dAMP hydrolysate nism does have deoxycytidylate deaminase (15, with solvent system I (not shown) as well as 16), whereas no dCyd deaminase activity has with system II indicated that the amount of been detected. The most probable reaction net- label to be found as dAMP, deoxyadenosine, or work is presented in Fig. 3. It is not known adenine was negligible; therefore, the label in whether in this organism deamination precedes the radioactive peak of Fig. 2D can be assumed methylation as shown for S. typhimurium (11) to be in the sugar only, as in the case of the or whether a methylated deoxycytidylate deriv- deoxyguanosine 5'-monophosphate (dGMP) hy- ative is deaminated. drolysate. Both base and sugar were labeled in The alternative route for deoxythymidylate the pyrimidine nucleotides. From the data in synthesis, that ofglycosyl transfer to unlabeled Fig. 2, The sugar/base ratios were computed uracil or thymine, followed by phosphorylation and compared with the generally labeled pre- with thymidine kinase (reactions 1 and 5), cursor (Table 1). The ratios were sugar/base TABLE 1. Radioactivity of nucleotides isolated from approximately equal in deoxycytidine 5'-mono- DNA ofL. acidophilus R-26 after 190 min ofgrowth phosphate (dCMP) and deoxythymidine 5'- in B12 assay medium containing [G- 4C]dCyda monophosphate (dTMP), with average values of 3.01 and 3.15, respectively, but were signifi- Nucleo- Sugar/ Determi- side or nu- Sugar Base base ra- cantly higher than the ratio of 1.21 found for nation cleotide (dpm) (dpm) tio the sugar/base ratio of the original ['4C]dCyd (dpm) added to the medium. [G-14C] 57,600 31,700 26,000 1.22 DISCUSSION dCyd 60,000 32,800 27,200 1.21 In contrast to other bacteria, L. acidophilus dCMP 94,900 72,200 22,700 3.18 R-26 readily incorporates dCyd into its DNA 91,300 67,500 23,800 2.84 when generally labeled dCyd is provided as the only source ofdeoxyribose. Ofthe radioactivity dTMP 169,500 128,000 41,600 3.07 incorporated into DNA, a fifth moved directly 158,200 121,000 37,300 3.24 into DNA deoxycytidylate. Earlier work from this laboratory (2) has shown that this orga- dAMP 126,000 124,000 '528 nism has dCyd kinase activity; therefore, the 124,600 121,900 <821 presence of this enzyme must serve to pernit dGMP 61,000 60,700 241 this unusual direct route of incorporation into 54,200 53,000 1,135 DNA. The other three nucleotides of DNA were also labeled but with the purine deoxyribonu- a The radioactivity is expressed in disintegrations cleotides being labeled only in the sugar moie- per minute, determined with a 14C-labeled internal ties. This result indicates that nucleoside deox- standard, and was corrected to 100% hydrolysis of yribosyl transferase (EC 2.4.2.6), an enzyme the nucleotides. A 5-A,l amount from the total 35 ul that of DNA was used in each of the two separate experi- is active in lactobacilli (5, 9), is involved in ments. the synthesis of deoxynucleotides from a com- mon pool of deoxyriboside. Adenine and gua- DNA assay nine available from the B12 medium pre- " It sumably accept labeled sugar from the ['4C]dCyd, and the resulting purine deoxyribo- AdR-P, GdR-P CdR-P UdR-P TdR-P nucleosides can then be phosphorylated by the 4 purine deoxynucleoside kinase activity also 2 2 :5 known to be present (2). Once a labeled deoxy- AdR, GdR = A,G CdR v UdR 1 1 nucleoside is phosphorylated, it would be pro- tected from further glycosyl exchange catalyzed FIG. 3. Predominant pathways of deoxynucleo- by nucleoside deoxyribosyl transferase. side metabolism in L. acidophilus. Enzymes involved The base moiety of the labeled dCyd was are: (1) nucleoside deoxyribosyl transferase, (2) deox- ynucleoside kinases, (3) dCMP aminohydrolase, (4) partially diluted by exchange with the pyrimi- thymidylate synthetase, and (5) thymidine kinase. dine pool before incorporation into DNA, as AdR-P, deoxyadenosine monophosphate; GdR-P, reflected in the increase sugar/base ratio of deoxyguanosine monophosphate; A, adenine; G, DNA deoxycytidylate shown in Table 1. How- guanine; CdR-P, deoxycytidine monophosphate; ever, no further dilution ofthe thymine base of UdR-P, deoxyuridine monophosphate; TdR-P, de- DNA thymidylate was observed. This strongly oxythymidine monophosphate. 1140 DAVIS AND IVES J. BACTERIOL. seems unlikely. Thymine has been found to be graphic data for purines, pyrimidines and derivatives in a variety ofsolvents. J. Chromatogr. 22:118-129. a very poor DNA precursor in this strain (14), 4. Hoff-Jorgensen, E. 1952. A microbiological assay of and there is little reason to suppose that uracil deoxyribonucleosides and deoxyribonucleic acid. Bio- would be a better precursor than thymine un- chem. J. 50:400-403. der these conditions. 5. Holguin, J., R. Cardinaud, and C. A. Salemink. 1975. Trans-N-deoxyribosylase: substrate specificity stud- Excess adenosine greatly enhances dCyd up- ies. Purine bases as acceptors. Eur. J. Biochem. take in the lactobacilli, an effect first observed 54:515-520. with thymidine uptake in E. coli. (10). The 6. Hough, L., and J. K. N. Jones. 1962. Chromatography adenosine is thought to compete with degrada- on paper, p. 21-31. In R. L. Whistler, M. L. Wolfrom (ed.), Methods in carbohydrate chemistry, vol. I. Aca- tive pathways, which might otherwise destroy demic Press Inc., New York. the nucleoside precursors. One possible degra- 7. Ives, D. H., J. P. Durham, and V. Tucker. 1969. Rapid dative route involves the hydrolysis ofdeoxynu- determination of nucleoside kinase and nucleotidase cleosides, either directly or through phospho- activities with tritium-labeled substrates. Anal. Bio- chem. 28:192-205. rylic cleavage, followed by rigid hydrolysis of 8. Karlstrom, 0. 1970. Inability of Escherichia coli B to deoxyribose-1-phosphate. Sawula et al. (13) incorporate added deoxycytidine, deoxyadenosine, have reported a thymidine phosphorylase or and deoxyguanosine into DNA. Eur. J. Biochem. hydrolase in L. acidophilus R-26 but did not 17:68-71. 9. MacNutt, W. S. 1952. The enzymically catalyzed trans- report trying other substrates. In our hands the fer of the deoxyribosyl group from one purine or py- hydrolytic activity of the ammonium sulfate rimidine to another. Biochem. J. 50:384-397. fraction is effective towards all four deoxynu- 10. Munch-Petersen, A. 1968. On the catabolism ofdeoxyri- cleosides, leading us to suspect the activity is a bonucleosides in cells and cell extracts ofEscherichia coli. Eur. J. Biochem. 6:432-442. relatively nonspecific hydrolase or else a mix- 11. Neuhard, J., and E. Thomassen. 1971. Deoxycytidine ture of hydrolases. Further work is needed to triphosphate deaminase: identification and function characterize this activity, however. in Salmonella typhimurium. J. Bacteriol. 105:657- 665. ACKNOWLEDGMENTS 12. Reeves, W. J., Jr., A. S. Seid, and D. M. Greenberg. 1969. A new paper chromatography solvent system This work was supported by Public Health Service grant resolving pyrimidine-pyrimidine riboside-pyrimidine CA-06913 from the National Cancer Institute and grant GB- deoxyriboside mixtures. Anal. Biochem. 30:474-477. 38084 from the National Science Foundation. 13. Sawula, R. V., S. Zamenhof, and P. J. Zamenhof. 1974. We also are indebted to Thomas R. Hutchinson for carry- Degradation of thymidine by Lactobacillus acidophi- ing out preliminary studies on which this work is based. lus. J. Bacteriol. 117:1358-1360. 14. Sawula, R. V., S. Zamenhof, and P. J. Zamenhof. 1975. LITERATURE CITED Participation ofexogenous thymine and thymidine in 1. Bean, B., and A. Tomasz. 1973. Selective utilization of deoxyribonucleic acid synthesis in Lactobacillus aci- pyrimidine deoxyribonucleosides for deoxyribonu- dophilus. Can. J. Microbiol. 21:501-509. cleic acid synthesis in Pneumococcus. J. Bacteriol. 15. Sergott, R. C., L. J. Debeer, and M. J. Bessman. 1971. 113:1356-1362. On the regulation of a bacterial deoxycytidylate de- 2. Durham, J. P., and D. H. Ives. 1971. The metabolism of aminase. J. Biol. Chem. 246:7755-7758. deoxyribonucelosides in Lactobacillus acidophilus: 16. Siedler, A. J., and M. T. Holtz. 1963. Regulatory mech- regulation of deoxyadenosine, deoxycytidine, deoxy- anisms in the deoxyribonucleic acid metabolism of guanosine and deoxythymidine kinase activities by Lactobacillus acidophilus R-26. J. Biol. Chem. nucleotides. Biochim. Biophys. Acta 228:9-25. 238:697-701. 3. Fink, K., and W. S. Adams. 1966. Paper chromato-