Improved Anaerobic Use of Arginine by Saccharomyces cerevisiae

Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Δ¹-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.     

Arginine Biosynthesis by Streptococcus bovis

The pathway of arginine biosynthesis in Streptococcus bovis was studied by radioactive tracer techniques. Cells were grown anaerobically with (14)CO(2) in a synthetic medium containing NH(4) (+) as the sole nitrogen source except for the trace present in nitrogen-containing vitamins. The protein fraction isolated from the labeled cells was acid-hydrolyzed, and (14)C-arginine was isolated from the protein hydrolysate by ion-exchange chromatography. The carboxyl carbon of the isolated arginine was removed with arginine decarboxylase, and the guanidino carbon was removed by simultaneous arginase-urease degradation. By manometric measurement and liquid scintillation counting of the CO(2) released by enzymatic degradation, 50% of the label was found in the carboxyl carbon and 50% in the guanidino carbon. Specific radioactivity determinations indicated that growth on (14)CO(2) resulted in twice as much label in arginine as with aspartate, glutamate, or lysine. These results are consistent with a glutamate --> ornithine --> citrulline pathway of arginine biosynthesis in S. bovis and provide further evidence for the synthesis of glutamate via the tricarboxylic acid cycle reactions from citrate through alpha-ketoglutarate.     

Utilization of arginine by Klebsiella aerogenes.

Klebsiella aerogenes utilized arginine as the sole source of carbon or nitrogen for growth. Arginine was degraded to 2-ketoglutarate and not to succinate, since a citrate synthaseless mutant grows on arginine as the only nitrogen source. When glucose was the energy source, all four nitrogen atoms of arginine were utilized. Three of them apparently did not pass through ammonia but were transferred by transamination, since a mutant unable to produce glutamate by glutamate synthase or glutamate dehydrogenase utilized three of four nitrogen atoms of arginine. Urea was not involved as intermediate, since a unreaseless mutant did not accumulate urea and grew on arginine as efficiently as the wild-type strain. Ornithine appeared to be an intermediate, because cells grown either on glucose and arginine or arginine alone could convert arginine in the presence of hydroxylamine to ornithine. This indicates that an amidinotransferase is the initiating enzyme of arginine breakdown. In addition, the cells contained a transaminase specific for ornithine. In contrast to the hydroxylamine-dependent reaction, this activity could be demonstrated in extracts. The arginine-utilizing system (aut) is apparently controlled like the enzymes responsible for the degradation of histidine (hut) through induction, catabolite repression, and activation by glutamine synthetase.     

Occurrence of succinyl derivatives in the catabolism of arginine in Pseudomonas cepacia.

Pseudomonas cepacia NCTC 10743 utilizes arginine as the sole source of carbon and nitrogen for growth. Arginine is degraded to glutamate via succinyl derivatives. The catabolic sequence in this pathway is L-arginine----N2-succinylarginine----N2-succinylornithine--- -N2-succinylglutamate semialdehyde----N2-succinylglutamate----glutamate + succinate. The formation of the enzymes responsible for arginine degradation is regulated not only by induction but also by both carbon and nitrogen catabolite repression.     

Evidence for a role of glutamine synthetase in assimilation of amino acids as nitrogen source in the cyanobacterium Nostoc muscorum.

Methylammonium/ammonium ion, glutamine, glutamate, arginine and proline uptake, and their assimilation as nitrogen sources, was studied in Nostoc muscorum and its glutamine synthetase-deficient mutant. Glutamine served as nitrogen source independent of glutamine synthetase activity. Glutamate was not metabolised as a nitrogen source but still inhibited nitrogenase activity and diazotrophic growth. Glutamine synthetase activity was essential for the assimilation of N2, ammonia, arginine and proline as nitrogen sources but not for the control of their transport, heterocyst formation, and production of ammonia or aminoacid dependent repressor signal for N2-fixing heterocysts. These results also suggest that glutamine synthetase serves as the sole route of ammonia assimilation and glutamine synthesis, and ammonia per se as the repressor signal for N2-fixing heterocysts and methylammonium (ammonium) transport.     

The effect of lactate addition on the growth of Penicillium camembertii on glutamate

The effect of an additional carbon source, lactate, on Penicillium camembertii growth on glutamate as both carbon and nitrogen sources was examined. Glutamate (and lactate) was present in excess in both media. Throughout the whole culture, similar growth time-courses were recorded on both media, indicating the absence of a lactate effect on growth. During the first part of growth, corresponding to an increasing amount of viable biomass, the rate of glutamate consumption remained high, as well as the related ammonium production, indicating its use as a carbon source in addition to being nitrogen source. The low growth rates recorded during the last part of growth resulted in low glutamate consumption, while lactate consumption continued mainly by a maintenance mechanism for the energy supply. A clear differentiation appeared therefore between the carbon source and the energy source: glutamate was mainly used as C source (and N source) for biosynthesis, while lactate was mainly assimilated for energy supply. Carbon and nitrogen yield examinations confirmed this result. Indeed, the C/N ratio found for P. camembertii cellular material (8.14) was about twice that of glutamate (4.29). From this, about half of the available nitrogen was used for biomass formation during growth on glutamate-lactate based medium, as experimentally confirmed (constant yield nitrogen from biomass on nitrogen from glutamate was found (0.49), while the excess nitrogen was released as ammonium). The constant and close to unit (0.99) yield carbon from CO2 on carbon from lactate, also recorded during growth on glutamate-lactate based medium, confirmed that lactate was mainly used as an energy source.     

Glutamate synthesis in Streptomyces coelicolor.

Both glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are involved in glutamate synthesis in Streptomyces coelicolor. The highest levels of GDH were seen in extracts of cells grown with high levels of ammonium as the nitrogen source. GOGAT activity was reduced two- to threefold in extracts of cells grown with good sources of glutamate. S. coelicolor mutants deficient in GOGAT (Glt-) required glutamate for growth with L-alanine, asparagine, arginine, or histidine as the nitrogen source but grew like wild-type cells when ammonium, glutamine, or aspartate was the nitrogen source. The glt mutations were tightly linked to hisA1. Mutants deficient in both GOGAT and GDH (Gdh-) required glutamate for growth in all media. The gdh-5 mutation was mapped to the left region of the S. coelicolor chromosomal map, between proA1 and uraA1.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Comparison of Hup Trait and Intrinsic Antibiotic Resistance for Assessing Rhizobial Competitiveness Axenically and in Soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.     

Effect of glutamine on growth and heterocyst differentiation in the cyanobacterium Anabaena variabilis.

Mutants of the cyanobacterium Anabaena variabilis that were capable of increased uptake of glutamine, as compared with that in the parental strains, were isolated. Growth of these mutants and their parental strains was measured in media containing N2, ammonia, or glutamine as a source of nitrogen. All strains grew well with any one of these sources of fixed nitrogen. Much of the glutamine taken up by the cells was converted to glutamate. The concentrations of glutamine, glutamate, arginine, ornithine, and citrulline in free amino acid pools in glutamine-grown cells were high compared with the concentrations of these amino acids in ammonia-grown or N2-grown cells. All strains capable of heterocyst differentiation, including a strain which produced nonfunctional heterocysts, grew and formed heterocysts in the presence of glutamine. However, nitrogenase activity was repressed in glutamine-grown cells. Glutamine may not be the molecule directly responsible for repression of the differentiation of heterocysts.     

Role of 4-aminobutyrate aminotransferase in the arginine metabolism of Pseudomonas aeruginosa.

4-Aminobutyrate aminotransferase (GABAT) from Pseudomonas aeruginosa was purified 64-fold to apparent electrophoretic homogeneity from cells grown with 4-aminobutyrate as the only source of carbon and nitrogen. Purified GABAT catalyzed the transamination of 4-aminobutyrate, N2-acetyl-L-ornithine, L-ornithine, putrescine, L-lysine, and cadaverine with 2-oxoglutarate (listed in order of decreasing activity). The enzyme is induced in cells grown on 4-guanidinobutyrate, 4-aminobutyrate, or putrescine as the only carbon and nitrogen source. Cells grown on arginine or on glutamate contained low levels of the enzyme. The regulation of the synthesis of GABAT as well as the properties of the mutant with an inactive N2-acetyl-L-ornithin 5-aminotransferase suggest that GABAT functions in the biosynthesis of arginine by convertine N2-acetyl-L-glutamate 5-semialdehyde to N2-acetyl-Lornithine as well as in catabolic reactions during growth on putrescine or 4-guanidinobutyrate but not during growth on arginine.     

Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.     

Salmonella typhimurium Mutants with Altered Glutamate Dehydrogenase and Glutamate Synthase Activities

Although glutamate is a key compound in nitrogen metabolism, little is known about the function or regulation of its two biosynthetic enzymes, glutamate dehydrogenase and glutamate synthase. To begin the characterization of glutamate formation in Salmonella typhimurium, we isolated mutants having altered glutamate dehydrogenase and glutamate synthase activities. Mutants which failed to grow on media with glucose as the carbon source and less than 1 mM (NH₄)₂SO₄ as the nitrogen source (Asm⁻) had about one-fourth the normal glutamate synthase activity and one-half the glutamine synthetase activity. The asm mutations also prevented growth with alanine, arginine, or proline as nitrogen sources and conferred resistance to methionine sulfoximine. When a mutation (gdh-51) causing the loss of glutamate dehydrogenase activity was transferred into a strain with an asm-102 mutation, the resulting asm-102 gdh-51 mutant had a partial requirement for glutamate. A strain isolated as a complete glutamate auxotroph had a third mutation, in addition to the asm-102 gdh-51 lesions, that further decreased the glutamate synthase activities to 1/20 the normal level. Both the asm-102 and gdh-51 mutations were located on the S. typhimurium linkage map at sites distinct from those found for mutations causing similar phenotypes in Klebsiella aerogenes and Escherichia coli.     

Roles of arginine in growth of Clostridium botulinum Okra B.

Group I strains of Clostridium botulinum are known to degrade arginine by the arginine deiminase pathway. We have found that C. botulinum Okra B consumed a level of arginine (20 g/liter) higher than the basal requirement for growth (3 g/liter). Arginine was probably the preferred source of nitrogen for biosynthesis but did not serve as a major source of energy. Citrulline and proline were produced as major fermentation products in media containing high levels of arginine, but in media with basal amounts of arginine these products were produced in lower quantities during growth and were later reassimilated. The results indicate that C. botulinum Okra B changes its metabolism during consumption of surplus arginine, and this change is associated with toxin repression, formation of citrulline and proline as end products, and possibly resistance to environmental stresses such as increased acidity and osmolarity.     

Roles of arginine in growth of Clostridium botulinum Okra B.

Group I strains of Clostridium botulinum are known to degrade arginine by the arginine deiminase pathway. We have found that C. botulinum Okra B consumed a level of arginine (20 g/liter) higher than the basal requirement for growth (3 g/liter). Arginine was probably the preferred source of nitrogen for biosynthesis but did not serve as a major source of energy. Citrulline and proline were produced as major fermentation products in media containing high levels of arginine, but in media with basal amounts of arginine these products were produced in lower quantities during growth and were later reassimilated. The results indicate that C. botulinum Okra B changes its metabolism during consumption of surplus arginine, and this change is associated with toxin repression, formation of citrulline and proline as end products, and possibly resistance to environmental stresses such as increased acidity and osmolarity.     

Metabolism of [2-14C]acetate and its use in assessing hepatic Krebs cycle activity and gluconeogenesis

To examine the fate of the carbons of acetate and to evaluate the usefulness of labeled acetate in assessing intrahepatic metabolic processes during gluconeogenesis, [2-14C]acetate, [2-14C]ethanol, and [1-14C]ethanol were infused into normal subjects fasted 60 h and given phenyl acetate. Distributions of 14C in the carbons of blood glucose and glutamate from urinary phenylacetylglutamine were determined. With [2-14C]acetate and [2-14C]ethanol, carbon 1 of glucose had about twice as much 14C as carbon 3. Carbon 2 of glutamate had about twice as much 14C as carbon 1 and one-half to one-third as much as carbon 4. There was only a small amount in carbon 5. These distributions are incompatible with the metabolism of [2-14C]acetate being primarily in liver. Therefore, [2-14C]acetate cannot be used to study Krebs cycle metabolism in liver and in relationship to gluconeogenesis, as has been done. The distributions can be explained by: (a) fixation of 14CO2 from [2-14C]acetate in the formation of the 14C-labeled glucose and glutamate in liver and (b) the formation of 14C-labeled glutamate in a second site, proposed to be muscle. [1,3-14C]Acetone formation from the [2-14C]acetate does not contribute to the distributions, as evidenced by the absence of 14C in carbons 2-4 of glutamate after [1-14C]ethanol administration.     

The Correlation of Physiological Differences and Varions Leaf Forms of an Aqnatic Plant

An analysis of the celluar constituents of the leaves of an aquatic fern, Marsilea vestita, was done after and during the formation of different leaf forms. Land leaves of Marsilea produced almost twice as much dry material, soluble carbohydrate, insoluble protein and soluble peptides compared to submerged leaves on a fresh weight basis, but on a dry weight basis there is tittle difference between the two types of leaves. the submerged leaf soluble nitrogen fraction consisted mostly of ammonia and free amino acids, while that of the land leaf probably con tained mostly peptides. Free arginine was present in large quantities in megaspores and as the plant matured, at the expense of the megaspore, the amount of arginine left in the pool diminished. A correlation was found between soluble sugar increase and leaf elongation, Just prior to the production of submerged leaves the developing leaves contained low levels of protein and soluble sugar, but high levels of soluble nitrogen and an increased amount of starch. A theory which involves metabolism was proposed to account for the leaf form changes in Marsilea.