UTILIZATION OF PYRIMIDINES AND PYRIMIDINE DEOXYNUCLEOSIDES BY THERMOBACTERI UM ACIDOPHILUMII (LACTOBACILLUS ACIDOPHILUS) SOREN L0VTRUP AND DAVID SHUGAR Department of Histology, University of G6teborg, Gbteborg, Sweden, and Institute of Biochemistry 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 metabolism 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 halogen- mainly by the supplementary use of a number of substituted uracil 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 enzyme 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 microorganism. 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 deoxyuridines had a certain inhibitory effect on 1959). the addition of guanine as the only purine 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, deoxyguanosine was used as the bond source. The criterion for thymine as the only pyrimidine source. The deoxyribose 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. spectroscopy. N-MIethylthymidine was prepared by methylation of thymidine 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 organism 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 hydrolysis 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 bacteria. 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 Orotic acid.......................... 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. Cytosine ............................ 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- deoxyuridine, 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 uridine 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, deoxycytidine, 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, adenine, uracil, and cytidylic acid, but different from what is at present known for no thymine, all at the high concentration. It is other organisms, 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