Nucleotide Fermentation Iv. Effect of Gmp Reductase

J. Gen. Appl. Mierobiol., 15, 399-411 (1969)

GENETIC AND BIOCHEMICAL STUDIES ON 5'-NUCLEOTIDE FERMENTATION

IV. EFFECT OF GMP REDUCTASE AND PURINE ANALOGUE RESISTANCE ON PURINE NUCLEOSIDE ACCUMULATION PATTERN IN ADENINE AUUXOTROPHS OF BACILLUS SUBTILIS

HARUO MOMOSE AND ISAMU SHIIO

Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki, Japan

(Received December 26, 1968)

1. The presence or absence of GMP reductase had no effect on nucleoside production in adenine-requiring mutants of Bacillus subtilis K, which produced a large amount of inosine when cultured in an adenine-limited medium. 2. Mutants resistant to 8-azaxanthine were derived from strain 38-3, a GMP reductase-positive inosine producer, and from strain 30-12, a GMP reductase-negative inosine producer. About 60% of the resistant mutants from strain 38-3 were found to produce xanthosine as well as inosine, while 70% of the resistant mutants from strain 30-12 produced guanosine as well as inosine. Loss of the adenine requirement in these 8-azaxanthine-resistant mutants reduced the accumulation of xanthosine or guanosine and completely halted the accumulation of inosine. Many more mutants with an altered nucleoside accumulation pattern were obtained when 8-azaxanthine was used as a selective agent than when 8-azaguanine was used. 3. Guanine-requiring mutants, GU-16 and 308-18, were isolated respectively from an 8-azaxanthine-sensitive inosine producer and from an 8- azaxanthine-resistant xanthosine-inosine producer, and the effects of guanosine on xanthosine and inosine formation were compared in these two guanine auxotrophs ; strain GU-16 produced a large amount of xanthosine only when grown in media containing limited amounts of guanosine, and the production was sharply reduced by higher amounts of guanosine. In strain 308-18, however, xanthosine formation was not so greatly affected by exogenous guanosine as in strain GU-16. There was no difference in the effect of guanosine on the total amount of purine nucleosides produced, including both xanthosine and inosine, in these two strains.

Although the production of purine nucleosides such as inosine, xanthosine, or guanosine by mutants of Bacillus subtilis has been dealt with in several

399 400 MOMosE ANDSHIIO VOL. 15

papers (1-9), there is as yet very little information concerning the mechanism by which each of these products is accumulated. However, for the industrial production of large amounts of individual purine nucleosides, it is important to know what kinds of genetic traits determine the pattern of accumulation. The results presented in this paper clearly demonstrate the roles of GMP reductase, a key enzyme in the interconversion of purine nucleotides, of succinyl AMP synthetase, and of resistance to purine analogues, especially to 8-azaxanthine, in determining the accumulation pattern. The subject was studied with a typical inosine-producing strain 38-3, an adenine-requiring mutant of Bacillus subtilis K, and several kinds of mutants derived from it. Experiments were designed for the selection of the following four types of adenine-requiring mutants from strain 38-3 and for the analysis of products accumulated by these mutants : (1) GMP reductase-positive 8-azaxanthine- sensitive (original), (2) GMP reductase-negative 8-azaxanthine-sensitive, (3) GMP reductase-positive 8-azaxanthine-resistant, and (4) GMP reductase-negative 8-azaxanthine-resistant mutants. The role of adenine requirement will also be described.

MATERIALS AND METHODS

Bacterial strains. An adenine-requiring mutant of Bacillus subtilis K, strain 38-3, was employed as an original strain. This strain produces a large amount of inosine in media such as AA or AAT (see Media). Media. Three media were employed. The complete medium contained 10 g polypeptone, 10 g yeast extract, and 5 g NaCI in 1,000 ml of distilled water (pH 7.2). The MC medium contained 8.5 g KH2P04i 0.5 g Na citrate 2H20, 1 g (NH4)2S04, 0.2 g MgSO4.7H2O, 5 g glucose, and 1 g Difco casamino acids in 1,000 ml of distilled water (pH 7.2). MgS04.7H20 and glucose were sterilized separately. The AA medium contained 70 g glucose, 15 g NH4C1, 0.4 g MgS04.7H20, 2 ppm Fe2+ (as FeSO4.7H2O), 2 ppm Mn2+ (as MnSO4.4H2O), 1.5 g KC1, 1 g KH2P04i 2 g amino acid mixture (5) (a mixture of pure amino acids having a composition similar to Difco vitamin-free casamino acids), 300 mg adenine (NBC), and 25 g CaC03 (sterilized separately) in 1,000 ml of distilled water. The pH was adjusted to 7.2. When tryptophan-requiring mutants were employed, 50 or 300 ,ug/ml of L-tryptophan was added to the MC or AA medium, respectively. These supplemented media are indicated as MCT and AAT, respectively, in this paper. Agar plates were prepared by adding 2 % agar to the complete, MC, or MCT medium. Genetic techniques. All mutants used in this work were derived by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (10) : About 5 X 108/ml of vegetative cells in 0.05 M phosphate buffer, pH 7.2, were treated with 500- 2,000 pg/ml of this drug for 40 min in an ice bath. The treated cells were grown overnight in the complete medium, then washed, and used for the selection of auxotrophic or drug-resistant mutants (see RESULTS). Complete, 1969 Purine Nucleoside Accumulation Pattern 401

MC, MCT, or other supplemented plates were employed for the selection of mutants. When necessary, prototrophic strains were constructed from the mutants by transducing wild type markers from strain K into the test aux- otroph using the temperate phage sp-10 (11). Cultivation for production of nucleosides. To detect the ability of the mutants to produce nucleoside(s), 3 ml of either AA or AAT medium were poured into a test tube and inoculated with a loopful of mutant cells grown overnight on complete agar. Cultivation was carried out on a shaker at 30° for three or four days. To investigate the production of nucleosides more precisely, 20-ml cultures in the same medium were grown in a 500-ml flask. In the latter case, a streak of overnight culture on a slant of complete medium was used to inoculate each flask. Cultivation was carried out on a shaker at 30°, and nucleoside (s) production, growth, and pH were periodically observed. Determination of nucleosides. Inosine, xanthosine, and guanosine ex- creted into the culture medium were identified by comparison with authentic samples of the nucleosides using the following techniques : (1) Measurement of Rf values by paper chromatography in propanol-ammonia-water (20: 10 : 3), butanol-acetic acid-water (4: 1:1), or water-saturated butanol ; (2) evaluation of electrophoretic mobilities by paper electrophoresis in 10% acetic acid; and (3) analysis of UV absorption spectra (2, 12). Quantitative determination of nucleosides was carried out as follows : 2 ml of the culture were added to an equal volume of 0.2 N NaOH and the mixture was heated at 100° for 2 min in a water bath. After the cells were precipitated, 0.01-0.02 ml of the super- natant was spotted on a filter paper (Toyo Roshi No. 51). Following chro- matographic separation, each nucleoside was eluted from the paper with 0.1 N HCI, and estimated spectrophotometrically (12). Measurement of growth. An aliquot of each culture in AA or AAT medium was diluted 26 times with distilled water and the optical density at 540 mp was measured. Measurement of GMP reductase activity. The assay was carried out according to the method of MAGAsANIK (13) with a slight modification : The reaction mixture contained 6 pmoles GMP, 0.5 pmole NADPH2i 10 pmoles GSH, 150 pmoles Tris buffer (pH 7.4), and 2.2 ml of enzyme extract in a final volume of 3 ml. The reaction was initiated at 21.5° by the addition of NADPH2 and followed by observing the decrease in optical density at 340 m,cc. A blank control devoid of GMP was used to correct for the enzymatic oxidation of NADPH2. The specific activity was defined as -1034E/min/mg protein.

RESULTS

Derivation of GMP reductase-negative mutants and their characteristics GMP reductase, which catalyzes conversion of GMP to IMP, plays an 402 MOMOSEAND S3IIO VOL. 15 important role in the interconversion of purine nucleotides (14) (see Fig. 1). To investigate whether or not this enzyme has any effect on the accumulation pattern of purine nucleosides, GMP reductase-negative adenine-requiring mutants were derived from strain 38-3, an adenine-requiring inosine producer, according to the procedure indicated in Table 1, and the products accumulated by these mutants were determined. Tryptophan requirement was used as a genetic marker throughout the isolation process, because it had no effect on the inosine productivity (Expt. 1 in Table 2). Strain TD-9, an ade+-transductant derived from strain TR-101, completely lost the ability to produce inosine. This loss can be attributed exclusively to the acquisition of the wild type adenine gene, because several adenine auxotrophs derived from TD-9 showed the same level of inosine production as strain TR-101 (Expt. 2 in Table 2). A mutant which grew on adenosine or inosine but not on guanosine (15) was isolated from strain PU-17, a non-exacting purine aux- otroph derived from strain TD-9 (see Table 1). This mutant, strain RD-37, was proved to be devoid of GMP reductase activity as shown in Table 3. Several adenine-requiring mutants lacking succinyl-AMP synthetase (see Fig. 1) were finally derived from this GMP reductase-negative strain via a pur+- transductant TP-3. All of the adenine auxotrophs obtained in this way, for example, RA-23, RA-30, and RA-60, produced inosine in amounts equal to that of the original strain 38-3 or strain TR-101 (Expt. 3 in Table 2). These GMP reductase-negative mutants did not accumulate any other product be-

Fig, 1. Biosynthesis and interconversion of purine nucleotides in B. subtilis (18). 1: PRPP amidotransferase, 2 and 8: sAMP lyase, 3: AICAR trans- formylase and IMP cyclohydrolase, 4 : IMP dehydrogenase, 5 : GMP synthetase, 6: GMP reductase, 7: sAMP synthetase, 9: AMP deaminase. 1969 Purine Nucleoside Accumulation Pattern 403

Table 1. Derivation of GMP reductase-negative mutants from strain 38-3.

Table 2. Inosine productivity in various mutants. 404 MOMOSE AND SHIIO V&L. 15

Table 3. GMP reductase activities in strains PU-17 and RD-37.

sides inosine (see Table 6).

Derivation of mutants resistant to purine analogues and their characteristics Attempts were made to isolate mutants resistant to purine analogues from two types of inosine-producing strains ; a GMP reductase-positive adenine requirer, strain 38-3, and a GMP reductase-negative adenine requirer, strain 30-12, a clone of strain RA-30 randomly selected by single colony isolation. Table 4 shows the sensitivity of these two strains to 1 mg/ml of 19 purine analogues. Similar patterns of sensitivity were observed in these two strains with one exception. They were equally sensitive to 2,6-dichloropurine, 2,8-dimercapto-6-hydroxypurine, 2-amino-6-mercaptopurine, 8-azaxanthine, and 8-azaguanine. In the case of 8-azaxanthine and 8-azaguanine, growth inhibi- tion was overcome by guanosine. This fact suggested that these two analogues specifically inhibit some step (s) in GMP synthesis (see Fig. 1). It might be expected, therefore, that the accumulation pattern of the original strains 38-3 and 30-12 could be altered by mutational events expressed as resistance to 8-azaxanthine or 8-azaguanine. Table 5 shows the accumulation patterns in mutants resistant to 1 mg/ml of 8-azaxanthine derived from strain 38-3 and 30-12. About 60% of the 256 resistant mutants obtained from GMP reductase-positive strain 38-3 were found to produce xanthosine as well as inosine, but none of the mutants produced guanosine. On the other hand, 70% of the 400 resistant mutants obtained from GMP reductase-negative strain 30-12 were found to produce guanosine as well as inosine, and none of these mutants produced xanthosine. As a control experiment, 20 clones were randomly selected from strains 38-3 and 30-12, and the accumulation pattern was investigated. The strains tested were all sensitive to 8-azaxanthine and produced inosine alone. The productivities of purine nucleosides in several resistant mutants obtained from strains 38-3 and 30-12 are shown in Table 6 (Exp. 1,2). Loss of the tryptophan requirement did not affect the production of either inosine or guanosine in strain 12-3 (Expt. 3 in Table 6). On the other hand, loss of the adenine requirement had remarkable effects on both productivities. In an ade+-transductant derived from strain 12-3, inosine was no longer produced and guanosine was produced only in small amounts. An investigation was made to see whether 8-azaxanthine-resistant mutants 1969 Purine Nucleoside Accumulation Pattern 405

Table 4. Sensitivity of strains 38-3 and 30-12 to purine analogues.

Table 5. Accumulation of purine nucleosides in 8-azaxanthi ne-resistant mutants derived from strains 38-3 and 30-12. 406 MoMosE AND SHIIo VOL. 15

Table 6. Production of purine nucleosides by various mutants resistant to 8-azaxanthine.

could be obtained directly from ade+-strain TD-9 which possessed GMP reductase activity and produced none of purine nucleosides. Among the 100 strains isolated as mutants resistant to 1 mg/ml of 8-azaxanthine, three strains were found to produce comparatively small amount of xanthosine. Strain 9- 73, the most potent xanthosine producer among them, produced 1.5 g/liter of this substance after four days' cultivation (Expt. 4 in Table 6). No inosine was produced by these three strains as in the case of 123-At Fig. 2 shows the time course of purine nucleosides formation by the original strain, 38-3, a typical xanthosine producer, 383-308, and a typical guanosine producer, 12-3. In strain 383-308, the amount of xanthosine accumulated after four days' cultivation was 8 g/liter. The amount of total purine nucleosides accumulated by this strain was equivalent to the amount of inosine accumulated by strain 38-3. Strain 12-3 accumulated up to 5 g/liter guanosine after four days' cultivation. The amount of total purine nucleosides accumulated by this strain was 15 g/liter, considerably greater than the accumulation of inosine by strain 38-3. 1969 Purine Nucleoside Accumulation Pattern 407

Fig. 2. Time course of purine nucleoside accumulation by strains 38-3, 383-308, and 12-3. -D total nucleosides 0-0 Inosine, •-• xanthosine, A-L guanosine, , x -- x growth (see MATERIALS AND METHODS).

Attempts were also made to isolate mutants resistant to 8-azaguanine. However, all of the 100 strains derived from strain 38-3 as mutants resistant to 1 mg/ml of this drug displayed the same accumulation pattern as strain 38-3 (inosine alone). Mutants resistant to various concentrations of this drug (200-1,000pg/ml) were derived from strain 30-12. None of the 400 strains tested produced significant amounts of xanthosine or guanosine, i.e., more than 2 g/liter, although some strains were found to produce 0.5-1.5 g/liter of guanosine. The 20 strains that were resistant to 1 mg/ml of 8-azaguanine and were able to produce small amounts of guanosine (0.5-1.5 g/liter) were all sensitive to 1 mg/ml of 8-azaxanthine by the criterion of colony-forming ability. Conversely, the 20 strains which were resistant to 1 mg/ml of 8- azaxanthine and which produced guanosine (1-4 g/liter) were all sensitive to 1 mg/ml of 8-azaguanine. Effect of guanosine on purine nucleoside formation It is possible that the 8-azaxanthine-resistant strains exhibiting accumulation patterns different from that of the original strain may have arisen from a mutation in one of the genes regulating GMP synthesis. To test this hypothesis, guanine auxotrophs were isolated from both the original and resistant strains and the effects of guanosine on xanthosine formation by these auxotrophs were compared. Strains GU-16 and 30848 were selected 408 MOMOSE AND SHIIO VOL. 15

Fig. 3. Effect of guanosine on nucleoside formation by strains GU-16 and 308-18.

0-0 Inosine, •-• xanthosine, A---A total nucleosides, X -- X growth. as the test strains ; the former was one of the guanine auxotrophs derived from original strain 38-3, and the latter from strain 383-308, a typical xanthosine producer resistant to 8-azaxanthine (see Table 6 and Fig. 2). Various concentrations of guanosine (0-1,000 pg/ml) were added to each 20 ml of AA medium in a 500 ml flask. After GU-16 or 308-18 cells were inoculated into supplemented medium, cultivation was carried out for three days and the amount of xanthosine or inosine accumulated in the culture as well as the cell density was determined. The result is indicated in Fig. 3. In strain GU-16, the amount of xanthosine accumulated increased sharply with growth level and this increase was correlated with the concentration of guanosine in the range of 0-200 pg/ml. The production of xanthosine rapidly decreased at concentrations above 200 ,gig/ml although the cell density reached the same saturating level at these concentrations. On the other hand, the amount of inosine gradually increased in a wide range of 0-750 ~~g/ml of added guanosine and then decreased at higher concentrations (Fig. 3a). In contrast to strain GU-16, strain 308-18 was less sensitive to the inhibitory effect of higher concentrations of guanosine on the accumulation of xanthosine (Fig. 3b). However, there was little difference between these strains in the effect of guanosine on the accumulation of total purine nucleosides (xanthosine plus inosine).

DISCUSSION

From the present results, it may be concluded that an adenine-requiring 1959 Purine Nucleoside Accumulation Pattern 409 strain sensitive to 8-azaxanthine produces inosine alone, irrespective of the presence or absence of GMP reductase, and the genetic acquisition of resistance to this analogue can alter the accumulation pattern from inosine alone to inosine plus xanthosine for a GMP reductase-positive strain or to inosine plus guanosine for a GMP reductase-negative strain. This result suggests that GMP reductase may play an important role in qualitatively determining the accumulation pattern. The fact that 8-azaxanthine-resistant mutants derived from a GMP reductase-negative strain produced guanosine instead of xanthosine agrees well with the finding by KONISHI and SHIRO (7). They report- ed that a guanosine-inosine producing strain, S-5G-22, isolated spontaneously from an 8-azaguanine-resistant xanthosine-inosine producer of B, subtilis K was devoid of GMP reductase activity. In addition to GMP reductase and resistance to purine analogues, adenine requirement may be regarded as a significant factor in the determination of the accumulation pattern as indicated in Table 6 (Expt. 3 and 4). A much lower level of xanthosine or guanosine was produced as a sole product in GMP reductase positive- or GMP reductase negative-, 8-azaxanthine-resistant prototrophic strains, respectively. The relationship between the three above- mentioned genetic traits and the accumulation of purine nucleosides is sum- marized in Table 7. The reason why there exists such a relationship is an interesting problem to be solved. As for the role of resistance to purine analogues, the difference between 8-azaxanthine-resistant and sensitive mutants is clearly demonstrated by the effect of guanosine on xanthosine formation (see Fig. 3). It may be considered that in strain GU-16, isolated from the

Table 7. Relationship between genetic trait and accumulation of purine nucleoside. 410 MOMOSEAND SHIIO VOL. 15 sensitive strain, exogenous guanosine, a derivative of the end product in GMP synthesis, is taken up by the cells and converted to some effective derivative (s) (probably nucleotide (16, 17)) which in turn exerts a stronger inhibitory effect on the synthesis of GMP from IMP than on IMP synthesis. Thus, according to this hypothesis, the accumulation product is converted from xanthosine to inosine as the concentration of guanosine increases (see Fig. 1). On the other hand, such a product conversion was scarecely observed in strain 308-18 which was isolated from the resistant strain. How- ever, the effect of guanosine on the total amount of purine nucleosides (xanthosine and inosine) produced in this strain was almost identical with that in the sensitive strain. These findings suggest that the mutational event phenotypically expressed as resistance to 8-azaxanthine specifically af- fects the control mechanism related to GMP synthesis from IMP. It was proved in the present work that 8-azaxanthine is a more potent agent than 8-azaguanine for the selection of mutants altered in the accumulation pattern. It may be that 8-azaxanthine can be converted to a more specific inhibitor against GMP synthesis than 8-azaguanine can. Although it is possible that some of the resistant mutants obtained by use of either 8-azaxanthine or 8-azaguanine are identical, it is true nonetheless that many of the 8-azaxanthine-resistant guanosine producers are different from 8- azaguanine-resistant guanosine producers, since the former strains are still sensitive to 8-azaguanine, and conversely, the latter to 8-azaxanthine.

The authors are indebted to Dr. T. Yoshida, Dr. T. Tsunoda, and Dr. N. Katsuya of the laboratories for their helpful encouragement. The authors are also indebted to Mr. S. Miyashiro of the laboratory for his technical assistance. This study was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, held on April 1, 1968.

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