Oxygen and nitrate in utilization by Bacillus licheniformis of the arginase and arginine deiminase routes of arginine catabolism and other factors affecting their syntheses.

Bacillus licheniformis has two pathways of arginine catabolism. In well-aerated cultures, the arginase route is present, and levels of catabolic ornithine carbamoyltransferase were low. An arginase pathway-deficient mutant, BL196, failed to grow on arginine as a nitrogen source under these conditions. In anaerobiosis, the wild type contained very low levels of arginase and ornithine transaminase. BL196 grew normally on glucose plus arginine in anaerobiosis and, like the wild type, had appreciable levels of catabolic transferase. Nitrate, like oxygen, repressed ornithine carbamoyltransferase and stimulated arginase synthesis. In aerobic cultures, arginase was repressed by glutamine in the presence of glucose, but not when the carbon-energy source was poor. In anaerobic cultures, ammonia repressed catabolic ornithine carbamoyltransferase, but glutamate and glutamine stimulated its synthesis. A second mutant, derived from BL196, retained the low arginase and ornithine transaminase levels of BL196 but produced high levels of deiminase pathway enzymes in the presence of oxygen.     

L-ornithine transaminase synthesis in Saccharomyces cerevisiae: Regulation by inducer exclusion

Summary The ornithine transaminase (EC.2.6.1.13) of Saccharomyces cerevisiae is induced by arginine, ornithine, and their analogs. Genetic regulatory elements which are involved in this induction process have been defined due to the isolation of specific mutants. Two classes of OTAse operator mutants have previously been described; three unlinked genes are presumed to code for a specific repressor, CARGR of both of the arginine catabolic enzymes, arginase, and ornithine transaminase. The level of transaminase of cells grown on ammonia plus arginine is much lower than it is when arginine is the sole nitrogen source. Ammonia thus seems to limit the amount of enzyme synthesized when arginine is present in the growth medium. Nevertheless, all attempts to disclose a nitrogen catabolite repression process in OTAse synthesis have failed; neither the action of mutations that release this regulation on arginase and other catabolic enzymes, nor the use of derepressing growth conditions, affect OTAse synthesis. A decrease of the cells' arginine pool when amonia or aminoacids (serine, glutamate) are added to arginine as a nitrogen nutrient results in a progressive reduction of transaminase synthesis. This suggests that arginine is the only physiological effector in those conditions: ammonia or some aminoacids would reduce the enzyme synthesis because of an inducer exclusion. The first stage of OTAse induction would then be operated by the CARGR repressor, and an additional regulatory element might take part in the full scale process. Preliminary data favoring the involvment of such an element are presented.     

Induction and derepression of arginase and ornithine transaminase in different strains ofSaccharomyces cerevisiae

The syntheses of arginase and ornithine transaminase were studied in two strains ofSaccharomyces cerevisiae, viz. strain B and strain α-Σ1278b. Derepression of both enzymes during nitrogen starvation was shown only by strain B, non-specific induction of arginase only by strain α-Σ1278b. This different response of both strains studied reveals substantial differences in the regulation of enzyme synthesis among yeast strains of one and the same species. The specific enzyme activities observed in chemostat cultures with arginine as the nitrogen source and different sugars, at variable carbon to nitrogen ratios, did not indicate the involvement of carbon catabolite repression in the regulation of arginase and ornithine transaminase syntheses. Specific arginase activities observed in the continuous cultures varied widely and did not show a correlation with the intracellular arginine concentration. Extracellular steady-state arginine concentrations higher than about 0.1mm, in addition to abundant energy supply, were found to be required for high production of arginase. It is suggested that, besides intracellular arginine, extracellular arginine may provide an induction signal necessary for full-scale induction of arginase synthesis. A possible intermediary role of arginine permeases or of other membrane proteins is discussed.     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

Enzyme repression in the arginine pathway ofSaccharomyces cerevisiae

Enzyme repression in the arginine pathway ofSaccharomyces cerevisiae was demonstrated by comparison of specific enzyme activities in yeast grown with and without arginine in various mineral salts media. Of the enzymes tested only ornithine transcarbamoylase was found to be repressed by exogenous arginine. Acetylornithine-glutamate transacetylase and argininosuccinate lyase were not affected. No relationship between specific enzyme activities and intracellular arginine concentration was observed.During the adaptation of yeast grown in a medium supplemented with amino acids to a mineral salts medium, the enzymes ornithine transcarbamoylase and argininosuccinate lyase were not derepressed beyond their specific activities normally present in yeast grown in mineral salts media. Neither were the arginine-degrading enzymes arginase and ornithine transaminase broken down during this adaptation.Thanks are due to Professor E. G. Mulder and to Professor H. Veldkamp for stimulatory discussions; to the Heineken's Brouwerij, Rotterdam, and to the Landbouwhogeschoolfonds for research grants.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis.

Specific activities of arginase and ornithine aminotransferase, inducible enzymes of arginine catabolism in Bacillus subtilis 168, were examined in cells grown with various carbon and nitrogen sources. Levels of these enzymes were similar in arginine-induced cultures whether glucose or citrate was the carbon source (in contrast to histidase), suggesting that carbon source catabolite repression has only limited effect. In media with combinations of nitrogen sources, glutamine strongly repressed induction of these enzymes by proline or arginine. Ammonium, however, only repressed induction by proline and had no effect on induction by arginine. These effects correlate with generation times in media containing these substances as sole nitrogen sources: growth rates decreased in the order glutamine-arginine-ammonium-proline. Similar phenomena were observed when glutamine or ammonium were added to arginine- or proline-grown cultures, or when arginine or proline were added to glutamine- or ammonium-grown cultures. In the latter cases, an additional feature was apparent, namely a surprisingly long transition between steady-state enzyme levels. The results are compared with those for other bacteria and for eucaryotic microorganisms.     

Regulation of arginine and proline catabolism in Bacillus licheniformis

The enzymes in the arginine breakdown pathway (arginase, ornithine-delta-transaminase, and Delta'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Delta'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Delta'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.     

Arginine metabolism in insects. Role of arginase in proline formation during silkmoth development

1. Ornithine delta-transaminase (l-ornithine-2-oxo acid aminotransferase, EC 2.6.1.13) and Delta(1)-pyrroline-5-carboxylate reductase [l-proline-NAD(P) 5-oxidoreductase, EC 1.5.1.2] were demonstrated in fat-body and flight-muscle tissues of the silkmoth Hyalophora gloveri. Arginase (l-arginine ureohydrolase, EC 3.5.3.1) is also present in these tissues. 2. Arginase, ornithine transaminase and pyrroline-carboxylate reductase are generally considered to make up the catabolic pathway for the conversion of arginine into proline. The conversion of l-[U-(14)C]arginine into [(14)C]proline by intact fat-body tissue was used to show that the enzymes in insect fat body also function in this capacity. 3. Of the three enzymes of the catabolic pathway, only arginase increased during adult development and the increase coincided with the emergence of the winged adult moth. Since proline appears to be a major substrate utilized in insect flight metabolism, the increase in arginase activity at this stage suggests a major role for arginase in proline formation.     

Induction and repression of arginase and ornithine transaminase in baker's yeast

The arginase and the ornithine transaminase of baker's yeast are induced byl-arginine. Both enzymes have been shown to be repressed by nitrogen compounds. This is evidenced primarily by the decrease in specific enzyme activities caused by the addition of readily assimilable nitrogen compounds to a yeast culture with arginine, secondly by the derepression of both enzymes during nitrogen starvation of the yeast grown in various arginine-free media. This derepression equals both in rate and in amount the enzyme synthesis during the adaptation of the yeast to a medium withl-arginine as the sole nitrogen source. It is inhibited by various assimilable and non-assimilable amino acids. The derepression is the result of the nitrogen deficiency itself, since during the starvation of the yeast for sulphate, phosphate or magnesium, neither of the two enzymes is derepressed, and since it is independent of the nature of the carbon source in the nitrogen starvation medium, provided the latter is immediately assimilable.The enzymes are not subject to catabolite repression by glucose metabolites.It is concluded that the synthesis of arginase and ornithine transaminase in yeast is regulated by induction and repression. Arginine induces the enzymes; they are repressed by nitrogen compounds, probably in cooperation with one or more vitamins.Thanks are due to Professor E. G. Mulder for his frequent encouragement, to the Heineken's Brouwerij, Rotterdam and to the Landbouwhogeschoolfonds for research grants, and to Miss H. P. M. Klinkers, to Mr. P. J. Buysman and to Mr. G. J. K. Pesch for their skilful technical assistance.     

Polyamine Metabolism in Acanthamoeba: Polyamine Content and Synthesis of Ornithine,Putrescine, and Diaminopropane1

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Selection and characterisation of mutants lacking arginase in Neurospora crassa

Summary A Neurospora mutant (aga) lacking arginase was selected by virtue of its inability to utilize arginine as a source of ornithine, using a strain in which ornithine was needed to satisfy a proline requirement. It mapped in linkage group VII (right arm), close to wc. The most important characteristic of the mutant was its extreme sensitivity to arginine. Inclusion of 1 mM arginine in the medium lead to a 40-fold increase in the arginine pool and a 90% inhibition of growth. This inhibition was relieved by the addition of ornithine or proline. The high arginine pool was associated with only a slight repression of two biosynthetic enzymes examined and with a five-fold induction of ornthine transaminase, the second enzyme of arginine catabolism. It is expected that the aga mutant will be of value in further work on the regulation of arginine biosynthesis in Neurospora.     

Evidence for urea cycle activity in Sporosarcina ureae

Sporosarcina ureae BS 860, a motile, sporeforming coccus, possesses the enzymes required for a functioning urea (ornithine) cycle. This is only the second known example of urea cycle activity in a prokaryote. Specific activities are reported for ornithine carbamoyltransferase, argininosuccinase, arginase, and urease. Although argininosuccinate synthetase activity could not be detected directly in crude cell extracts, indirect evidence from radiocarbon tracing data for arginine synthesis from the substrate, l-[1-¹⁴C]-ornithine, strongly suggest the presence of this or other similar enzyme activity. Furthermore, good growth in defined media containing either 1.0% glutamine, ornithine, or citrulline as sole carbon sources suggests argininosuccinate synthetase activity is necessary for arginine synthesis. The effect of varying pH on arginase and urease activities indicate that these two enzymes may function within the context of the urea cycle to generate ammonia for amino acid synthesis, as well as for raising the pH of the growth micro-environment.     

Induction of arginase and ornithine transaminase in the fission yeast Schizosaccharomyces pombe.

The induction of arginase and ornithine transaminase in the fission yeast Schizosaccharomyces pombe requires the absence of ammonia and the presence of the inducer arginine. It seems that immediate arginase degradation is initiated by starved cells or ones from which arginine has been removed.     

Polyamine metabolism in Acanthamoeba: polyamine content and synthesis of ornithine, putrescine, and diaminopropane

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

Urea formation in the green vegetable bug Nezara viridula

Urea comprises 7·7 per cent of the total nitrogen excretion of Nezara viridula. The bug is capable of oxidizing uric acid to allantoin, which is also excreted, but the uricolytic pathway is not active beyond this point. Of the enzymes of the ornithine cycle, arginase and ornithine transcarbamalase are active, but there is no evidence for the arginine synthetase system. Carbamyl phosphate synthetase has a low activity detectable only by the use of radioactive substrates. Confirmation of the operation of only part of the ornithine cycle is seen in the incorporation of bicarbonate carbon into citrulline, but not into arginine or urea, by homogenates of bug tissue. It is concluded that urea in the excreta is derived from excess arginine in the diet by the action of the enzyme arginase. Free arginine is present in the cell sap of the bean pods on which the bugs feed in amounts sufficient to account for the urea excreted.     

Simultaneous production of inulase and lactase in batch and continuous cultures of Kluyveromyces fragilis

Under different induction conditions, the industrial yeast Kluyveromyces fragilis is an excellent producer of the enzymes inulase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26) and lactase (β-d-galactoside galactohydrolase, EC 3.2.1.23), producing 27 and 1.6 U mg^?1dry cell weight, respectively. In order to improve overall enzyme yields, conditions for the simultaneous production of both enzymes in a one-stage fermentation have been examined. Techniques employed include carbon-limited batch and continuous culture, single and mixed carbon substrates, and the use of a mutant semi-constitutive for inulase production. Synthesis of both enzymes suffered strongly from carbon catabolite repression in batch cultures grown on single and mixed inducing substrates. Only glycerol and dl-malate did not repress either enzyme. The non-metabolizable analogues of lactose, isopropyl-β-d-thiogalactoside and methyl-β-d-thiogalactoside induced lactase in glycerol grown batch cultures, but were ineffective in sucrose grown continuous cultures. They also depressed the normally high levels of inulase in such continuous cultures. The highest simultaneous inulase and lactase activities in the wild-type yeast were obtained in continuous culture on an equal mixture of d-fructose and d-galactose; 25 and 0.78 U mg^?1dry cell weight, respectively. In this fermentation the combined yield per unit carbon substrate of the two enzymes was 141%, compared to a reference value of 100% for the highest yield of each enzyme in separate fermentations. On the same mixture of d-fructose and d-galactose, the mutant produced ～60 and 0.70 U mg^?1dry cell weight, respectively. The combined enzyme yield per unit of carbon substrate was 172%.     

Arginine catabolism in wine lactic acid bacteria: is it via the arginine deiminase pathway or the arginase-urease pathway?

The wine lactic acid bacteria Leuconostoc oenos OENO and Lactobacillus buchneri CUC-3 catabolize L-arginine to ornithine and ammonia as major end-products, with 1 mole of arginine converted into 2 moles of ammonia and 1 mole of ornithine. Some citrulline was also excreted into the medium. The excreted citrulline was reassimilated and catabolized by the lactobacillus strain, though not by the leuconostoc. Urea was not detected during arginine degradation. The activities of all three enzymes of the arginine deiminase pathway (arginine deiminase, ornithine transcarbamylase and carbamate kinase) increased significantly over time in the presence of arginine. On the other hand, arginase and urease activities were undetectable in cell extracts of cultures grown in the presence of arginine. The results show that the arginine deiminase pathway, and not the arginase-urease pathway, is the route for arginine degradation in wine lactic acid bacteria.