APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1977, p.

337-341 Copyright 1977 American Society for Microbiology

Vol. 34, No. 4 Printed in U.S.A.

Mutation of an Inosine-Producing Strain of Bacillus subtilis to DL-Methionine Sulfoxide Resistance for Guanosine Production
HIROSHI MATSUI,* KATSUAKI SATO, HITOSHI ENEI,
AND

YOSHIO HIROSE

Central Research Laboratories, Ajinomoto Company, Inc., Kawasaki, Japan Received for publication 28 March 1977

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.
Studies on the microbial production of guanosine so far indicate that the keys to increased guanosine production are: (i) genetic lack of succino-adenosine 5'-monophosphate (AMP) synthetase and guanosine 5'-monophosphate (GMP) reductase; (ii) deficiency in the purine nucleoside hydrolyzing activity; (iii) avoidance of regulation of inosine 5'-monophosphate (IMP) dehydrogenase and GMP synthetase by GMP; and (iv) deregulation of IMP synthetic enzymes to feedback effects of AMP or GMP. Konishi and Shiro (4) derived 8-azaguanineresistant mutants lacking succino-AMP synthetase and GMP reductase (from Bacillus subtilis) that produced 4 g of guanosine per liter. Momose and Shiio (8) obtained 8-azaxanthineresistant mutants lacking succino-AMP synthetase and GMP reductase (from B. subtilis) that produced 5 g of guanosine per liter. Nogami et al. (9) derived adenosine-resistant mutants from Bacillus sp. that produced 5 g of guanosine per liter and found that the activity of the GMP synthetase of these mutants was not inhibited by adenosine. Komatsu et al. (3) obtained mutants of B. subtilis that were deficient in nucleoside phosphorylase and produced 10 g of guanosine per liter. We have derived methionine sulfoxide (MSO)-resistant mutants from an inosine-producing strain, 1411, that was devoid of GMP reductase and found that these mutants excreted more guanosine and less inosine than the parent. The present paper deals with the

derivation of improved guanosine-producing mutants from strain 1411 and the clarification of some of their properties, especially the correlation of MSO resistance with guanosine productivity and the activities of enzymes involved in the conversion of IMP to GMP.
MATERIALS AND METHODS Bacterial strains. The MSO-resistant mutants were derived from an inosine-producing auxotroph of B. subtilis 1411. The genetic characteristics of this parental strain were adenine- and histidineless, lacking GMP reductase and succino-AMP synthetase. Culture media. Medium 1 contained: polypeptone, 10 g; yeast extract, 10 g; NaCl, 5 g; glucose, 5 g; and distilled water, in a total volume of 1 liter. The pH was adjusted to 7.0 with NaOH. Medium 2 contained: glucose, 20 g; NH4Cl, 5 g; KH2PO4, 4 g; MgSO4-7H2O, 0.2 g; FeSO4 7H2O, 0.01 g; MnSO4 4H20, 0.01 g; sodium citrate, 0.5 g; L-glutamic acid, 1 g; L-histidine, 0.5 g; adenine, 0.1 g; and distilled water, in a total volume of 1 liter. The pH was adjusted to 7.0 with KOH. Medium 3 contained: glucose, 80 g; NH4NO3, 15 g; KH2PO4, 0.2 g; MgSO4 * 7H2O, 0.4 g; FeSO4*7H2O, 0.01 g; MnSO4 4H20, 0.01 g; CaCl2- 2H20, 2 g; DL-methionine, 0.3 g; L-glutamic acid, 10 g; L-histidine, 0.3 g; Ajieki (soybean hydrolysate), 40 ml; ribonucleic acid, 1.2 g; CaCO3, 15 g; and distilled water, in a total volume of 1 liter. The pH was adjusted to 7.5 with KOH. Calcium carbonate was sterilized inde-

pendently. Derivation of mutants. Cells (1 x 109 to 5 x 109) from the logarithmic growth phase in medium 1

337

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were

MATSUI ET AL.

APPL. ENVIRON. MICROBIOL. (pH 7.0) and treated with 500 gg of lysozyme (EC 3.2.1.17) per ml and 5 ,ug of deoxyribonuclease (EC 3.1.4.5) per ml in the same phosphate buffer for 30 min at 37C. The lysate was centrifuged at 12,000 x g for 20 min, and the supernatant was used to assay the activity of the glutamine synthetase. Enzyme activity assay. The activities of IMP dehydrogenase, GMP synthetase, 5'-nucleotidase, and glutamine synthetase were assayed by the methods of Konishi and Shiro (4), Magasanik et al. (7), Heppel and Hilmoe (2), and Elliott and Gale (1), respectively. Specific activities of the enzymes were expressed as nanomoles per minute per milligram of dried cells or protein. The protein content was estimated by the method of Lowry et al. (6).

harvested, washed with 50 mM KH2PO4K2HPO4 buffer (pH 7.0), and exposed to 250 or 500 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine per ml in the same phosphate buffer for 30 to 50 min in an ice bath. The N-methyl-N'-nitro-N-nitrosoguanidine-treated cells were washed, diluted, and placed on medium 2 agar plates supplemented with DLmethionine and DL-MSO. After incubation for 2 to 5 days at 34C, colonies appeared on the plates and were picked. Fermentation tests. Cells that grew overnight on medium 1 agar plates were inoculated into 20 ml of medium 3 in a 500-ml flask. After cultivation for 72 h at 34C under agitation, 10 ,ul of the culture broth was spotted on filter paper (Toyo Roshi no. 51). After separation by paper chromatography, using isopropanol-ammonia-water (7:2:1) as the solvent, inosine, xanthosine, and guanosine were extracted from the paper with 10 ml of 0.1 N HCl for 5 h at room temperature and were estimated spectrophotometrically. Growth inhibition tests. Bacterial cells grown overnight in 3 ml of medium 2 were washed with 50 mM KH2PO4-K2HPO4 buffer (pH 7.0), and about 106 cells were inoculated into 3 ml of medium 2 supplemented with methionine analogs. The growth was measured by absorbance at 540 nm after culturing for 24 h at 34C. Enzyme preparation. The cells that had grown overnight on medium 1 agar plates were transferred to 20 ml of the inoculum medium, the composition being the same as medium 3 except CaCO3 was omitted. After cultivation for 16 to 20 h at 34C, the cells were harvested from the culture broth by centrifugation, washed twice with a 0.85% NaCl solution and three times with chilled acetone, and dried in a vacuum. For GMP synthetase preparation, the harvested cells were washed with a 0.85% NaCl solution containing 0.1 M ethylenediaminetetraacetic acid. The acetone-dried cells were used to assay the activities of IMP dehydrogenase, GMP synthetase, and 5'-nucleotidase. For the glutamine synthetase preparation, the harvested cells were washed twice with 50 mM KH2PO4-K2HPO4 buffer

RESULTS

Growth inhibition by methionine analogs and recovery of inhibition by purine bases and amino acids. The growth of strain 1411 on medium 2 was inhibited by the addition of 30 mg of L-methionine or 10 mg of DL-MSO per ml. The growth inhibition was partially reversed by 10 mg of L-glutamic acid or L-glutamine per ml, but not by 100 ,ug of hypoxanthine, xanthine, or guanine per ml. Furthermore, 30 mg of DL-methionine, 20 mg of DLMSO, or 20 mg of L-MSO per ml or a mixture of 30 mg of DL-methionine and 5 mg of DL-MSO per ml strongly inhibited the growth of strain 1411, and the inhibition could not be reversed by 10 mg of L-glutamic acid or L-glutamine per ml. The results are presented in Table 1. Derivation of MSO-resistant mutants from strain 1411 and their guanosine productivity. The N methyl -N' nitro -N nitrosoguanidinetreated cells were placed on medium 2 agar plates containing 5 to 10 mg of DL-MSO per ml plus 30 mg of DL-methionine per ml, and the colonies that appeared were transferred to test tubes containing medium 3; these were cul-

TABLE 1. Growth inhibition by methionine analogs and recovery of the inhibition by purine bases and amino acidsa
Growth response (%) Addition to medium 2 (mg/ml) None

(0.1 mg/

Hyp
88
0 0 0 0 0
0

Xan Gua (0.1 mg/ (0.1 mg/

78
0 0 0 0 0
0
0

L-Met

1 mg/
ml

10 mg/
ml

L-Glu 1 mg/ 10 mg/

ml ml

L-Gln 1 mgl 10 mg/

ml ml

None L-Met (30) DL-Met (30) L-MSO (20) DL-MSO (10) DL-MSO (20) DL-Met (30) + DLMSO (5) DL-Met (30) + DLMSO (10)

100 0 0 0 0 0
0 0

69
0 0 0 0 0
0 0

78
0 0 0

58
0 0 0

88
25 0 0 57 0
0
0

0 0
0 0

0
0
0

84 33 0 0 53 0
0 0

84 31 3 0 3 0
0

93 29 4 0 30 0
0

a B. subtilis 1411 was used. Hyp, Hypoxanthine; Xan, xanthine; Gua, guanine; Met, methionine; Glu, glutamic acid; Gln, glutamine.

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tured for 72 h at 34C. Those that produced more than 7 g of guanosine and 5 g of inosine per liter were selected (first screening test). Parental strain 1411 produced 5 g of guanosine and 10 g of inosine per liter under these conditions. Mutants improved in guanosine productivity were obtained from these MSO-resistant mutants with a frequency of 16% (32 of 200 colonies). The selected mutants were then purified by the single-colony isolation technique, and their guanosine productivity was determined in medium 3 variations containing ribonucleic acid at 1.0, 1.2, and 1.4 g/liter (second screening test). The parent accumulated 5.5 g of guanosine and 11.0 g of inosine per liter at its optimal ribonucleic acid concentration. Mutant 14119 accumulated 9.6 g of guanosine and 4.8 g of inosine per liter (Table 2). Growth inhibition of parent 1411 and mutant 14119 by MSO is presented in Fig. 1. Strain 14119 was more resistant to MSO than the parent, 1411. Furthermore, mutants more resistant to MSO were derived from strain 14119. The cells treated with N-methyl-N'-nitro-N-nitrosoguanidine were incubated and enriched in medium 2 containing 30 mg of DL-methionine plus 10 mg of DL-MSO per ml and placed on agar plates containing the same antagonist. The colonies that appeared were cultured in medium 3 in test tubes for 72 h. Strain AG169, which produced large amounts of xanthosine and guanosine but did not produce inosine,
TABLE 2. Purine nucleoside productivity of strains 1411, 14119, and AG169
Productivity (glliter)

Strain

XanthoInoaine sine

. Guanosine

Total purine nucleoside

1411 14119 AG169

11.0 4.8 0

0 0 6.0

5.5 9.6 8.0

16.5 14.4 14.0

was selected. The guanosine productivity of strain AG169 was lower than that of the parent, 14119 (Table 2). Growth inhibition of strain AG169 by MSO was less than that of its parent, 14119 (Fig. 1). Glutamine synthetase activity and its inhibition by DL-MSO in MSO-resistant mutants. Since MSO is generally recognized as an antagonist of glutamic acid or glutamine, it was assumed that the MSO-resistant mutants 14119 and AG169 might be different in glutamine synthetase activity or its inhibition by MSO. Therefore, the glutamine synthetase activities of strains 1411, 14119, and AG169 were compared, but there was practically no difference in the specific activity or the degree of inhibition (Table 3). GMP synthetase activity and its inhibition by GMP in MSO-resistant mutants. Since GMP synthetase combines xanthosine 5'-monophosphate (XMP) and glutamine to give GMP and MSO may serve as an antagonist of glutamine, it was assumed that the GMP synthetase ofthe MSO-resistant mutants might differ from that of the parent. As a matter of fact, no differences were observed among the three strains in specific activity of the GMP synthetase and the degree of inhibition by GMP (Table 4). Therefore, GMP synthetase was not the key in solving this problem. IMP dehydrogenase activity and its inhibition by GMP in MSO-resistant mutants. IMP dehydrogenase, which converts IMP to XMP, was investigated. Table 5 shows that the specific activity of this enzyme in the MSO-resistant mutants 14119 and AG169 increased slightly in comparison with that of the parent, 1411. Inhibition of enzyme activity by GMP was not observed in any of the three strains. 5'-Nucleotidase activity in MSO-resistant mutants. The activity of 5'-nucleotidase was compared in the three strains. The specific activity of 5'-nucleotidase, which decomposes IMP, XMP, and GMP, decreased remarkably in the MSO-resistant mutants (Table 6). Km value of IMP dehydrogenase and 5'nucleotidase in the MSO-resistant mutants. The substrate affiniity of IMP dehydrogenase
TABLE 3. Glutamine synthetase activity and its inhibition by DL-MSO in MSO-resistant mutants
Strain

to

DL-4MSO (mg/mi)

1. Growth inhibition of strains 1411 (0), 14119 (0), and AG169 (A) by DL-MSO.
FIG.

1411 14119

AG169

Sp act of glutamine synthetase (nmol/min per mg of protein) 10 mM DL-MSO No addition 80.8 55.8 80.0 55.8 81.5 56.5

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APPL. ENVIRON. MICROBIOL.

TABLE 4. GMP synthetase activity and its inhibition by GMP in MSO-resistant mutants
Strain Sp act of GMP synthetase (nmollmin per mg of dried cells)
No addition
0.6 mM GMP
4 mM GMP

1411 14119 AG169

0.580 0.568 0.563

0.577 0.572 0.560

0.464

0.426 0.450

TABLE 5. IMP dehydrogenase activity and its inhibition by GMP in MSO-resistant mutants
Sp act of IMP dehydrogenase
Strain

(nmol/min per mg of dried cells)

No addition
0.55 mM GMP

1411 14119 AG169

9.53 12.5 13.9

9.39 12.3 13.9

and 5'-nucleotidase was investigated in MSOresistant mutants 14119 and AG169. No differences were found 'in the substrate affinity of the two enzymes (Table 7). It is notable, however, that the affinity of the IMP dehydrogenase to IMP is approximately 10 times greater than that of 5'-nucleotidase to IMP and GMP.

DISCUSSION A high concentration of MSO inhibited the growth of the adenine auxotroph of B. subtilis 1411, which lacked GMP reductase, and the inhibition was overcome by glutamine or glutamic acid. Among mutants resistant to MSO, those producing more guanosine than their TABLE 6. 5'-Nucleotidase activity of MSO-resistant mutants parent were found with high frequency; the reason for this is not known at present. The Sp act of 5'-nucleotidase (nmol/min per mg best mutant, 14119, produced 9.6 g of guanosine of dried cells) Strain per liter at a weight yield of 12% from con5'-GMP 5'-XMP 5'-IMPa sumed sugar in comparison to that of 6.9% for 3.43 the parent 1411. Inosine production decreased 1411 0.686 6.86 1.89 0.343 2.83 concomitantly. Since the growth of this mutant 14119 1.11 0.129 0.815 is still inhibited by 50% by 10 mg of DL-MSO AG169 plus 30 mg of DL-methionine per ml, mutants a Substrate. that might produce much more guanosine were expected to be found among strains resistant TABLE 7. Km value of the IMP dehydrogenase and 5'-nucleotidase in MSO-resistant mutantsa to a higher concentration of MSO. Surprisingly, more potent guanosine-producing mutants K,, (mM) were not found among the mutants resistant to 5'-Nucleotidase Strain IMP dehydrogenase a higher concentration of MSO, but a mutant that produced xanthosine and guanosine in (IMP)b IMP GMP place of inosine was found. This suggests that 3.2 2.5 0.26 the peculiarities of these mutants were not 1411 2.4 0.25 3.0 changed by changes in glutamine synthetase 14119 2.7 4.0 0.33 or GMP synthetase. To grasp the significance AG169 of the MSO resistance more clearly, these rea Km value was calculated by the method of Linesistant mutants were compared with their par- weaver and Burk (5). b Substrate. ent with respect to the activities of enzymes

involved in the conversion of IMP to GMP, glutamine synthetase, GMP synthetase, IMP dehydrogenase, and 5'-nucleotidase. In glutamine synthetase and GMP synthetase, variations were not observed. In the MSO-resistant mutants, the specific activity of the IMP dehydrogenase slightly increased as the degree of resistance increased. The specific activity of 5'nucleotidase decreased remarkably in parallel with the acquisition of MSO resistance. Although the affinity of IMP dehydrogenase to IMP was 10 times higher than that of 5'-nucleotidase to IMP, the differences were not recognized among these MSO-resistant mutants. These findings indicate that the key to effective guanosine accumulation in MSO-resistant mutants is that the breakdown of IMP to inosine is limited due to the decrease of 5'-nucleotidase activity and the increase of IMP dehydrogenase activity, and the main metabolic flow of the IMP conversion is directed to GMP by way of XMP. Since the lack of GMP reductase blocks the conversion of GMP to IMP, the degradation of GMP to guanosine is caused by 5'-nucleotidase, even though the specific activity of 5'nucleotidase on GMP in the MSO-resistant mutants is 55 or 32% of that of the parent. Especially in strain AG169, the extreme decrease of 5'-nucleotidase activity causes an increase in intracellular GMP concentration, and the GMP synthetase activity is inhibited strongly by GMP. As a result, the intracellular XMP concentration increases and is degraded gradually to xanthosine by 5'-nucleotidase.

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ACKNOWLEDGMENTS We thank M. Takahashi, H. Okada, and T. Shiro, Directors of the Central Research Laboratories, Ajmomoto Co. Inc., for their encouragement during the course of this work. LITERATURE CITED 1. Elliott, W. H., and E. F. Gale. 1948. Glutamine-synthesizing system of Staphylococcus aureus: its inhibition by crystal violet and methionine sulphoxide. Nature (London) 161:129-130. 2. Heppel, L. A., and R. J. Hilmoe. 1951. Purification and properties of 5'-nucleotidase. J. Biol. Chem. 188:665-676. 3. Komatsu, K., A. Saijo, R. Kodaira, and T. Osawa. 1970. Derivation of purine nucleoside-producing strains from an adenine-lacking mutant of Bacillus subtilis. Amino Acid Nucleic Acid (Japan) 22:54-58. 4. Konishi, S., and T. Shiro. 1968. Fermentative production of guanosine by 8-azaguanine resistant of Bacillus subtilis. Agric. Biol. Chem. 32:396-398.

5. Lineweaver, H., and D. Burk 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56:658-666. 6. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. Magasanik, B., H. S. Moyed, and L. B. Gehring. 1957.

Enzymes essential for the biosynthesis of nucleic acid guanine; inosine 5'-phosphate dehydrogenase of Aerobacter aerogenes. J. Biol. Chem. 226:339-350. 8. Momose, H., and I. Shiio. 1969. Genetic and biochemical studies on 5'-nucleotide fermentation. IV. Effect of GMP reductase and purine analogue resistance on purine nucleotide accumulation pattern in adenine auxotrophs of Bacillus subtilis. J. Gen. Appl. Microbiol. 15:399-411. 9. Nogami, I., M. Kida, I. Iijima, and M. Yoneda. 1969. Studies on the fermentative production of purine derivatives. Part I. Derivation of guanosine and inosine-producing mutants from aBacillus strain. Agric. Biol. Chem. 32:144-152.