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Home , Cytosine, Deoxycytidine, Deoxyguanosine, Guanosine, Pyrimidine, Thymine

UTILIZATION OF AND DEOXYNUCLEOSIDES BY THERMOBACTERI UM ACIDOPHILUMII (LACTOBACILLUS ACIDOPHILUS) SOREN L0VTRUP AND DAVID SHUGAR Department of Histology, University of G6teborg, Gbteborg, Sweden, and Institute of and Biophysics, Academy of Sciences, Warsaw, Poland Received for publication February 20, 1961

ABSTRACT terium. Some other reports have been published L0VTRUP, S0REN (University of G6teborg, concerning the pyrimidine requirements of this Sweden) AND DAVID SHUGAR. Utilization of strain (Jeener and Jeener, 1952; Siedler, Nayder, pyrimidines and pyrimidine deoxynucleosides by and Schweigert, 1957; L0vtrup and Roos, 1957a). Thermobacterium acidophilum (Lactobacillus acid- Two aspects of pyrimidine have ophilus). J. Bacteriol. 82:623-631. 1961.-The been investigated in the present work: the utili- utilization of pyrimidine deoxynucleosides was zation of (i) deoxyribosides and (ii) free pyri- investigated by means of deoxyribosides of midines. Both problems have been attacked unnatural pyrimidines, especially by - mainly by the supplementary use of a number of substituted derivatives. All investigated unnatural pyrimidines and pyrimidine deoxyri- deoxyribosides could be used, except that of bosides. N-methylthymidine. It was concluded that this MATERIALS AND METHODS substance cannot be a substrate for the trans-N-deoxyribosylase, which has been shown The microbiological technique used in this to be active in the utilization of deoxyribosides study has been described previously (L0vtrup in this . With uracil as the only and Roos, 1957a), and additional modifications pyrimidine source, the halogen-substituted have also been published (L0vtrup and Roos, A further modification introduced here is had a certain inhibitory effect on 1959). the addition of as the only source growth. basal Contrary to previous findings, it was observed in the double strength medium. Unless that normal growth occurs in the presence of otherwise stated, was used as the bond source. The criterion for as the only pyrimidine source. The utilization of this substance is less efficient than utilization of added substances was growth, All that of uracil; a 1:10 dilution leads to a decrease as measured turbidimetrically. experiments in which an in the extent of growth with the former, but not reported here, effect, enhancing or was been with the latter. From these results, complemented inhibiting, observed, have repeated to establish the with experiments in which halogen-substituted several times, significance of the uracil derivatives and the corresponding ribosides results. the were were used as it MIost of compounds obtained from or deoxyribosides inhibitors, where has been possible to account for most of the commercial sources and, considered neces- metabolic interconversions of pyrimidines in the sary, checked by chromatography or absorption investigated microorganism. . N-MIethylthymidine was prepared by of with diazomethane, according to MIiles (1957). Thermobacterium acidophilum (Lactobacillus All the deoxyribosides and other inhibitors acidophilus) Orla Jensen strain R26 will grow tested were added in a concentration corre- only in the presence of deoxyribosides. For this sponding to the highest deoxyriboside concentra- reason the has been used in a micro- tion in our standard curves, i.e., 4 m/uM. In a biological assay for deoxyribonucleic acid (DNA) few instances, the effect of a higher concentration and its products (Hoff-J0rgensen, has been investigated. In these cases the concen- 1952, 1954). During work on the development of tration 40 ,ug per ml double strength basal me- this method, Hoff-J0rgensen also had to establish dium, the concentration of pyrimidines in the the general nutritional requirements of the bac- basal medium, was used for convenience. 623 624 L0VTRUP AND SHUGAR [VOL. 82

RESULTS TABLE 2. Utilization of pyrimidines by Thermobacterium Utilization of deoxyribosides. A number of acidophilum deoxyribosides were tested with respect to their Pyrimidine concn per ml ability to support growth of our . Solu- of medium tions of the substances listed in Table 1, and of the same concentration as those of deoxyribosides 40 Ag 4 jug at the highest point on our standard curves (4 mtum), were prepared. The pyrimidine content ...... 323 285 was varied as indicated, and the concentration Thymine...... 316 95 of the individual pyrimidines was 40 per ml Uracil ...... 325 308 jig 5-Bromouracil ...... 53 37 of double strength basal medium. ...... 44 39 It will be seen that the bacteria are quite 5-Methylcytosine ...... 33 36 unable to utilize the deoxyriboside bond in 5-Hydroxymethylcytosine .. 43 39 N-methylthymidine. The remaining compounds may all support growth, although there is a significant difference in efficiency between thy- TABLE 3. Effect of various combinations midine, which gives the highest, and 5-bromo- of pyrimidines on the growth of Thermo- , which gives the lowest, growth. bacterium acidophilum. Thymine in low The results with the unnatural deoxyuridines concentration (4 ,ug/ml) in all cases make it possible to distinguish between two 5-Methyl- 5-Hydroxy- Growth types of inhibition. When the results with deoxy- 5-Bromn- Cytosine cytosine methyl- response and its iodo derivative are compared, it is cytosine seen that growth proceeds to the same extent ag/ml in the presence of either thymine plus uracil 64 or thymine alone. The deoxyriboside bond of 128 5-iododeoxyuridine may thus be freely utilized. + + 83 This does not hold for the fluoro and bromo + 93 derivatives, which under the same circumstances + + 69 permit somewhat lower growth. + 65 + 57 Another kind of inhibition is observed when + 141 the results with uracil as the only pyrimidine + + + 123 source are considered; in this case inhibition + 110 occurs, which is specific for uracil. This inhibi- + + 87 tion is seen to decrease as the atomic weight of + + 76 the halogen substituent increases. + + 76 It has been observed that 5-bromouracil, a + + 155 thymine analogue, may be incorporated into the + 79 DNA of bacteria in place of thymine. Once

TABLE 1. Utilization of natural and unnatural the unnatural DNA has been formed, further deoxyribosides by Thermobacterium multiplication of the bacteria may be inhibited. acidophilum (jsg/ml of medium) We have investigated this question by repeated subeultivation of our bacteria on 5-bromodeoxy- Thy- Thy- No mine + Uracil addi- uridine. The first three subcultures gave growth uracil mine tion responses corresponding to 233, 223, and 245, respectively, indicating no increased inhibition of N-Methylthymidine .... 32 40 37 25 the bacteria. Thymidine ...... 398 379 384 27 Utilization of pyrimidines. In a medium 5-Fluorodeoxyuridine 316 304 48 38 supplied with deoxyguanosine, various natural 5-Bromodeoxyuridine. 292 276 82 31 5-Iododeoxyuridine .... 361 334 122 30 and unnatural pyrimidines were added, in two Deoxyuridine ...... 358 349 357 56 different concentrations. It appears (Table 2) that only three of the seven tested substances 1961] UTILIZATION OF PYRIMIDINE DERIVATIVES BY 7'. ACIDOPHILUM 625 may sustain normal growth, namely, orotic acid, thesized from both thymine and uracil. To thymine, and uracil. Of these, thymine is least estimate whether the synthesis of cytosine may efficient; growth is substantially decreased when limit growth, an experiment was performed in the thymine concentration is decreased from 40 which cytosine was added to tubes containing to 4 Mg. one or two of the other pyrimidines. Thymidine, To test the possible effect of combining two or deoxyuridine, , and deoxyguanosine more of the inefficient pyrimidines, these were were added in the respective experiments and added (Table 3) to a medium containing thymine two different concentrations of pyrimidines were in the lower concentration (Table 2), which used (Table 4). gave a low but significant growth response. The These results will be dealt with in the Dis- concentration of the other pyrimidines was 40 cussion; it should only be mentioned here that ,ug per ml of basal medium. Growth was inhibited deoxycytidine is seen in some instances (thymine in all cases where 5-bromouracil was added. + uracil and uracil in low concentration, uracil Apart from this, it appears that none of the in high concentration + cytosine) to give combinations will sustain full growth. Cytosine maximal growth. This does not agree with gives a significant stimulation, and a slight previous observations concerning the efficiency effect is apparently also obtained with the of different deoxyribosides (L0vtrup and Roos, combination of the two other cytosine deriva- 1959). It should be noted, however, that in the tives. previous experiments the medium contained Unless a synthetic pathway exists, entirely guanine, , uracil, and cytidylic acid, but different from what is at present known for no thymine, all at the high concentration. It is other , it must be concluded from the likely that this difference suffices to explain the results in Table 2 that cytosine may be syn- observed discrepancy.

TABLE 4. Growth response of Thermobacterium acidophilum to various combinations of pyrimidines and deoxyribosides (ug/ml of medium) Concn Thymine + per ml Pyrimidine uracil + Thmnuracil + hm~ee+ rcl+NoCytosi+eUracil medium cytosine urcl Thmiycosn Thtoneymn cyoie Ual Cysne addtionditn

jAg 40 Thymidine 328 279 305 269 317 307 42 39 Deoxyuridine 320 280 288 280 301 290 83 66 Deoxycytidine 332 315 306 310 360 300 44 36 Deoxyguanosine 314 283 278 278 295 296 35 27 4 Thymidine 332 315 114 99 333 291 42 37 Deoxyuridine 340 353 145 135 331 283 73 69 Deoxycytidine 395 399 136 125 373 333 39 40 Deoxyguanosine 312 323 86 86 288 248 30 27

TABLE 5. Comparison of the inhibitory effect of 5-fluoro- and 5-bromodeoxyuridine on the growth of Thermobacterium acidophilum (pug/ml) Thymine Thymine Uai Concof mediumlperiumlyimdnPyrimidine + cyto-uracil Thymine+ uracil +sinecyto- Thymine Uracilcytosine+ Uracil Cyto-sine Noditionad- sine

jAg 40 5-Fluorodeoxyuridine 359 316 320 304 54 48 34 48 5-Bromodeoxyuridine 319 306 300 276 149 82 42 31 4 5-Fluorodeoxyuridine 65 81 48 47 49 39 37 34 5-Bromodeoxyuridine 167 168 73 61 94 80 38 30 626 L0VTRUP AND SHUGAR [VOL. 82

Similar results were observed when 5-fluoro- uridine (Table 5) shows directly that, as far as and 5-bromodeoxyuridine were used (Table 5). the specific uracil inhibition is concerned, the One difference to be noted was the inferior free is much less potent than the deoxy- utilization of the deoxyriboside bond in the . When the inhibitor concentration bromo derivative. In addition, specific uracil was raised, however, considerable inhibition was inhibition was observed with both substances. found, and this occurred even in the presence In this respect 5-fluorodeoxyuridine is seen to be of thymine. A rather large effect of cytosine the most potent inhibitor. addition was observed, even in the presence of When an unnatural deoxyriboside is added, thymine, except when deoxycytidine was present. the deoxyribose moiety will be transferred to Such a distinct stimulation was not observed in other bases, liberating the unnatural pyrimidine the presence of thymine in any of the results re- during the course of this reaction. The observed ported above, and may thus be taken to indicate inhibitions might therefore equally well be due that 5-bromouracil may interfere with the synthe- to the free base. Some experiments were de- sis of cytosine from thymine. With dexoyguano- signed to test this question. With either deoxy- sine, and uracil in high concentrations and inhibi- or mixtures of this with pyrimidine tor in low concentration, growth was considerably deoxynucleosides as the deoxyribose source, stimulated by cytosine addition. A similar, but 5-bromouracil was added in two different concen- slighter effect was observed when thymidine was trations, namely, that of the pyrimidine additions also present, but not when deoxycytidine was (40 ,ig per ml of medium), and that of the added. deoxyribose addition (4 mAm per ml or about With fluoro-substituted derivatives we have 0.8 ,ug per ml of medium). The results are shown been able to compare not only base and deoxy- in Table 6. riboside, but the riboside as well, with regard to A comparison of the effect of the inhibitor in their inhibitory ability. The results of these low concentration with those of 5-bromodeoxy- experiments, in which the inhibitors were added

TABLE 6. Effect of 5-bromouracil on the growth of Thermobacterium acidophilum in the presence of various combinations of pyrimidines and deoxyribosides (,ug/ml of medium) Pyrim- 5-Bromo idine uraci Thymine conlcn Subtrtecnnle + uracil Thymine Thymine Uracil Cyto- N a-- permlmolSubstrate of + cyto- + uracil + cyto- Thymine + cyto- Uracil sine dition of me- medium sine ci sine +sinesie dto dium mdu

jAg 40 Deoxyguanosine 40 jug 157 102 169 110 53 44 36 31 4 111Mm 329 280 335 286 283 142 36 24 Deoxyguanosine (3/4) + 40 jig 170 146 167 122 73 63 48 48 thymidine (1/4) 4 mjiM 288 259 308 265 266 212 36 33 Deoxyguanosine (3/4) + 40 o,g 116 121 110 117 35 57 39 43 deoxycytidine (1/4) 4 m,AM 300 290 311 297 226 201 27 35 Deoxyguanosine (1/2) + 40,g 148 159 137 151 55 68 45 50 thymidine (1/4) + 4 m,uM 308 315 313 340 270 278 29 32 deoxycytidinie (1/4)

4 Deoxyguanosine 40 ,ug 49 40 58 53 58 49 44 37 4 mMAM 252 180 73 61 73 61 31 26 Deoxyguanosine (3/4) + 40 Mg 63 68 57 64 60 49 53 46 thymidine (1/4) 4 m1uM 260 219 75 72 209 191 27 33 Deoxyguanosine (3/4) + 40,ug 44 50 40 52 43 45 43 47 deoxycytidine (1/4) 4 m,M 216 211 123 97 146 149 25 30 Deoxyguanosine (1/2) + 40,ug 51 78 55 68 54 65 41 46 thymidine (1/4) + 4 m,uM 260 272 79 82 185 217 27 29 deoxycytidine (1/4) 19611 UTILIZATION OF PYRIMIlINE DERIVATIVES BY T. ACIDOPHILUM 627 TABLE 7. Conmparison of the effects of a free pyrimidine base and the corresponding riboside on the growth of Thermobacterium acidophilum (,g/ml of medium) Pyrim- idine Thymine concn er Substrate Thymil Thmn+ uracilcytosmine+ hmn Ym + rclNoUracil Cytosine ditionad- ml + cytosine+urcl+ctin+cyonedincytosine medium

pg 40 5- 348 260 298 291 56 45 47 47 5-Fluorouridine 321 286 327 302 105 50 48 34

4 5-Fluorouracil 101 103 58 51 56 48 48 41 5-Fluorouridine 265 212 89 87 67 70 42 39

TABLE 8. Effect of 6-methyluracil on the growth of Thermobacterium acidophilulm (,ug/ml of medium)

Pyrimidine 6-Methyluracil Thymine . . concn per ml concn per ml + uracil +Thymine Thymine + Uracil Uracil Cytosine of medium of medium + cytosine +uhyml d- 'Ag 40 40 ,ug 322 227 330 228 313 279 31 28 4 mium 343 297 351 283 328 262 37 24

4 40 ,ug 282 265 82 73 246 195 27 24 4 mjuM 330 254 84 81 257 170 29 23 only in the low concentration, are shown in TABLE 9. Influence of on the Table 7. growth of 7'hermobacterium acidophilum A comparison with Table 6 shows that 5- Aminopterin fluorouracil is a more potent inhibitor than the (1.6 pg per m of medium) corresponding bromo derivative, which agrees with the findings for the deoxyribosides reported + Cytosine - Cytosine in Table 5. Both the free base and the nucleoside give nearly complete inhibition with uracil, hence it is rather difficult to decide which is the Thymine ...... 129 150 353 337 more potent inhibitor. Only with thymine + Uracil ...... 45 50 356 312 uracil in low concentration was a difference 5-Methylcytosine ...... 33 25 33 23 observed, showing that the deoxyriboside has Thymine + uracil ...... 125 127 343 340 the strongest effect. This result agrees with that Thymine + 5-methyl- found for the corresponding bromo compounds. cytosine ...... 142 93 301 210 In contrast to this, 5-fluorouridine was seen to Uracil + 5-methyl- exert a much slighter inhibiting effect than the cytosine ...... 79 59 296 232 other two substances. The influence of the thymine isomer, 6- The synthesis of thymine from uracil is methyluracil, was also investigated (Table 8). presumed to involve a methylation in which This compound is seen to have little influence tetrahydrofolic acid takes part. To test whether on the growth of the bacteria, and the slight this reaction occurs also in our microorganism, inhibition observed affects the utilization of both some experiments were performed (Table 9) thymine and uracil for synthesis of cytosine. with aminopterin, the folic acid inhibitor. The The former effect is seen most clearly in the pyrimidine concentration was 40 ,ug per ml of experiments with high concentration of both basal medium. inhibitor and pyrimidines (thymine + uracil and Growth on uracil was suppressed by thymine). aminopterin, but this inhibition was partly 628 LOVTRUP AND SHUGAR [VOL. 82 relieved by the addition of thymine. Even the remaining pyrimidines at the or addition of 5-methylcytosine had a slightly level, the bases thus formed being stimulating effect. Aminopterin was also seen liberated and incorporated into deoxynucleo- to exert an inhibitory influence on the conversion sides by the enzymatic reaction suggested above. of both thymine and uracil to cytosine. The The most likely mechanism for utilization of the influence on growth of 5,6-dimethyluracil was desoxyriboside is therefore that a trans-N- also investigated. No effect whatsoever was glycosylation occurs with the pyrimidines added, observed. and that transformation to other pyrimidine deoxynucleosides occurs on the deoxyriboside DISCUSSION level. Utilization of deoxyribosides. Recent work has The slight inhibition of growth by 5-fluoro- demonstrated that in mammalian cells deoxy- and 5-bromodeoxyuridine in the presence of compounds are formed from the corre- thymine without cytosine addition is of the sponding ribose compounds, on the nucleotide same order as that observed with the free bases, level (review by Reichard, 1960). Such a meta- and may therefore be an effect of the free halogen bolic link between ribonucleic acid (RNA) and pyrimidines liberated by the trans-N-glycosyla- DNA is evidently absent in this microorganism. tion. As shown by Hoff-J0rgensen (1952), only deoxy- It has been reported by Dunn and Smith or deoxynucleosides will support the (1957) and by Zamenhof, Rich, and DeGio- growth of T. acidophilum. Neither deoxyribose vanni (1958) that 5-bromouracil may be in- nor deoxyribose 1- could replace these corporated into bacterial DNA. By this in- substances. corporation the bacteria may lose their ability The added bound deoxyribose must therefore to multiply. The subcultivation experiments be distributed between the various bases in which we have carried out show that no such DNA in the correct pattern. The mechanism of loss occurs. this distribution in the present microorganism Metabolism of pyrimidines. The original and other bacteria was investigated medium employed by Hoff-J0rgensen (1952, by MacNutt (1952). He demonstrated an 1954) included adenine, guanine, thymine, and enzyme, deoxyribose tramn-N-glycosylase, or cytidylic acid. In later work it was found by trans-N-deoxyribosylase (Roush and Betz, 1958), Jeener and Jeener (1952) that even uracil had which catalyzes the transfer of the deoxyribosyl to be supplied to achieve bacterial growth. The group from one purine or pyrimidine base to same result was reported by Siedler et al. (1957) another. The specificity of this enzyme was and by L0vtrup and Roos (1957a). It was found found to be rather low, since its substrates in the latter work that omission of guanine and include various bases which normally do not uracil (in the absence of thymine) led to inhibi- occur in deoxyribosides. The activity of the tion of growth, whereas the absence of adenine, enzyme may account for the observed utilization cytidylic acid (cytosine), and thymine (in the of unnatural deoxyribosides, although there may presence of uracil) had no serious effect. A be a somewhat less efficient utilization of the systematic study of the pyrimidine requirements deoxyriboside linkage. Since N-methylthymidine was not carried out at that time. The present cannot support growth, it appears that this results show, however, that the bacteria may substance cannot be a substrate for the enzyme. grow in the presence of thymine as the only The utilization of uracil, as opposed to that of pyrimidine source. The reason for the repeated thymine, was more strongly inhibited by the observation of the necessity of uracil is obscure. halogen-substituted deoxyribosides than by It remains undisputed, though, that in a medium the corresponding free bases. The effect is thus supplied with uracil and cytosine, addition of presumably exerted at the deoxynucleoside level, thymine generally has a very slightly stimulating i.e., the exchange between uracil and the halogen effect; in many cases it may even depress growth derivative is inhibited to a much greater degree to some extent. than the corresponding exchange with thymine. It is thus obvious from our results that thymine This finding seems to show that the added and uracil are interconvertible, and that they pyrimidine source is not transformed to the both may give rise to cytosine. (It is assumed in 19611 UTILIZATION OF PYRIMIDINE DERIVATIVES BY T. ACIDOPHILUM 629 the present discussion that no pyrimidines other with low pyrimidine concentration (Table 4). than these three are present in the bacteria.) First of all, irrespective of the source of deoxyri- Before discussing the metabolism of pyri- boside, thymine + cytosine always gave much midines, the consequences of the fact that these lower growth than uracil + cytosine. This must substances are present in both DNA and RNA mean that thymine -+ uracil occurs, but limits may be considered. As mentioned previously, the growth. It is not possible to demonstrate there is reason to believe that the synthetic that the conversion uracil -* thymine is limiting. pathways of the two nucleic acids are com- The results with thymidine and thymine show pletely separated. The transformations of DNA that thymine -* cytosine may be limiting, and pyrimidines which must occur probably take all the experiments with uracil show that uracil place at the nucleoside level (Reichard, 1960). -* cytosine limits growth. Particularly, the Thus, if uracil is added, the deoxyuridine formed results with addition of deoxycytosine show will give rise to thymidine and deoxycytidine; that it is at the RNA level that this limitation if thymine is added, only the formation of occurs. deoxycytidine is needed. With RNA, the added It is not possible to decide with certainty pyrimidine(s) is presumed to be transferred to whether the transformation thymine -* cytosine ribosides or ribotides, at which level similar occurs via uracil, but the results do suggest an transformations may occur (uracil -+ cytosine or intermediate position of uracil. When the thymine thymine -- uracil + cytosine). requirements are fulfilled (thymine + uracil), This circumstance obviously obscures the deoxyuridine is seen to be a much more efficient interpretation of the inhibition experiments source of deoxycytidine than is thymidine. In carried out by us, because a limited supply of the absence of thymine (with or without cyto- RNA pyrimidines may inhibit growth just as sine), they are equally efficient, but here deoxy- well as the supply of deoxyriboside (Jeener and uridine must supply two other deoxyribosides, Jeener, 1952; Siedler et al., 1957). This fact whereas thymidine has to take part only in the must be taken into account in the interpretation production of deoxycytidine. Even at the DNA of our results. level there is much to be said in favor of the The separation of DNA and RNA metabolism uracil derivative taking an intermediate position. is indicated by the experiments with uracil The conversion cytosine -- uracil is not (Table 4). From the high concentration experi- possible, because if it occurred to only a very ments (without cytosine), it was seen that slight extent growth would occur in the presence growth is independent of the added deoxyribo- of cytosine and a deoxyriboside. Such growth side. Addition of cytosine had no significant occurs only with deoxyuridine, demonstrating effect on growth, except with deoxycytidine. It the extremely efficient utilization of this pyri- thus appears that in the presence of cytosine, midine. the synthesis of deoxycytidine limits growth; There remain many unanswered questions and in the presence of deoxycytidine, the supply about the pyrimidine metabolism of T. acido- of cytosine is the limiting factor. This shows philum, but the main outline of the possible that added cytosine is not easily transformed to conversions is probably as represented in Schema deoxycytidine, i.e., the metabolism of cytosine 1. (The symbol R indicates that the pyrimidines in RNA and DNA occurs along separate path- react either as ribosides or ribotides. The broken ways. lines represent uncertain reactions.) Another fact which emerged from the results With halogenated deoxyuridines, a specific shown in Table 4 was the much more efficient inhibition of uracil utilization was observed. utilization of uracil as compared to thymine. The combined results of Tables 1 and 5 showed When the lower concentration of pyrimidine was that this inhibition increased with decreasing used, growth was significantly reduced in the molecular weight. A similar regularity was not presence of thymine, but not when uracil was observed when the chloro, bromo, and iodo added. derivatives act as thymine inhibitors in thy- Certain suggestions as to the possible path- mineless strains of (Zamenhof ways of pyrimidine conversion may be obtained et al., 1958; Dunn and Smith, 1957). from these experiments, particularly from those As the halogenated bases are liberated by 630 LQVTRUP AND SHUGAR [VOL. 82

Deoxyribosides

r--Thymidine X Thymine - *) Thymine Re---I II1-1I

Deoxyuridine ( - Uracil - Uracil R 5-Methylcytosine Orotic acid - OroticOroticacidacIdR L0Deoxycytidine e-- --- Cytosine - ) Cytosine R*--

DNA RNA SCHE:IMA 1 the action of trans-N-deoxyribosylase, some ex- specific inhibitor, was better utilized in the periments were performed to test the action enzymatic deoxyribose transfer. of 5-bromouracil. It was found that the free In our experiments with 6-methyluracil, we pyrimidine was less active than 5-bromodeoxy- found this substance to possess very little inhib- uridine. The inhibitory effect was antagonistic itory action. The effect seemed to be concerned mainly to uracil, but with high inhibitor concen- particularly with synthesis of cytosine, from tration even thymine utilization was affected. both thymine and uracil. In the experiments we added not only deoxy- Friedkin and Kornberg (1957) have shown guanosine, but also three mixtures of deoxyri- that the transformation uracil -- thymine in bosides. The results of these experiments give E. coli occurs at the deoxyriboside level, in a some information about the reactions which tetrahydrofolic acid-dependent reaction. Folic were inhibited. In the experiments with low acid is a necessary component of the medium uracil and low 5-bromouracil concentrations, it for T. acidophilum (Hoff-J0rgensen, 1952; was seen that the synthesis of thymidine, par- L0vtrup and Roos, 1957b). In the presence of ticularly, was affected. When this substance was uracil alone, growth was completely blocked by added, a substantial increase in growth was the folic acid , aminopterin. achieved. Addition of cytosine had no effect, and This inhibition was partly relieved by thymine, even the addition of deoxycytidine gave onlv showing that among the reactions influenced by very slight stimulation. aminopterin must be the formation of thymine. With the fluoro derivatives a comparison was Also, the addition of 5-methylcytosine counter- made of the activity of both the free pyrimidine acted, to a very slight extent, the inhibition by and the riboside with that of the deoxyriboside. aminopterin. This suggests the possibility that As with the bromo derivatives, the deoxy- 5-methylcytosine is an intermediate between and as not nucleoside was a stronger inhibitor than the thymine cytosine, if, is quite certain from our results, there is a pathway between base, whereas the riboside was less efficient. This latter observation indicates that the free base is these two substances not involving uracil. only partially liberated from the riboside, or not ACKNOWLEDGMENTS at all. The inhibition by the deoxyribosides, Our sincerest thanks are due to Inger Janson over and above that of the free pyrimidines, for technical assistance. This study was supported must presumably hit the DNA synthesis proper. by grants from the Swedish Society. This effect cannot, however, be a mere inhibition of the utilization of the deoxyribose linkage in LITERATURE CITED the trans-N-glycosylation reaction, since 5- DUNN, D. B., AND J. D. SMITH. 1957. Effects of fluorodeoxyuridine, which was the strongest 5-halogenated on the growth of Escher- 1961] UTILIZATION OF PYRIMIDINE DERIVATIVES BY T. ACIDOPHILUM 631

ichia coli and their incorporation into deoxy- L0VTRUP, S., AND K. Roos. 1959. Microbiological ribonucleic acids. Biochem. J. 67:494-506. determination of deoxyribonucleic acid. FRIEDKIN, M., AND A. KORNBERG. 1957. The Influence of deoxyribosides on the standard enzymatic conversion of deoxyuridylic acid curves. J. Bacteriol. 78:114-116. to thymidylic acid and the participation of MAcNuTT, W. S. 1952. Enzymically catalyzed tetrahydrofolic acid, p. 609-614. In W. D. transfer of the deoxyribosyl group from one McElroy and B. Glass, [ed.], The chemical purine or pyrimidine to another. Biochem. basis of heredity. The Johns Hopkins Press, Baltimore. J. 50:384-397. JEENER, H., AND R. JEENER. 1952. Cytological MILES, H. T. 1957. Synthesis of some model study of Thermobact.erium acidophilus R 26 pyrimidine . J. Am. Chem. Soc. cultured in absence of 79:2565-2568. or uracil. Exptl. Cell Research 3:675-680. REICHARD, P. 1960. of pyrimidine HOFF-J0RGENSEN, E. 1952. A microbiological , p. 70-76. In J. S. assay of deoxyribonucleosides and deoxyri- Mitchell, [ed.], The cell nucleus. Butterworth bonucleic acid. Biochem. J. 50:400-403. and Co., London. HOFF-J0RGENSEN, E. 1954. Deoxynucleic acid in ROUSH, A. H., AND F. R. BETZ. 1958. Purification some gametes and embryos, p. 79-88. In and properties of trans-N-deoxyribosylase. J. A. Kitching, [ed.], Recent developments in J. Biol. Chem. 233:261-266. cell physiology. Academic Press, Inc., New SIEDLER, A. J., F. A. NAYDER, AND B. S. York. SCHWEIGERT. 1957. Studies on improvements L0VTRUP, S., AND K. Roos. 1957a. Microbiological in the medium for Lactobacillus acidophilus determination of DNA. The growth of Ther- in the assay for deoxyribonucleic acid. J. mobacterium acidophilum on a chemically defined medium. Exptl. Cell Research, Suppl. Bacteriol. 73:670-675. 4:269-278. ZAMENHOF, S., K. RICH, AND R. DEGIOVANNJ. L0VTRUP, S., AND K. Roos. 1957b. The vitamin 1958. Further studies on the introduction of requirements of Thermobacterium acidophi- pyrimidines into deoxyribonucleic acids of lum. Arch. Mikrobiol. 26:89-92. Escherichia coli. J. Biol. Chem. 232:651-657.