Addition Of Uracil Research Articles

Evidence for the existence of a novel component of biological water stress (anhydrotic stress) inEscherichia coli

The pathways for biosynthesis of pyrimidines, L-arginine and the polyamines are intimately interrelated in many microorganisms. We discovered in this study that growth of wild-typeEscherichia coli in low-water-activity minimal media is inhibited by the addition of uracil. Uracil sensitivity was observed irrespective of whether the dissolved solute(s) contributing to decreased water activity was ionic (e.g. NaCl, K2SO4), nonionic and impermeable (e.g. sucrose), nonionic and freely permeable (e.g. glycerol), or any mixture of these types. A mutant resistant to such growth inhibition was isolated and was shown to harbour a bradytrophic mutation inargA, the gene encoding the first step in the L-arginine biosynthetic pathway. Mutations inargR, whose product is the aporepressor of the same pathway, or exogenous supplementation with L-arginine or L-citrulline, also conferred resistance to uracil inhibition in low-water-activity media. A similar uracil-sensitivity phenotype, which was reversible byargA, argR, or L-arginine addition, was exhibited even in media with a more moderate reduction in water activity in two different situations: for aspeC mutant (which is defective in the enzyme ornithine decarboxylase required for biosynthesis of the polyamines) and for the wild-type strain in media additionally supplemented with L-ornithine. On the basis of these observations, we propose a model in which high cytoplasmic levels of the intermediary metabolite L-ornithine are inhibitory to growth ofE. coli in media of low water activity. Our results also provide the first evidence for the existence of a third component of physiological water stress, which is elicited by both impermeable and permeable dissolved solutes (the other two known components are ionic stress, which is elicited only by ionic solutes, and osmotic stress, which is elicited only by impermeable solutes either ionic or nonionic). We propose the term anhydrotic stress to refer to this novel component of water stress.

First-pass metabolism of 5-fluorouracil in the perfused rat small intestine.

The first-pass intestinal metabolism of 5-fluorouracil (5-FU) was investigated by single-pass perfusion of the rat small intestine. At the low concentration of 0.06 mg/ml, the fraction of 5-FU absorbed into (i.e., appeared in) the mesenteric venous blood (Fa,b) was about 50% smaller than the fraction absorbed (disappeared) from the intestinal lumen (Fa), indicating the first-pass extraction of 5-FU in the intestinal mucosa. By addition of uracil (6 mg/ml), the Fa of 5-FU was reduced presumably by competition for the pyrimidine carrier at the process of intestinal uptake (entry into the mucosa). The Fa,b was also reduced, but to a lesser extent, resulting in insignificant first-pass extraction. These results suggest that the extraction of 5-FU in the absence of uracil is caused by metabolism and that uracil is a competitor for this pathway. When 5-FU concentration was raised from 0.06 to 0.6 mg/ml in the absence of uracil, the Fa was reduced by about 50%, consistent with the suggestion of the involvement of saturable uptake by the pyrimidine carrier, and thereafter remained unchanged at 6 mg/ml. However, since Fa,b was also reduced by a similar extent, the intestinal availability (FI=Fa,b/Fa) was unchanged at about 0.5, indicating that the intestinal first-pass extraction of 5-FU is independent of concentration with the extraction ratio (difference between unity and FI) of about 0.5 over the wide range of concentration from 0.06 to 6 mg/ml. Thus, the present study demonstrates that the significant first-pass metabolic extraction of 5-FU occurs in the mucosa of the small intestine, supporting our previous suggestion that 5-FU undergoes first-pass metabolism not only in the liver but also in the small intestine after oral administration. Considering that the oral bioavailability of 5-FU in the human (28%) is reportedly comparable with that in the rat (28%), it is likely that intestinal first-pass metabolism may be significant also in the human. Intestinal first-pass metabolism should be taken into account to explore more efficient and controlled oral 5-FU therapy.

Complete Inactivation of Escherichia coli Uridine Phosphorylase by Modification of Asp5 with Woodward's Reagent K

Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addition of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, respectively) and k2 (to 6.1 or 4.8 min-1, respectively) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addition of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J.C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, respectively. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.

Molecular characterization of pyrimidine biosynthesis genes from the thermophile Bacillus caldolyticus.

The genes encoding the six pyrimidine biosynthesis enzymes from the thermophile Bacillus caldolyticus were characterized by cloning and complementation in Escherichia coli, and by nucleotide sequence analysis. Nine cistrons are clustered within an 11 kb region of the chromosome, the gene order being: orf1-pyrB-pyrC-pyrAa-pyrAb-orf2-p yrD-pyrF-pyrE. This organization of the cluster is very similar to that of the pyr operon of Bacillus subtilis. Different parts of the B. caldolyticus cluster were cloned in two orientations in the expression shuttle vector pHPS9. Complementation studies in B. subtilis established that expression of the pyr genes was dependent on the vector-borne promoter, suggesting that they are part of an operon, and that the native promoter of the operon had not been cloned. The deduced amino acid sequence of the individual cistrons showed 49 to 78% identity with the corresponding B. subtilis cistrons. Measurements of the aspartate transcarbamylase (pyrB), orotidine monophosphate decarboxylase (pyrF) and orotate phosphoribosyltransferase (pyrE) levels in cells grown under different conditions indicated that expression of the operon is repressed 7-9-fold by addition of uracil to the growth medium. Based on the nucleotide sequence in the intercistronic region between orf1 and pyrB a regulatory mechanism involving transcriptional termination and antitermination is proposed to control expression of the operon.

Mutants of Toxoplasma gondii Resistant to Atovaquone (566C80) or Decoquinate

Mutants of Toxoplasma gondii resistant to drugs that appear to affect the mitochondrial bc1 complex were isolated with the aid of mutagenesis with ethylnitrosourea. Mutant DeqR-1 was > 1,000-fold more resistant to decoquinate than was the wild type but more sensitive to atovaquone (formerly called 566C80). Mutant AtoR-1 was 20-fold more resistant to atovaquone than was the wild type and was also partially cross-resistant to decoquinate. Both drugs rapidly inhibited oxygen uptake by freshly prepared extracellular parasites, and the mutants were resistant to this inhibition. Neither the addition of uracil to the medium nor the use of a mutant of T. gondii with a defect in pyrimidine salvage had a substantial effect on the in vitro anti-parasitic activity of these drugs, suggesting that de novo pyrimidine synthesis was not the major biochemical target of either drug.

Role of transcription pausing in the control of the pyrE attenuator in Escherichia coli

Expression of the Escherichia coli pyrE gene is regulated by transcription attenuation in the intercistronic orfE-pyrE region and modulated by the distance between the transcribing RNA polymerase and the leading ribosome as a function of the supply of UTP and GTP. In this communication we show that pyrE expression is hyper-repressed in vivo following addition of uracil in strains carrying the nusAcs10 mutation. This phenotype, previously seen in rpsL1204 strains whose ribosomes are pseudodependent on streptomycin and work at suboptimal elongation rate, indicates that RNA polymerase escapes from the ribosomes in the pyrE attenuator region in the nusA mutant. In vitro transcription studies revealed that the build-up of the full-length attenuated orfE transcript occurred more slowly in the presence of the NusA protein than in its absence. Moreover, the NusA protein enhanced several transcription pauses through the orfE gene. These effects were more pronounced when low concentrations of either UTP or GTP were used than at low concentrations of either CTP or ATP. The results indicate that the NusA protein is required for proper regulation of pyrE gene expression and is involved, together with the NTP pools, in maintaining the coupling between transcription and translation in the pyrE attenuator region by inhibiting RNA chain elongation.

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5'-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the level of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5'-triphosphate and uridine 5'-phosphate.

Ribose‐transfer Activity from Uridine to 5‐Fluorouracil in Ehrlich Ascites Tumor Cells

Anabolism of 5-fluorouracil (5-FU) in the presence of uracil was examined using the cell-free extract of Ehrlich ascites tumor cells. FU-nucleoside formation from 5-FU with ribose 1-phosphate (R-1-P) or 2'-deoxyribose 1-phosphate was not readily inhibited even by the addition of uracil at 100 times higher concentration than 5-FU. FU-nucleotide formation from 5-FU with R-1-P and adenosine 5'-triphosphate or with 5-phosphoribosyl 1-pyrophosphate was slightly reduced as the concentration of uracil was increased. It was also found that 5-fluorouridine (5-FUR) was produced by "nucleoside N-ribosyltransferase," transferring a ribose moiety from uridine (UR) to 5-FU directly. This activity might play a role in the preferential formation of 5-FUR. However, 5-fluoro-2'-deoxyuridine was not produced by directly transferring a deoxyribose moiety. On the basis of several column chromatographies and characterization of kinetics, pH dependency, and response to inhibitors, the enzyme protein of the ribosyltransferase could not be distinguished from that of the phosphorylase.

Intestinal 5-fluorouracil absorption: use of Ussing chambers to assess transport and metabolism.

We have employed an in vitro system to study transport and metabolism of organic molecules by gastrointestinal tissues. Such a system would aid in the evaluation of the potential for oral delivery of organic molecules. Transport and metabolism of 5-fluorouracil (5-FU) were studied using rabbit intestinal preparations. Unidirectional fluxes and metabolism were measured in vitro in Ussing chambers under short-circuit conditions. Results from these studies reveal that in ileum, proximal, and distal colon, steady-state fluxes of 5-FU (10 microM added to both bathing solutions) are established after 30 min and remain constant for at least 110 min. Transport of 5-FU under "sink" conditions with 10 microM 5-FU present in the mucosal or serosal bathing solution alone demonstrated similar rates of transport as under "nonsink" conditions. The concentration dependence of 5-FU fluxes indicates that the mucosal (m)-to-serosal (s) flux is composed of both a saturable and a linear component over the range of 1-100 microM in the ileum, whereas the s-to-m flux in the ileum and both fluxes in the colon are linear functions of concentration. Over the concentration range employed and the time course of these studies, 5-FU had no effect on the electrical properties of the ileum or colon. In the ileum, the m-to-s but not the s-to-m flux of 5-FU was reduced by (1) serosal ouabain (0.1 mM); (2) reduction of the bathing solution Na concentration; and (3) addition of uracil, thymine, thymidine, uridine, 2-deoxyuridine, or uridine-5'-monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

PHOTOREACTIONS OF TRIS(2,2′‐BIPYRIDYL)‐RUTHENIUM(II) WITH PEROXYDISULFATE IN DEOXYGENATED AQUEOUS SOLUTION IN THE PRESENCE OF NUCLEIC ACID COMPONENTS, POLYNUCLEOTIDES, AND DNA*

Abstract— The photobleaching of excited tris(2,2′‐bipyridyl)‐ruthenium(II), *Ru(bpy)32+, by peroxydis‐ulfate in the presence of DNA and a series of polynucleotides, mononucleosides (uridine, cytidine, adenosine, guanosine), and purine or pyrimidine bases was studied in deoxygenated aqueous solution at room temperature. A reaction scheme is proposed which is confirmed by data obtained from mixing experiments with Ru(III) and bleaching measurements of Ru(II) using either continuous visible light or a nanosecond laser pulse (353 nm). The primary photobleaching step is the formation of Ru(bpy)32+ and the SO4‐ radical anion. In the presence of nucleic acids the two oxidizing species are formed in close proximity to the strand, since we used conditions where the Ru(bpy) 32+ ion is bound to the strand. Concerning the secondary reactions two clear cases (and several more complex cases) can be distinguished. On addition of uracil to the Ru(bpy)32+/S2O82‐ system the quantum yield for photobleaching is not significantly changed (φrel⋍ 0.95), whereas it drops to virtually zero for guanosine‐containing substrates, including DNA. The former result is explained by a reaction of SO4‐ with uracil leading to theN–1 radical which oxidizes Ru(II) to Ru(IIl). In contrast, the guanine moiety reacts with Ru(III) converting it into Ru(II). Therefore, in the presence of S2O82‐ and a substrate carrying a guanine moiety, Ru(bpy32+ acts as a photocatalyst.

Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster.

A 10.5-kilobase PstI endonuclease fragment encoding the entire Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster was cloned in Escherichia coli by transformation of a carB strain to uracil-independent growth. The cloned fragment also complemented E. coli pyrB, pyrC, pyrD, pyrE, and pyrF mutants. From the ability of subclones to complement E. coli pyr mutants, the gene order was deduced to be pyrBCADFE. The B. subtilis pyrB gene was shown to be expressed in E. coli, but synthesis of the enzyme was not repressible by the addition of uracil to the growth medium. The approximate molecular weights of the polypeptides encoded by B. subtilis pyrA, pyrB, pyrC, pyrD, pyrE, and pyrF were found to be 110,000, 36,000, 46,000, 34,000, 25,000, and 27,000, respectively.

Genetic analysis of the 5-azacytidine sensitivity of Escherichia coli K-12

DNA containing 5-azacytidine (5-azaC) has been shown to form stable detergent-resistant complexes with cytosine methylases. We reasoned that if 5-azaC treatment causes protein-DNA cross-links in vivo, then mutations in DNA repair and recombination genes may increase the sensitivity of a cell to 5-azaC. We found that although recA (defective) and lexA (induction-negative) mutants of Escherichia coli were very sensitive to the drug, mutations in uvrA and ung genes had little effect on cell lethality. The sensitivity of recA strains to 5-azaC was dose dependent and was enhanced by the overproduction of a DNA cytosine methylase in the cell. Unexpectedly, a strain of E. coli carrying a recA mutation and a deletion of the DNA cytosine methylase gene (dcm) was found to be significantly sensitive to 5-azaC. Study of mutations in the pyrimidine salvage pathway of E. coli suggests that direct phosphorylation of 5-azaC, rather than phosphorylation of its degradation products, is largely responsible for the lethal effects of the drug. The addition of uracil to the growth medium has little effect on cell lethality of recA mutants, but it partially reversed the inhibition of cell growth caused by 5-azaC. This reversal of the bacteriostatic effects of the drug could not be achieved by adding cytosine or orotic acid to the growth medium and required the presence of functional UMP-pyrophosphorylase (gene upp) in the cell.

Effect of Dietary Nucleotides on Response to Bacterial Infections

A nucleotide-free diet (NFD) suppresses cellular immunity including suppression of both murine allograft rejection and murine graft-vs-host disease in H2 mismatched marrow recipients. To explore the effect of this diet upon response to a bacterial challenge, BALB/c mice were placed upon one of four diets: NFD, NFD supplemented with 0.25% RNA (NFR) and NFD reconstituted with either 0.06% adenine (NFA) or 0.06% uracil (NFU). After 8 weeks on the diets, which supported normal growth in all groups, each dietary group of 10 mice was challenged with an intravenous injection of 1 X 10(7) live Staphylococcus aureus (Strain ATCC 25923). By 120 hr postinoculation all mice in the NFD group had died; mortality was only 56% in the NFR group (p less than 0.05). Addition of uracil resulted in mortality similar to that of the NFR group. Addition of adenine resulted in mortality close to that of the NFD group. Thus, dietary nucleotide restriction increases the mortality from staphylococcal sepsis. Addition of certain dietary nucleotides appears to decrease this susceptibility to bacterial challenge.

In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo

The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID 50 values (concentration of compound causing 50% inhibition of [ 3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 ± 8.7, 6.4 ± 5.3 and 6.3 ± 0.5 μM, respectively. Comparative ID 50 values for chloroquine were 13.5 ± 4.2, 22.8 ± 7.6 and 343 ± 114 nM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 μM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5′-phosphate decarboxylase (E.C. 4.1.1.23).

Salmonella typhimurium mutants with altered expression of the pyrA gene due to changes in RNA polymerase.

Rifampicin-resistant mutants of Salmonella typhimurium were isolated and tested for pleiotropic defects in the regulation of pyr gene expression. Seven per cent of all the Rifr mutants were inhibited in growth by addition of uracil (uracil-sensitive). The uracil-sensitive phenotype ( UraS ) was reversed by arginine or citrulline, but not by ornithine, and it was suppressed by mutations in either argR or pyrH , which causes increased expression of pyrA . It was shown that the basal levels of carbamoylphosphate synthase (the pyrA gene product) was reduced to approximately 60% in the mutants, and that addition of arginine and/or uracil to the growth medium caused hyperrepression of pyrA expression. The expression of other genes of the arginine and pyrimidine biosynthetic pathways was not affected significantly in the mutants. The mutations were located in the rpoB gene coding for the beta-subunit of RNA polymerase, suggesting a regulatory function of RNA polymerase in the control of pyrA expression.

Purine and pyrimidine utilisation by chicks fed nitrogen‐limiting diets

AbstractThe responses of chicks to purine and pyrimidine supplementation of diets severely limiting in the dispensable amino acids have been measured in three experiments. In experiment 1, combined additions of adenine and uracil to a diet with a nitrogen (N) content of 19.84 g kg−1 DM enhanced growth and dry matter intakes to levels similar to those attained by chicks receiving an equinitrogenous supplement of glutamic acid. Chicks in the latter group, however, had the best efficiencies of carcass N deposition. In experiment 2, growth, dry matter intakes and efficiency of food conversion of chicks fed a diet with 23.36 g N kg−1 DM were improved markedly by combined supplements of adenine, uracil, guanine and cytosine. Equinitrogenous inclusions of guanine; guanine with thymine; uracil with thymine; or uracil with adenine were ineffective. Efficiency of carcass N deposition declined appreciably on supplementing the basal diet with purines and pyrimidines in different combinations, appreciable reductions occurring with additions of thymine; uracil with thymine and uracil with adenine. The low N efficiencies observed in the last two groups were improved by combined additions of adenine, uracil, guanine and cytosine. The highest efficiencies of N utilisation occurred in chicks fed a glutamic acid supplement. In experiment 3, the simultaneous addition of uracil, adenine and guanine was as effective in stimulating the growth performance of chicks fed a diet with 18.89 g N kg−1 DM as a more complex mixture including cytosine. The best growth and efficiency of food conversion values were recorded in the group receiving supplementary glutamic acid.

Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.

Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine.

The repressive effects of exogenous cytidine on growing cells was examined in a specially constructed strain in which the pool sizes of endogenous uridine 5'-diphosphate and uridine 5'-triphosphate cannot be varied by the addition of uracil and/or uridine to the medium. Five enzymes of the pyrimidine biosynthetic pathway and one enzyme of the arginine biosynthetic pathway were assayed from cells grown under a variety of conditions. Cytidine repressed the synthesis of dihydroorotase (encoded by pyrC), dihydroorotate dehydrogenase (encoded by pyrD), and ornithine transcarbamylase (encoded by argI). Moreover, aspartate transcarbamylase (encoded by pyrB) became further derepressed upon cytidine addition, whereas no change occurred in the levels of the last two enzymes (encoded by pyrE and pyrF) of the pyrimidine pathway. Quantitative nucleotide pool determinations have provided evidence that any individual ribo- or deoxyribonucleoside mono-, di-, or triphosphate of cytosine or uracil is not a repressing metabolite for the pyrimidine biosynthetic enzymes. Other nucleotide derivatives or ratios must be considered.

Control of metabolite secretion in Bacillus subtilis.

SUMMARY: Bacillus subtilis, grown in media containing amino acids and glucose or other carbohydrates (including glycerol), secreted pyruvate, acetate, acetoin, butanediol, and small quantities of isobutyrate and isovalerate. The enzymes producing acetoin and butanediol were specifically induced by acetate and weakly by propionate. Even when these enzymes were present, acetate accelerated the rate of acetoin production. Since acetoin and butanediol are readily interconverted (via butanediol dehydrogenase), they serve as an oxidation-reduction buffer for NAD/NADH. When carbohydrate was exhausted, acetate and, more slowly, acetoin were oxidized. Mutants unable to produce acetoin or acetate were blocked in the metabolic path from the carbohydrate to these compounds. Other mutants unable to re-utilize the compounds were either deficient in the necessary enzymes, e.g. of citric acid cycle, or they stopped growth so early that they could not use up the carbohydrate. In particular, a uracil-requiring mutant stopped growth precociously, did not oxidize acids or acetoin, and was unable to sporulate. Addition of uracil restored all these properties to normal. The differences in secretion and re-utilization of metabolites can be employed to classify unidentified sporulation mutants.

Inactivation of aspartic transcarbamylase in sporulating Bacillus subtilis: demonstration of a requirement for metabolic energy.

The aspartic transcarbamylase (ATCase) activity of Bacillus subtilis cells disappears rapidly from stationary-phase cells prior to sporulation. ATCase activity does not appear in the culture fluid during the stationary phase; hence the enzyme appears to be inactivated in the cells. The enzyme is inactivated normally in two different mutants lacking proteases; the activity is very stable in crude extracts of cells or in the culture fluid. These results suggest that ATCase is not inactivated by the general proteolysis that occurs in sporulating bacteria. The inactivation of ATCase can be completely inhibited after it has begun by oxygen starvation or addition of fluoroacetate. Inhibitors of oxidative phosphorylation and electron transport also interrupt the inactivation of ATCase. The inactivation of ATCase is very slow in two mutant strains that are deficient in enzymes of tricarboxylic acid cycle. Addition of gluconate to stationary cultures of the mutant strains, which is known to restore depleted adenosine 5'-triphosphate pools in these bacteria, also restores inactivation of ATCase. These experiments support the conclusion that the generation of metabolic energy is necessary for the inactivation of ATCase in stationary cells. ATCase activity is stable in growing cells in which ATCase synthesis is repressed by addition of uracil; the enzyme is inactivated normally, however, when such cells cease growing.