Agric. Biol. Chem., 53 (1), 97- 102, 1989 97

Regulation of Pyrimidine Nucleotide Biosynthesis in Cytidine Deaminase-negative Mutants of Bacillus subtilisf Satoru ASAHi, Muneharu Doi, Yutaka TSUNEMI and Shun-ichi AKIYAMA Applied Microbiology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka 532, Japan Received July ll, 1988

The role of uridine and cytidine compoundsin regulating pyrimidine nucleotide biosynthesis de novo was studied with cytidine deaminase-negative mutants of Bacillus subtilis. In the wild type strain, the formation of six enzymes for uridine 5'-monophosphate (UMP)biosynthesis was severely repressed by exogenous cytidine or uracil, whereas the formation of the enzymesin a cytidine deaminase-negative mutant was repressed only by uracil. On the other hand, the formation of cytidine 5'-triphosphate (CTP) synthetase was not affected by uracil. This enzynme was repressed only when a cytidine deaminase-negative mutant was grown in the presence of excess cytidine. Studies on feedback inhibition also showed that the activity of CTP synthetase was inhibited by cytidine nucleotides, but not by uridine nucleotides.

The synthesis de nove of uridine 5'-mono- distinguish between the effects of these two phosphate (UMP) in Bacillus subtilis is cata- compounds on gene expression, because they lyzed through the sequential actions of six are readily converted into each other through enzymes (Fig. 1). The expression of the pyr the sequential actions of several enzymes in- genes encoding these enzymesis repressed by volved in pyrimidine metabolism5) (Fig. 1). In adding uracil2) and appears to be coordinately the course of studies on the production of regulated.3) On the other hand, regulation of pyrimidine nucleosides by B. subtilis,l>6) we these enzymes through feedback inhibition or found that cytidine deaminase-negative mu- activation is focused on the first UMPbiosyn- tants unable to convert cytidine to uridine thetic enzyme, carbamyl phosphate synthetase are helpful in distiguishing between the effects P (CPSase P). This enzyme is strongly inhibited of uridine and cytidine. This paper deals with by uridine nucleotides, and is activated by 5- the isolation of such mutants ofB. subtilis and phospho-a-D-ribose 1-pyrophosphate (PRPP) with the regulation of pyrimidine nucleotide and guanosine nucleotides.2'4) In contrast to biosynthesis de novo. all this information on the UMPbiosynthetic pathway, nothing is known about the regu- Materials and Methods latory system for the conversion of uridine 5'- triphosphate (UTP) to cytidine 5'-triphos- Microorganisms. Bacillus subtilis No. 122n and mutants phate (CTP) by CTP synthetase. of it were used (Table I). The regulation of the pyrimidine pathway Media. The minimal medium (M-l) was that of from the viewpoint of gene expression can be Spizizen7) supplemented with 0.3% (w/v) Casamino acids demonstrated by adding uracil or cytidine to (Difco) and with 100/ig/ml L-arginine to repress the the growth medium. It is difficult, however, to synthesis ofcarbamyl phosphate synthetase A.4) The stock

Microbial Production of Uridine. Part II. For Part I, see ref. 1. 98 S. Asahi et ai culture medium (A-l) contained (w/v) 1% peptone, 1% and Switzer.4) ATCaseactivity was determined by the meat extract, 0.3% yeast extract and 0.5% NaCl; the pH method of Brabson and SwitzerU); the product formed in being adjusted to 7.2 with 1 n NaOH.The fermentation this reaction being determined by the method of Prescott medium (F-l) contained (w/v) 16%glucose, 4% corn steep and Jones.12) DHOaseactivity was assayed in the reverse liquor, 0.7% corn gluten meal, 2% urea and 0.5% CaCO3; of the biosynthetic direction13); the amount of product the pH being adjusted to 7.0 with 1 n NaOH. Glucose and being determined in the same way as for ATCase. DHO- urea weresterilized separately. DHase activity was determined by the method of O'Donovan and Gerhart.14) OPRTase and OMP-DCase Measurementof bacterial growth. Growth was deter- activities were assayed by the methods of Umezuet al.15); mined by measuring the absorbance at 590nm of the the amount of each product being determined by HPLC.1* culture broth. An optical density of 1.0 corresponded to The specific activity of each enzyme was expressed in about 4.0 x 108 cells/ml. units/mg protein. One unit of enzyme activity corre- sponded to 1 nmol of substrate consumed or product Orotic acid and orotidine excretion. Cells grown over- formed/min under the assay conditions. night on an A-l agar plate were inoculated into 3 ml ofA-l medium in a test tube. After cultivation at 37°C for 8hr, Chemicals. Carbamyl phosphate, N-carbamyl-D,L- 1ml of the culture was transferred to a 200-ml flask aspartic acid, dihydro-D,L-orotic acid, orotic acid, orot- containing 20ml of F-l medium supplemented with vari- idine, OMPand 6-diazo-5-oxo-L-norleucine (DON) were ous concentrations of cytidine or uracil. Incubation was purchased from Sigma Chemical Co., Ltd.; N-methyl-N'- carried out at 37°C for 3 days on a rotary shaker nitro-N-nitrosoguanidine (NTG) was from Aldrich (240rpm). The amounts of orotic acid and orotidine Chemical Co.; 3-(/?-iodophenyl)-2-(/?-nitrophenyl)-5- produced were determined by high performance liquid phenyl-2//-tetrazolium chloride, reduced glutathione and chromatography (HPLC).1* dithiothreitol were from Wako Pure Chemical Ind., Ltd.; and PRPPwas from PL-Biochemicals. Enzyme preparation. The buffers used for the crude enzymepreparation were: for cytidine deaminase, 0.1 m Results Tris-HCl (pH 7.5); for CPSase P (for harvesting by centrif- ugation), 0.05 m K-HEPES (pH 7.2) containing 10% (w/v) Isolation of B. subtilis mutants glycerol and 2 mML-glutamine; for CPSase P (for dialysis), For the efficient development of a cytidine 0.1 M K-HEPES (pH 7.2) containing 10% (w/v) glycerol deaminase-negative mutant, an OMP-DCase and 0. 1 mKC1; for aspartate transcarbamylase (ATCase), 0.1 mTris-acetate (pH 7.2); for dihydroorotase (DHOase) deficient strain (F-100)1* derived from the wild and dihydroorotate dehydrogenase (DHO-DHase), 0. 1 m type strain, B. subtilis No. 122, was used as the Tris-HCl (pH 8.5); and for orotate phosphoribosyltrans- ferase (OPRTase) and orotidine 5'-monophosphate de- starting strain. NTG-Treated cells of strain F- carboxylase (OMP-DCase), 0.1 m Tris-HCl (pH 8.5) con- 100 were spread on agar plates ofM-l medium taining 2mM dithiothreitol. The bacterial strains were supplemented with 10 //g/ml of uracil and then grown in 500-ml flasks containing 150ml of M-l medium incubated at 37°C for 2 days. Among the supplemented with various additions at 37°C on a rotary shaker (240rpm). Cells (1 x 108~5x 108 cells/ml) at the Table I. OMP-DCaseand Cytidine Deaminase mid-log growth phase were harvested by centrifugation at Activities of Various Strains Derived 20,000 x q at 4°C for 10min, washed twice with buffer and from B. subtilis No. 122 then disrupted with a sonic oscillator (Kubota Model Cells of each strain were grown in M-l medium 200M, 9kHz) at 4°C for 10min. The sonicates were centrifuged at 20,000 x g at 4°C for 30 min. When assaying supplemented with 10 /zg/ml of uracil. Enzyme activity is for DHO-DHaseactivity, this centrifugation process was expressed in units/mg protein. omitted. The supernatant or sonicates were immediately Enzyme activity dialyzed against buffer at 4°C for 6hr under gently Strain stirring. Protein was measured by the method of Lowryet alS) OMP-DCase Cytidine deaminase No. 122 8.0 74.7 Assaying of enzyme activities. Unless otherwise stated, F-100 <0.01 73.8 all procedures were carried out at 30°C. Cytidine de- FC-200 <0.01 <0.01 aminase activity was determined according to the pro- CD-300 7.6 <0.01 cedure of Cohen and Wolfenden.9) CTPsynthetase activity was determined by the method of Anderson10) at 38°C. Abbreviation: OMP-DCase, orotidine 5'-monophos- CPSase P activity was assayed by the method of Paulus phate decarboxylase. Regulation of Pyrimidine Nucleotide Biosynthesis 99

Table II. Relative Activities of Pyrimidine Nucleotide Biosynthetic Enzymes in B. subtilis No. 122 and Its Mutant Strains under Various Conditions The bacterial strains were grown in M-l medium with various additions. The specific activities (units/mg protein) of carbamyl phosphate synthetase P (CPSase P), aspartate transcarbamylase (ATCase), dihydroo- rotase (DHOase), dihydroorotate dehydrogenase (DHO-DHase), orotate phosphoribosyltransferase (OPRTase), orotidine S'-monophosphate decarboxylase (OMP-DCase) and CTP synthetase in strain No. 122 grown in M-l medium were 0.2, 3.4, 1.9, 0.7, 1.0, 8.0 and 0.2, respectively. The enzyme activities were normalized to the levels found in strain No. 122 grown in M-l medium.

Relative activity Strain Addition Mg/ml CPSaseP ATCase DHOase J^?° OPRTase DHase

No. 122 None -1.01.0 1.0 1.0 1.0 1.0 1.0 Uracil (U) 100 0.0 0.2 0.3 0.0 0.2 0.3 1.0 Cytidine (CR) 200 0.0 0.2 0.2 0.2 0.3 0.2 1.0 CD-300 U 100 0.0 0.3 0.3 0.1 0.2 0.3 1.0 CR 200 0.9 1.0 0.9 1.1 1.0 0.9 0.2 colonies appearing on the plates, strain FC- Table III. Effect of Uracil and Cytidine on Orotic Acid and Orotidine Excretion by 200, which failed to grow in the presence of the F-100 and FC-200 Strains cytidine but retained the ability to grow in the The F-100 (OMP-DCasO and FC-200 (OMP- presence of uracil, was isolated by the replica DCase", cytidine deaminase") strains were grown in F-l plating technique.16) To obtain a transformant medium supplemented with various concentrations of with the OMP-DCaseactivity, cells of strain uracil and cytidine. The amounts of orotic acid and FC-200 transformed1} with DNAfrom strain orotidine excreted were determined by high performance No. 122 were spread on M-l agar plates and liquid chromatography. then incubated at 37°C for 2 days. Among the Addition (jug/ml) Productivity (mg/ml) colonies appearing on the plates, strain CD- 300, which had recovered the OMP-DCase Uracil Cytidine Orotic acid+orotidine 0 4 0 F-100 activity, was selected. The cytidine deaminase 0 0 20 15 0 0 and OMP-DCaseactivities of these strains are 2 0 0 shown in Table I. 10 15 0 0 30 2 0 0 Repression ofpyrimidine nucleotide biosynthetic FC-200 4 0 0 15

enzymes 0 0 20 2 0 0 The activities of pyrimidine nucleotide bio- 10 15 0 0 synthetic enzymes from strain No. 122 and 30 15 CD-300 grown under various conditions were studied (Table II). The results can be sum- marized as follows, i) All six UMP biosyn- none of the UMPbiosynthetic enzymeswas thetic enzymes were repressed when uracil repressed, whereas CTPsynthetase was. These (100/ig/ml) was added to the growth medium results indicate that the formation of each for each strain; however, no influence was UMPbiosynthetic enzyme is regulated by uri- observed on CTP synthetase. ii) The same dine compounds, while the formation of CTP results were obtained whenwild type strain synthetase is regulated by cytidine compounds. No. 122 was grown with cytidine (200//g/ml). iii) When cytidine deaminase-negative strain Excretion of orotic acid and orotidine CD-300 was grown with cytidine (200 /ig/ml), In a previous paper,1) we showed that an 100 S. Asahi et al.

Table IV. Inhibition and Activation of Pyrimidine Nucleotide Biosynthetic Enzymes in B. subtilis No. 122 Strain No. 122 was grown in M-l medium. Each enzyme activity is expressed as a percentage of that obtained with no addition.

Addition Relative activity (5 him) CPSase P ATCase DHOase DHO-DHaseOPRTase OMP-DCaseCTP synthetase

None 00 100 100 100 100 100 100 Carbamyl phosphate 62 Nt 94 102 93 101 92 N-Carbamyl-D,L-aspartate 97 62 Nt 97 101 104 96 Dihydro-D,L-orotate 88 90 70 Nt 121 103 92 Orotate 00 111 93 48 Nt 97 103 Orotidine 5 '-monophosphate ll 108 98 82 63 Nt 98 UMP 22 103 101 89 63 75 102 UTP ll 104 103 93 72 72 Nt CMP 98 99 108 101 103 96 42 CTP 02 97 110 111 105 99 1 GMP 28 96 89 82 96 91 113 GTP 33 94 91 93 91 Nt AMP 42 91 95 102 100 92 62 ATP Nt 93 97 96 102 96 Nt PRPP 38 100 99 103 Nt 91 101 DON 2 Nt Nt Nt Nt Nt 1

Abbreviations : Nt, not tested; PRPP, 5-phospho-a-D-ribose 1-pyrophosphate; DON, 6-diazo-5-oxo-L-norleucine; others, see Table II.

OMP-DCase-deficient strain of B. subtilis Inhibition and activation of pyrimidine nucle- No. 122 excretes orotic acid and orotidine into otide biosynthetic enzymes the culture broth, and that the excretion is The effects of various compounds on the inhibited by an excessive amount of uracil. To activities of pyrimidine nucleotide biosynthetic assess the effect of uridine and cytidine com- enzymeswere examined. Theresults are sum- pounds on the excretion of orotic acid and marized in Table IV. The activity of CPSase P orotidine, strain F-100 with a defect in OMP- was severely inhibited by UMP, UTP and DCase activity and strain FC-200 with defects DON, and slightly activated by GMP, GTP in both OMP-DCaseand cytidine deaminase and PRPP. The activity ofCTPsynthetase was activities (Table I) were cultured in F-l me- completely inhibited by CTP and DON. The dium supplemented with various concen- activities of both CPSase P and CTP syn- trations of uracil or cytidine. As shown in thetase were weakly inhibited by AMP. The Table III, strain F-100 excreted orotic acid and activity of each UMPbiosynthetic enzyme was orotidine maximally in the presence of also weakly inhibited by its own product. 80jUg/ml uracil or lOOjUg/ml cytidine; the excretion was inhibited at a higher concen- Discussion tration of either. In the cytidine deaminase- negative strain (FC-200), the excretion was not Using cytidine deaminase-negative mutants inhibited at any concentration of cytidine; unable to convert cytidine into uridine com- however, uracil had the same effect as observ- pounds, we could distinguish between the ef- ed for strain F-100. fect ofexogenous uracil and that ofcytidine on the enzyme repression. Whencells of the wild type strain were grownin the presence ofuracil Regulation of Pyrimidine Nucleotide Biosynthesis 101

Fig. 1. Regulatory System for Pyrimidine Nucleotide Biosynthesis in B. subtilis. The individual reactions are catalyzed by (1) carbamyl phosphate synthetase P, (2) aspartate transcarba- mylase, (3) dihydroorotase, (4) dihydroorotate dehydrogenase, (5) orotate phosphoribosyltransferase, (6) OMPdecarboxylase, (7) CTP synthetase, (8) uracil phosphoribosyltransferase, (9) uridine phosphorylase, (10) cytidine deaminase and (1 1) 5'-nucleotidase (and non-specific phosphatases). or cytidine, the formation of all six UMP which was strongly inhibited by uridine nu- biosynthetic enzymes was repressed. Such re- cleotides, and was activated by PRPP and pression was not observed when a cytidine guanosine nucleotides; cytidine nucleotides deaminase-negative strain (CD-300) was were not involved in this regulation. These grown with cytidine (Table II). On the other observations agree with the results obtained hand, CTPsynthetase, responsible for convert- for the Marburg strain of B. subtilis.2AM) It is ing UTP to CTP, was not repressed in the notable, however, that the activity of CTP presence of uracil. This enzyme was repressed synthetase was not inhibited by uridine nu- only when the cytidine deaminase-negative cleotides, but was by cytidine nucleotides strain was grown in the presence of excess (Table IV). cytidine (Table II); the lack of repression in the On the basis of these results, an overall case of wild type strain No. 122 may be due to regulatory system for pyrimidine nucleotide the conversion of cytidine, added to the me- biosynthesis in B. subtilis No. 122 was de- dium, to uridine compounds by cytidine de- veloped (Fig. 1). Although the biosynthetic aminase (Table I). These results suggest that route for pyrimidine nucleotides is commonin the formation of UMPbiosynthetic enzymes a wide range of microorganisms,17) the reg- in B. subtilis is regulated by uridine com- ulatory system for the route appears to vary; pounds, while that of CTPsynthetase is reg- for example, in enteric bacteria, such as ulated by cytidine compounds. Further evi- Escherichia coli and Salmonella typhimurium, dence for this is that the excretion of orotic acid UMPbiosynthesis de novo is regulated by the and orotidine by a strain with defects in both feedback inhibition of ATCase18) and CPSase OMP-DCaseand cytidine deaminase activities by pyrimidine nucleotides,19) and by the feed- (FC-200) depended on the concentration of back repression of each of the UMPbiosyn- uracil, but not on cytidine (Table III). thetic enzymes by uridine or cytidine deriva- Studies on feedback inhibition and acti- tives.20^2^ vation revealed the following features (Table Acknowledgments. Weare grateful to Drs. K. Morita, IV). Among the UMPbiosynthetic enzymes, Y. Sugino, Y. Nakao and H. Okazaki for their interest and regulation was focused onlv on CPSase P. encouragement throughout this work. We also wish to 102 S. Asahi et al. thank Dr. R. J. Miller for the helpful comments on the ll) J. S. Brabson and R. L. Switzer, J. Biol. Chem., 250, manuscript. Thanks are also due to Miss M. Nakano for 8664 (1975). her skillful technical assistance. L. M. Prescott and M. E. Jones, Anal. Biochem., 32, 408 (1969). J. B. Beckwith, A. B. Pardee, R. Austrian and F. Referenc es Jacob, J. Mol. BioL, 5, 618 (1962). M. Doi, Y. Tsunemi, S. Asahi, S. Akiyama and Y. G. A. O'Donovan and J. C. Gerhart, /. BacterioL, 109, 1085 (1972). Nakao, Agric. Biol. Chem., 52, 1479 (1988). B. W. Potvin, R. J. JCelleher, Jr. and H. Gooder, /. K. Umezu, T. Amaya, T. Yoshimoto and K. Tomita, Bacteriol, 123, 604 (1975). /. Biochem., 70, 249 (1971). T. J. Paulus, T. J. McGarry, P. G. Shekelle, S. J. Lederberg and E. M. Lederberg, J. BacterioL, 63, 399 (1952). Rosenzweig and R. L. Switzer, J. Bacteriol., 149, 775 (1982). G. A. O'Donovan and J. Neuhard, BacterioL Rev., 34, 278 (1970). T. J. Paulus and R. L. Switzer, J. Bacteriol., 137, 82 (1979). J. C. Gerhart and A. B. Pardee, Cold Spring Harbor B. K. Rima and I. Takahashi, /. Bacteriol., 129, 574 Symposium, Quant. BioL, 28, 491 (1963). (1977). A. Pierard, N. Glansdorff, M. Mergeay and M. S. Asahi, Y. Tsunemi and M. Doi, Japan Kokai Wiame, J. Mol. BioL, 14, 23 (1975). Tokkyo Koho, 61-135597, June 23, 1986. R. A. Kelln, J. J. Kinahan, K. F. Folterman and G. J. Spizizen, Proc. Natl Acad. Sci. U.S.A., 44, 1072 A. O'Donovan, J. BacterioL, 124, 764 (1975). (1958). M. Schwartz and J. Neuhard, J. BacterioL, 121, 814 (1975). O. H. Lowry, N. J. Rosebrough, A. L. Farrand R. J. Randall, /. Biol. Chem., 193, 265 (1951). P. W. Thomas and G. A. O'Donovan, J. Gen. R. M. Cohen and R. Wolfenden, J. Biol. Chem., 246, MicrobioL, 128, 895 (1982). 7561 (1971). P. W. Thomas, S. A. Herlick and G. A. O'Donovan, D. M. Anderson, Biochemistry, 22, 3285 (1983). FEMS MicrobioL Lett., 18, 275 (1983).