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l-Theanine is a nonproteinogenic amino acid present almost exclusively in tea plants and is beneficial for human health. For industrial production, l-theanine is enzymatically or chemically synthesized from glutamine/glutamate (or a glutamine/glutamate derivative) and ethylamine. Ethylamine is extremely flammable and toxic, which complicates and increases the cost of operational procedures. To solve these problems, we developed an artificial biosynthetic pathway to produce l-theanine in the absence of supplemental ethylamine. For this purpose, we identified and selected a novel transaminase (NCBI:protein accession number AAN70747) from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase (NCBI:protein accession number AAY37316) from Pseudomonas syringae pv. syringae B728a, which catalyzes the condensation of l-glutamate and ethylamine to produce l-theanine. Expressing these genes in Escherichia coli W3110S3GK and enhancing the production capacity of acetaldehyde and l-alanine achieved successful production of l-theanine without ethylamine supplementation. Furthermore, the deletion of ggt , which encodes γ-glutamyltranspeptidase (EC 2.3.2.2), achieved large-scale production of l-theanine by attenuating its decomposition. We show that an alanine decarboxylase-utilizing pathway represents a promising route for the fermentative production of l-theanine. Our study reports efficient methods to produce l-theanine in the absence of supplemental ethylamine. IMPORTANCE l-Theanine is widely used in food additives and dietary supplements. Industrial production of l-theanine uses the toxic and highly flammable precursor ethylamine, raising production costs. In this study, we used Escherichia coli to engineer two biosynthetic pathways that produce l-theanine from glucose and ammonia in the absence of supplemental ethylamine. This study establishes a foundation for safely and economically producing l-theanine., (Copyright © 2021 American Society for Microbiology.)

L-glutamate overproduction in Corynebacterium glutamicum, a biotin auxotroph, is induced by biotin limitation or by treatment with certain fatty acid ester surfactants or with penicillin. We have analyzed the relationship between the inductions, 2-oxoglutarate dehydrogenase complex (ODHC) activity, and L-glutamate production. Here we show that a strain deleted for odhA and completely lacking ODHC activity produces L-glutamate as efficiently as the induced wild type (27.8 mmol/g [dry weight] of cells for the ohdA deletion strain compared with only 1.0 mmol/g [dry weight] of cells for the uninduced wild type). This level of production is achieved without any induction or alteration in the fatty acid composition of the cells, showing that L-glutamate overproduction can be caused by the change in metabolic flux alone. Interestingly, the L-glutamate productivity of the odhA-deleted strain is increased about 10% by each of the L-glutamate-producing inductions, showing that the change in metabolic flux resulting from the odhA deletion and the inductions have additive effects on L-glutamate overproduction. Tween 40 was indicated to induce drastic metabolic change leading to L-glutamate overproduction in the odhA-deleted strain. Furthermore, optimizing the metabolic flux from 2-oxoglutarate to L-glutamate by tuning glutamate dehydrogenase activity increased the l-glutamate production of the odhA-deleted strain.

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog alpha-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.

Escherichia coli has two primary pathways for glutamate synthetase-glutamate synthase pathway is known to be essential for synthesis at low ammonium concentrations and for regulation of the glutamine pool, but the necessity for glutamate dehydrogenase (GDH) has been uncertain. The results of competition experiments between the wild type and a GDH-deficient mutant during nutrient-limited growth and of direct enzyme measurements suggest that GDH is used in glutamate synthesis when the cell is limited for energy (and carbon) but ammonium and phosphate are present in excess, while the glutamine synthetase-glutamate synthase pathway is used when the cell is not under energy limitation. The use of alternative routes for glutamate synthesis implies that the energy cost of biosynthesis may be less when energy is limited than when energy is unlimited.

The murI gene of Escherichia coli, whose inactivation results in the inability to form colonies in the absence of D-glutamic acid, was identified in the 90-min region of the chromosome. The complementation of an auxotrophic E. coli B/r strain by various DNA sources allowed us to clone a 2.5-kbp EcoRI chromosomal fragment carrying the murI gene into multicopy plasmids. The murI gene corresponds to a previously sequenced open reading frame, ORF1 (J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller. J. Bacteriol. 148:107-127, 1987), located between the btuB gene, encoding the vitamin B12 outer membrane receptor protein, and the rrnB operon, which contains the genes for 16S, 23S, and 5S rRNAs. The murI gene product is predicted to be a protein of 289 amino acids with a molecular weight of 31,500. Attempts to identify its enzymatic activity were unsuccessful. Cells altered in the murI gene accumulate UDP-N-acetylmuramyl-L-alanine to a high level when depleted of D-glutamic acid. Pools of precursors located downstream in the pathway are consequently depleted, and cell lysis finally occurs when the peptidoglycan content is 25% lower than that of normally growing cells.

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.

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.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.

Nitrogen source regulation of glutamate synthase activity in Bacillus subtilis occurs at the level of transcription of the gltA and gltB genes, which encode the two subunits of the enzyme. We show here that transcription of gltA requires the product of gltC, a gene whose transcription is divergent from that of gltA and whose transcriptional control sequences overlap those of gltA. gltC mutants had decreased, aberrantly regulated levels of glutamate synthase activity and decreased gltA mRNA. The gltC gene product could act in trans to complement both these defects. In addition, the gltC gene product repressed its own transcription. The DNA sequence of gltC revealed that its putative product is very similar to a number of positive regulatory proteins from gram-negative bacteria (the LysR family).

Glutamine phosphoribosylpyrophosphate amidotransferase (ATase) activity is rapidly inactivated in stationary-phase cells of Bacillus subtilis. The inactivation of APase requires both the cessation of rapid cell growth and the presence of oxygen. ATase is inactivated in two protease-deficient mutant strains at a rate similar to that seen in the wild type, and is stable in anaerobic cell-free extracts of the parent strain. These results suggest that the inactivation of ATase is not the result of general proteolysis. The inactivation of ATase in stationary-phase cultures can be inhibited by oxygen starvation. This oxygen requirement does not reflect a dependence on the generation of metabolic energy, but appears to be a direct requirement for molecular oxygen. ATase synthesis is repressed by the addition of adenosine, and is inactivated only after the cessation of exponential growth. Addition of chloramphenicol or rifampin to exponential- and stationary-phase cells does not inhibit ATase inactivation, suggesting that protein or ribonucleic acid synthesis is not required for inactivation. ATase is inactivated at the end of exponential growth in cells that have exhausted a required amino acid.

In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.

In the dimorphic bacterium Caulobacter crescentus, ammonia assimilation occurs only via the combined action of the enzymes glutamine synthetase and glutamate synthase. Mutants auxotrophic for glutamate lacked glutamate synthase activity, and the mutations leading to the glutamate auxotrophy appeared to lie at two distinct genetic loci. Both glutamate synthase and glutamine synthetase activities were subject to regulation by repression. Glutamate synthase activity was highest in cultures grown in minimal medium with ammonia as sole nitrogen source and was about fivefold lower in rich broth. Glutamine synthetase activity was highest in cells grown with growth-rate-limiting amounts of ammonia as nitrogen source and was about fourfold lower in rich broth. In addition, glutamine synthetase activity appeared to be regulated by an adenylylation system like that described for Escherichia coli.

Selenomonas ruminantium was found to possess two pathways for NH4+ assimilation that resulted in net glutamate synthesis. One pathway fixed NH4+ through the action of an NADPH-linked glutamate dehydrogenase (GDH). Maximal GDH activity required KCl (about 0.48 M), but a variety of monovalent salts could replace KCl. Complete substrate saturation of the enzyme by NH4+ did not occur, and apparent Km values of 6.7 and 23 mM were estimated. Also, an NADH-linked GDH activity was observed but was not stimulated by KCl. Cells grown in media containing non-growth-rate-limiting concentrations of NH4+ had the highest levels of GDH activity. The second pathway fixed NH4+ into the amide of glutamine by an ATP-dependent glutamine synthetase (GS). The GS did not display gamma-glutamyl transferase activity, and no evidence for an adenylylation/deadenylylation control mechanism was detected. GS activity was highest in cells grown under nitrogen limitation. Net glutamate synthesis from glutamine was effected by glutamate synthase activity (GOGAT). The GOGAT activity was reductant dependent, and maximal activity occurred with dithionite-reduced methyl viologen as the source of electrons, although NADPH or NADH could partially replace this artificial donor system. Flavin adenine dinucleotide, flavin mononucleotide, or ferredoxin could not replace methyl viologen. GOGAT activity was maximal in cells grown with NH4+ as sole nitrogen source and decreased in media containing Casamino Acids.

A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase.

Methanomonas methanooxidans incorporates both carbon atoms of acetate into the glutamate and aspartate families of amino acids during growth on methane; carbon dioxide is also evolved from both carbon atoms of acetate. The distribution of carboxyl-labeled acetate incorporated into convalently bound glutamate is consistent with the operation of the tricarboxylic acid cycle in this species, and the presence of alpha-ketoglutarate dehydrogenase was demonstrated in cell-free extracts.

Alpha-Ketoglutaratedehydrogenase was undetectable in extracts of Methylococcus capsulatus. Cells incorporated [1-14-C] acetate into only four protein amino acids (glutamate, proline, arginine, and leucine) and the C5, but not C1, of glutamate.

The principal initial product of metabolism of [13N]N2 and 13NH4+ by five diverse cyanobacteria is glutamine. Methionine sulfoximine inhibits formation of [13N]glutamine except in the case of Gloeothece sp., an organism with a thick sheath through which the inhibitor may not penetrate. Thus, glutamine synthetase appears to catalyze the initial step in the assimilation of N2-derived or exogenous NH4+ by these organisms. [13N]Glutamate is, in all cases, the second major product of assimilation of 13N-labeled N2 and NH4+. In all of the N2-fixing cyanobacteria studied, the fraction of 13N in glutamine declines and that in glutamate increases with increasing times of assimilation of [13N]N2 and 13NH4+, and (Gloeothece again excepted) methionine sulfoximine reduces incorporation of 13N into glutamate as well as into glutamine. Glutamate synthase therefore appears to catalyze the formation of glutamate in a wide range of N2-fixing cyanobacteria. However, the major fraction of [13N]glutamate formed by Anacystis nidulans incubated with 13NH4+ may be formed by glutamic acid dehydrogenase. The formation of [13N]alanine from 13NH4+ appears to be catalyzed principally either by alanine dehydrogenase (as in Cylindrospermum licheniforme) or by a transaminase (as in Anabaena variabilis).

The large and small subunits of Escherichia coli glutamate synthase were isolated. The small subunit catalyzes the NH(3)-dependent synthesis of glutamate. The large subunit exhibits glutaminase activity.

The biosynthesis of isotopically labeled L-glutamic acid by the microorganism Brevibacterium flavum was studied with a variety of carbon-13-enriched precursors. The purpose of this study was twofold: to develop techniques for the efficient preparation of labeled L-glutamate with a variety of useful labeling patterns which can be used for other metabolic studies, and to better understand the metabolic events leading to label scrambling in these strains. B. flavum, which is used commercially for the production of monosodium glutamate, has the capability of utilizing glucose or acetate as a sole carbon source, an important criterion from the standpoint of developing labeling strategies. Unfortunately, singly labeled glucose precursors lead to excessive isotopic dilution which reduces their usefulness. Studies with [3-13C]pyruvate indicate that this problem can in principle be overcome by using labeled three-carbon precursors; however, conditions could not be found which would lead to an acceptable yield of isotopically labeled L-glutamate. In contrast, [1-13C]- or [2-13C]acetate provides relatively inexpensive, readily available precursors for the production of selectively labeled, highly enriched L-glutamate. The preparation of L-[15N]glutamate from [15N]ammonium sulfate was carried out and is a very effective labeling strategy. Analysis of the isotopic distribution in labeled glutamate provides details about the metabolic pathways in these interesting organisms.

High activity of taurine:alpha-ketoglutarate aminotransferase was found exclusively in cell-free extracts of Achromobacter superficialis and A. polymorph. The former was chosen for characterization of the enzymatic reaction. The enzyme activity was enhanced by addition of beta-alanine to the growth medium. The product from alpha-ketoglutarate was identified as l-glutamate. Another product has been isolated, purified, and identified as sulfoacetaldehyde (2-oxoethanesulfonate), a deamination product from taurine, by comparison between the 2,4-dinitrophenylhydrazones of the synthetic and enzymatic products on the basis of studies by paper chromatography, by visible, infrared, and nuclear magnetic resonance spectrophotometries, and by elemental analysis. This enzymatic transamination was found to proceed stoichiometrically and reversibly as follows: NH(2).CH(2).CH(2).SO(3)H + HOOC.CH(2).CH(2).CO.COOH right harpoon over left harpoon OHC.CH(2).SO(3)H + HOOC.CH(2).CH(2).CH(NH(2)).COOH.

At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resistant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthesis via glutamate synthetase enzyme.

The production of glutamic gamma-semialdehyde, an intermediate in the synthesis of proline, was inhibited in Escherichia coli by physiological concentrations of penicillin. Sucrose (0.6 m) and sodium chloride (0.1 m) prevented penicillin inhibition of glutamic gamma-semialdehyde synthesis. Cells which were in the stationary phase, or which had been permitted to metabolize without growth, were insensitive to the effects of penicillin on glutamic gamma-semialdehyde synthesis.

Citrate reversal of iodoacetate inhibition of glutamate synthesis is nonmetabolic. Reversal of fluoride inhibition is metabolic, occurring only at low Mg concentrations.

The addition of penicillin to cells of Corynebacterium glutamicum growing in 5-liter fermentors initiated the excretion of glutamic acid. The rate of glutamate production in fermentors declined continuously with time and reached 75% of the initial rate in 24 hr after penicillin had been added. The addition of glutamate to resting cell suspensions had only a slight effect on sugar utilization but caused a marked decrease in glutamate excretion. It is suggested that the high level of glutamate accumulating in the fermentation broth is responsible for inhibiting its own production.

Photoautotrophic metabolism of CO(2) was compared with glucose metabolism in the facultative unicellular blue-green alga, Aphanocapsa 6714. Glucose-fed cells incorporated more (14)C into phosphorylated sugar intermediates of the reductive and oxidative pentose phosphate cycles than autotrophic cells. The relative increases were: 140-fold in dark cells; 32-fold in dichlorophenylmethylurea (DCMU)-inhibited cells; and 16-fold in cells assumilating glucose during photosynthetic carbon reduction. On the other hand, incorporation of (14)C from glucose into 3-phosphoglycerate and the amino acid pools of glutamate and aspartate was reduced in dark cells. Rates of protein synthesis in dark and DCMU-inhibited cells were reduced 50 and 80% compared to photoautotrophic cells. In cells assimilating glucose during photosynthesis, rates of (14)C incorporation into the two amino acids and protein were the same as in photoautotrophic cells. Chase experiments, using an excess of (12)C-glucose and CO(2), revealed slow turnover of carbon in dark cells and intermediate turnover rates in DCMU-inhibited cells, when compared to cells assimilating glucose during photosynthesis.

The synthesis of citric and glutamic acids by extracts of Chloropseudomonas ethylicum was studied with labeled precursors. When acetyl-coenzyme A-1-(14)C was used as substrate, only 0.1% of the total radioactivity was found in the C-5 position of citric acid; whereas, with oxalacetate-4-(14)C as substrate, 100% of the total radioactivity was found in C-5. These results demonstrated that the Chloropseudomonas citrate synthetase had an absolute stereospecificity, identical to that of the pig heart synthetase. The distribution of radioactivity in the glutamic acid synthesized from acetyl-coenzyme A-1-(14)C was 0% in C-1 and 94.0% in C-5; whereas the glutamic acid formed from oxalacetate-4-(14)C contained 89.6% in C-1 and 0.5% in C-5. This distribution is entirely consistent with the biosynthesis of glutamic acid from citric acid via aconitase, d(s)-isocitrate, and l-glutamate dehydrogenases. The presence of l-glutamate dehydrogenase in extracts was demonstrated. The stereospecificity of the citrate synthetase and the pattern of glutamate labeling further establish that the aconitase of Chloropseudomonas is completely stereospecific.

Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered alpha-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 x g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to alpha-ketoglutarate which can then undergo a transamination to glutamate.

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.

By transduction with SP-15, we have mapped some of the genetic sites involved in glutamyl polypeptide (Pep) production by Bacillus licheniformis 9945A. Eleven peptide mutations were assigned to group 1 which lies between the gly-42 and his-6 markers. Three other mutations were assigned to group 2 which lies between the ade-2 and str-1 sites. A series of 28 additional Pep(-) mutants did not belong to either group 1 or 2; the chromosomal location of those peptide mutations is unknown. All three mutants of group 2 were highly transformable, but only two of group 1 transformed well under the conditions employed. Knowing the chromosomal markers that were linked to peptide mutations made it possible to investigate the effect peptide mutations might have on the development of competence for transformation. Nontransformable organisms were rendered transformable upon the introduction, by transduction, of Pep(-) mutations from transformable strains. These results supported the conclusion that the ability of cells to develop competence for transformation when grown under appropriate conditions was related to the physiological effects of particular peptide mutations.

The fate of aspartic acid used for proline fermentation by Kurthia catenaforma was traced by using aspartic acid-U-(14)C. The radioactivities of proline and glutamic acid increased with the disappearance of aspartic acid. After 40 hr, aspartic acid disappeared from the medium and radioactive alpha-ketoglutaric acid was detected. The radioactivity of proline reached 44% of aspartic acid radioactivity at 40 hr. The specific radioactivities of these amino acids and of alpha-ketoglutaric acid supported the notion that proline is produced mainly from aspartic acid via alpha-ketoglutaric acid and glutamic acid. Since the levels of glutamic acid dehydrogenases (EC 1.4.1.2 and EC 1.4.1.4) were low in this organism, it appears that the nitrogen atom of aspartic acid enters proline by the action of aspartate aminotransferase (EC 2.6.1.1). The mechanism of proline production is discussed on the basis of the role of aspartic acid in this fermentation.

icd Mutants of Escherichia coli K-12, selected for their resistance to nalidixic acid, are deficient in isocitrate dehydrogenase.

Uptake of valine by Arthrobotrys conoides was an active process and was independent of its incorporation into cellular protein. Chemical fractionation of cells supplied with (14)C-l-valine for different time intervals revealed that the amino acid initially entered a pool of metabolic intermediates and was extractable with cold trichloroacetic acid. After a 4-min interval, some intracellular valine was incorporated into cell proteins, but most underwent metabolic transformation to a variety of products that included carboxylic acids and other amino acids. Carbon derived from valine was not localized in the lipid or nucleic acid fraction of cells, but some was completely oxidized and recovered as metabolic (14)CO(2). Autoradiograms of paper and thin-layer chromatograms of acid hydrolysates of cellular protein identified the following amino acids as having originated from valine: glutamate, aspartate, alanine, and leucine. Similar analysis of cold trichloroacetic acid extracts established that (14)C supplied as l-valine had been transformed also to alpha-ketoisovalerate, isobutyrate, propionate, succinate, malate, oxalacetate, pyruvate, and alpha-ketoglutarate. Pathways for transformation of the carbon skeleton of valine to various metabolic products are proposed.

Aconitaseless glutamic acid auxotroph MO-1-9B of Saccharomyces grew in glutamic acid-supplemented minimal medium, but failed to grow when glutamic acid was substituted by proline, arginine, ornithine, or glutamine. This mutant was also unable to utilize lactate or glycerol as a carbon source. Under a glutamic acid-limiting condition, by using acetate-1-(14)C as tracer, the mutant accumulated rather large amounts of (14)C-citric acid and (14)C-succinic acid when compared with the wild-type strain. Under excess glutamic acid supplementation, accumulation of citric acid and succinic acid was considerably reduced. When (14)C-glutamic acid-(U) was used as tracer, (14)C-alpha-ketoglutaric acid, (14)C-citric acid, and (14)C-succinic acid were accumulated in the mutant. The citric acid peak was the largest, followed by alpha-ketoglutaric acid and succinic acid. In the wild-type strain under similar conditions, only small amounts of (14)C-citric acid and (14)C-succinic acid and no (14)C-alpha-ketoglutaric acid were accumulated.