By Ctpsynthetase

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 corresponded 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 centrifugation), 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 phosphoribosyltransferase (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), dihydroorotase (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.

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