Utilization of acetate by Methanomonas emthanooxidans.

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.     

Biosynthesis of α-Ketoglutarate by the Reductive Carboxylation of Succinate in Bacteroides ruminicola

Experiments with growing cells and with cell-free extracts of Bacteroides ruminicola indicate that this anaerobic bacterium can synthesize alpha-ketoglutarate by a reductive carboxylation of succinate. When the organism was grown in medium containing succinate-1,4-(14)C, most of the radioactivity in cells was in the protein fraction and most of the (14)C in protein was in the glutamic acid family of amino acids (glutamate, proline, and arginine). When unlabeled succinate was added to culture medium containing glucose-U-(14)C, incorporation of radioactivity into the glutamic acid family of amino acids was greatly reduced. This supports the concept that succinate is an intermediate in synthesis of alpha-ketoglutarate. Cell-free extracts of the organism incubated with succinate-1,4-(14)C incorporated (14)C into amino acids and most of this was found in glutamate. The cofactors which stimulate glutamate synthesis from succinate by extracts from these cells appear to be similar to the factors that have been demonstrated with extracts from photosynthetic bacteria. The position of label in glutamate synthesized from succinate-1,4-(14)C, the probable absence of isocitric dehydrogenase, and studies with labeled citrate and with inhibitors of citric acid cycle enzymes support the concept of a reductive carboxylation of succinate as the only, or at least a major, mechanism for synthesis of alpha-ketoglutarate in this organism. This appears to be the first evidence for a net synthesis of alpha-ketoglutarate by this reaction in a nonphotosynthetic heterotrophic organism.     

Keto acid metabolism in Desulfovibrio.

Four strains of Desulfovibrio each excreted pyruvate to a constant level during growth; it was re-absorbed when the substrate (lactate) was exhausted. Malate, succinate, fumarate and malonate also accumulated during growth. One of the strains (Hildenborough) excreted alpha-ketoglutarate as well as pyruvate when incubated in nitrogen-free medium; the former was re-absorbed on addition of NH4Cl. In a low-lactate nitrogen-free medium, strain Hildenborough rapidly re-absorbed the pyruvate initially excreted, but did not re-absorb the alpha-ketoglutarate. Arsenite (I mM) prevented the accumulation of alpha-ketoglutarate; I mM-malonate did not affect the accumulation of keto acids. Isocitrate dehydrogenase activity (NAD-specific) in all strains was lower than NADP-specific glutamate dehydrogenase activity. Alpha-Ketoglutarate dehydrogenase could not be detected in any strain. NADPH oxidase activity was demonstrated. This and previous work indicate that a tricarboxylic acid pathway from citrate to alpha-ketoglutarate exists in Desulfovibrio spp., and that succinate can be synthesized via malate and fumarate; however, an intact tricarboxylic acid cycle is evidently not present. The findings are compared with observations on biosynthetic pathways in clostridia, obligate lithotrophs, phototrophs, and methylotrophs, and various facultative bacteria.     

New Obligate Methylotroph

A new and unique obligate methylotroph was isolated from enrichment cultures with methanol as the sole source of carbon and energy. The organism grows only on methanol and methylamine and not on methane. It does not have a complex intracellular membrane system. (14)C-acetate was assimilated by growing cultures and cell suspensions but was incorporated into only a limited number of cell constituents. (14)C-acetate incorporation was strictly dependent on the oxidation of methanol or methylamine as a source of energy. Extracts had relatively low levels of enzymes of the tricarboxylic acid cycle, and alpha-ketoglutarate dehydrogenase was not detected. Comparisons were made with a facultative methylotroph isolated from the same enrichment cultures. The new obligate methylotroph contained hexose phosphate synthetase, a key enzyme in the ribose phosphate cycle of methyl metabolism.     

Analysis of conformationally restricted alpha-ketoglutarate analogues as substrates of dehydrogenases and aminotransferases.

Five synthetic, conformationally restricted alpha-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, L-leucine dehydrogenase, L-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and alpha-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several L-amino acids (e.g., phenylalanine, glutamate) and the alpha-ketoglutarate analogues of interest. Transamination between L-glutamate (or L-phenylalanine) and the alpha-ketoglutarate analogues was found to be 0.13 to 1.08 micromol/h/mg at 45 degrees C. The products resulting from transamination between L-phenylalanine and the alpha-ketoglutarate analogues were separated by reverse-phase HPLC, and the newly formed amino acid analogues were analyzed by LC-MS in an ion selective mode. In each case, the ions obtained were consistent with the expected product and a representative example is provided. The possibility existed that although the alpha-ketoglutarate analogues are not substrates of the dehydrogenases and most of the aminotransferases investigated, they might be good inhibitors. Weak inhibition of aminotransferases and glutamate dehydrogenase was found with some of the alpha-ketoglutarate analogues. The newly available thermostable aminotransferases may have general utility in the synthesis of bulky L-amino acids from the corresponding alpha-keto acids.     

Nutritional studies on Chloroflexus,a filamentous photosynthetic,gliding bacterium

Nutritional studies on four different strains of Chloroflexus, a new genus of filamentous, photosynthetic bacteria are described. This organism appears to be related to several different procaryotic groups, and in particular to the green sulfur bacteria and blue-green algae. Unlike these autotrophs, however, Chloroflexus is nutritionally diverse, being able to grow aerobically as a light-independent heterotroph, and anaerobically as a photoautotroph or photoheterotroph. Numerous organic carbon sources including hexoses, amino acids, short chain fatty acids, organic acids, and some alcohols are utilized under various growth conditions. These results suggest that this organism may be among the most nutritionally versatile organisms known.     

Nitrogen-assimilating enzymes in land plants and algae: phylogenic and physiological perspectives

An important biochemical feature of autotrophs, land plants and algae, is their incorporation of inorganic nitrogen, nitrate and ammonium, into the carbon skeleton. Nitrate and ammonium are converted into glutamine and glutamate to produce organic nitrogen compounds, for example proteins and nucleic acids. Ammonium is not only a preferred nitrogen source but also a key metabolite, situated at the junction between carbon metabolism and nitrogen assimilation, because nitrogen compounds can choose an alternative pathway according to the stages of their growth and environmental conditions. The enzymes involved in the reactions are nitrate reductase (EC 1.6.6.1-2), nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.13-14, 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.2-4), aspartate aminotransferase (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Many of these enzymes exist in multiple forms in different subcellular compartments within different organs and tissues, and play sometimes overlapping and sometimes distinctive roles. Here, we summarize the biochemical characteristics and the physiological roles of these enzymes. We also analyse the molecular evolution of glutamine synthetase, glutamate synthase and glutamate dehydrogenase, and discuss the evolutionary relationships of these three enzymes.     

Kinetics of Glucose Incorporation by Aphanocapsa 6714

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.     

Metabolism of organic acids by Thiobacillus neapolitanus

Summary A comparative study has been made of the metabolism in several strains of Thiobacillus neapolitanus of formate, acetate, propionate, butyrate, valerate and pyruvate. Conflicting reports in the literature concerning the mechanism of pyruvate assimilation in thiobacilli have been resolved. Pyruvate undergoes decarboxylation to yield acetyl coenzyme A, which is converted to glutamate, proline and arginine via reactions of the incomplete Krebs' cycle of this organism. Pyruvate is converted also to alanine, valine, isoleucine, leucine and lysine by mechanisms like those in heterotrophs. No aspartate is formed from the C-3 of pyruvate. Removal of the C-1 of pyruvate yields carbon dioxide, which is refixed into all cell constituents. Formate is not produced by this scission reaction, as formate itself is incorporated almost exclusively into purines. Aspartate can be synthesized by the activities of phosphoenolpyruvate carboxylase and oxaloacetate-glutamate transamination. Carbon from propionate is converted principally to lipids, although some amino acid production occurs with the same distinctive labelling pattern as is found after acetate assimilation by T. neapolitanus strains C and X. Butyrate and valerate also showed some distinctive patterns of incorporation into cell constituents. Fluoropyruvate and fluoropropionate inhibited the growth of T. neapolitanus and the mechanisms of this poisoning are discussed.Generally these compounds contributed only small proportions of the total cell carbon and tended to be converted to limited numbers of cell components. The thiobacilli thus tend to conserve carbon from these compounds and not to degrade them to carbon dioxide on a large scale when growing in an otherwise autotrophic medium.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Assimilation of exogenous fructose, aspartate and some organic acids during the growth of methylotrophs.

The percentage of bacterial carbon that was derived from exogenous labelled compounds present in the medium during the growth of some methylotrophs on trimethylamine or on non-C1 compounds was determined. Less than 10% of bacterial carbon was derived from acetate during the growth of the obligate methylotrophs 4B6 and C2A1, and of the restricted facultative methylotroph PM6; the other restricted facultative methylotroph W3A1 gave a value of 18%. Corresponding values for three typical facultative methylotrophs growing on trimethylamine were 41, 42 and 52%. Aspartate, fructose, pyruvate and succinate made only a small percentage contribution (0-4 to 12%) to bacterial carbon in 4B6, C2A1, W3A1 and PM6. Washed suspensions of 4B6, C2A1, W3A1 and PM6, all grown on trimethylamine, assimilated labelled acetate only in the presence of trimethylamine and there was a linear relationship between the amount of acetate assimilated and the amount of trimethylamine oxidized. Organisms 4B6, C2A1, W3A1 and PM6 assimilated 14C from labelled acetate predominantly into lipid (except PM6), glutamate, arginine, proline and leucine, whereas the typical facultative methylotrophs assimilated 14C from acetate into lipid, nucleic acid and all the protein amino acids. These results are consistent with the presence of a deficient tricarboxylic acid cycle in the obligate methylotrophs and in the restricted facultative methylotrophs.     

Metabolism of valine by the filamentous fungus Arthrobotrys conoides

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.     

Biochemical Basis of Obligate Autotrophy in Nitrosomonas europaea

The specific activities of isocitric dehydrogenase, alpha-ketoglutaric dehydrogenase, succinic dehydrogenase, malic dehydrogenase, and reduced nicotinamide adenine dinucleotide (NADH) oxidase were determined in extracts of Nitrosomonas europaea and compared with the corresponding values for Anacystis nidulans and autotrophically grown Hydrogenomonas eutropha. In common with other obligate autotrophs and in contrast to facultative autotrophs, Nitrosomonas extracts lacked alpha-ketoglutaric dehydrogenase and KCN-sensitive NADH oxidase activity and had low succinic dehydrogenase activity. The Nitrosomonas NADH oxidase appeared to be of the peroxidase type.     

A challenge for 21st century molecular biology and biochemistry: what are the causes of obligate autotrophy and methanotrophy?

We assess the use to which bioinformatics in the form of bacterial genome sequences, functional gene probes and the protein sequence databases can be applied to hypotheses about obligate autotrophy in eubacteria. Obligate methanotrophy and obligate autotrophy among the chemo- and photo-lithotrophic bacteria lack satisfactory explanation a century or more after their discovery. Various causes of these phenomena have been suggested, which we review in the light of the information currently available. Among these suggestions is the absence in vivo of a functional alpha-ketoglutarate dehydrogenase. The advent of complete and partial genome sequences of diverse autotrophs, methylotrophs and methanotrophs makes it possible to probe the reasons for the absence of activity of this enzyme. We review the role and evolutionary origins of the Krebs cycle in relation to autotrophic metabolism and describe the use of in silico methods to probe the partial and complete genome sequences of a variety of obligate genera for genes encoding the subunits of the alpha-ketoglutarate dehydrogenase complex. Nitrosomonas europaea and Methylococcus capsulatus, which lack the functional enzyme, were found to contain the coding sequences for the E1 and E2 subunits of alpha-ketoglutarate dehydrogenase. Comparing the predicted physicochemical properties of the polypeptides coded by the genes confirmed the putative gene products were similar to the active alpha-ketoglutarate dehydrogenase subunits of heterotrophs. These obligate species are thus genomically competent with respect to this enzyme but are apparently incapable of producing a functional enzyme. Probing of the full and incomplete genomes of some cyanobacterial and methanogenic genera and Aquifex confirms or suggests the absence of the genes for at least one of the three components of the alpha-ketoglutarate dehydrogenase complex in these obligate organisms. It is recognized that absence of a single functional enzyme may not explain obligate autotrophy in all cases and may indeed be only be one of a number of controls that impose obligate metabolism. Availability of more genome sequences from obligate genera will enable assessment of whether obligate autotrophy is due to the absence of genes for a few or many steps in organic compound metabolism. This problem needs the technologies and mindsets of the present generation of molecular microbiologists to resolve it.     

Acetate assimilation by Nitrobacter agilis in relation to its "obligate autotrophy"

Acetate (1 to 10 mm) had no effect on the rate of nitrite oxidation or exponential growth by Nitrobacter agilis. However, acetate-1-(14)C and -2-(14)C were both assimilated by growing cultures, and acetate carbon contributed 33 to 39% of newly synthesized cell carbon. Carbon from acetate was incorporated into all of the major cell constituents, including most of the amino acids of cell protein and poly-beta-hydroxybutyrate (PHB). Cultures grown in the presence of acetate showed a significant increase in turbidity, attributable in part to protein synthesis and the accumulation of PHB in the "post-exponential phase," when the supply of nitrite was completely exhausted. Cell suspensons of N. agilis assimilated acetate in the absence of bicarbonate and even in the absence of nitrite. However, the addition of nitrite increased the rate of acetate assimilation by cell suspensions. The distribution of (14)C-acetate incorporated by cell suspensions was qualitatively similar to that found with growing cultures. Cell suspensions of N. agilis slowly oxidized acetate to CO(2). Addition of nitrite suppressed CO(2) production from acetate but increased the assimilation of acetate carbon into cell material. N. agilis contained all the enzymes of the tricarboxylic acid cycle. Growth of N. agilis in the presence of acetate did not significantly affect the levels of the enzymes of the tricarboxylic acid cycle, but did result in a 100-fold increase in the specific activity of isocitratase. In contrast, carboxydismutase was partially repressed. N. agilis was grown heterotrophically through seven transfers on a medium containing acetate and casein hydrolysate. The addition of nitrite increased the rate of heterotrophic growth. Heterotrophically grown organisms still retained their ability to grow autotrophically with nitrite. However, these organisms oxidized nitrite at a slower rate. Organisms from autotrophic and heterotrophic cultures were analyzed to determine the mean guanine plus cytosine content of their deoxyribonucleic acid; in both cases this mean was 61.2 +/- 1%. We concluded that N. agilis is not an obligate autotroph; it appears to be a facultative autotroph which resembles the novel facultative autotroph, Thiobacillus intermedius, very closely.     

Incorporation of organic compounds into cell protein by lithotrophic,ammonia-oxidizing bacteria

Incorporation of organic compounds into cell protein by the obligate chemolithotrophs Nitrosomonas spec., Nitrosococcus oceanus, Nitrosococcus mobilis, Nitrosovibrio tenuis, Nitrosolobus spec., and Nitrosopira spec. was studied. In the presence of ammonia as energy source organic substrates were supplied. Distribution of ¹⁴C into cell amino acids arising from ¹⁴C-labelled glucose, Na-pyruvate, and Na-acetate was investigated. While carbon from glucose was distributed unrestricted, carbon from pyruvate preferably entered into the amino acids of the pyruvate and glutamate family and from acetate mainly into leucine and the glutamate family. Among the strains examined, slight differences were observed, but all should be included under group A of the scheme of Smith and Hoare (1977).     

Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds

The first step of amino acid degradation in lactococci is a transamination, which requires an alpha-keto acid as the amino group acceptor. We have previously shown that the level of available alpha-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding alpha-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce alpha-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added alpha-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added alpha-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding alpha-ketoglutarate to improve aroma development in cheese.     

Acetate assimilation pathway of Methanosarcina barkeri.

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.     

The metabolism of organic acids by a marine pennate diatom

Cocconeis diminuta, a marine benthic diatom, metabolizes acetate and lactate-¹⁴C. In the light, the major product was lipid, whereas in the dark, CO₂ was the major product. Analysis of proteins synthesized in the presence of acetate or lactate showed that radioactivity was incorporated predominantly into the glutamate family of amino acids and those amino acids related directly to the substrate. Light and dark assimilation of substrate was inhibited slightly by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea and 2,4-dinitrophenol. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea caused a pattern of metabolism of acetate in the light characteristic of that which occurs in the dark. Monofluoroacetic acid inhibited assimilation considerably in the dark, but less in the light. The level of enzymes of the tricarboxylic acid cycle and NADH-oxidase were found to be about the same as those in other autotrophs. The metabolism of acetate and lactate is discussed in relation to the autotrophic mode of nutrition of Cocconeis diminuta.     

Fine control of citrate synthase activity in blue-green algae

Summary The citrate synthases of four blue-green algae, two unicellular (Aphanocapsa spp.) and two filamentous (Nostoc sp., Phormidium sp.) were inhibited by -ketoglutarate but not by NADH. This control of citrate synthase activity reflects the lack of -ketoglutarate dehydrogenase in blue-green algae and the strictly biosynthetic role played by the glutamate branch of the tricarboxylic acid cycle. The citrate synthases were also inhibited by ATP and the enzyme of one of the unicellular organisms was also sensitive to inhibition by NADPH. These effectors may function in regulating the flow of fixed carbon into lipids rather than the glutamate family of amino acids.Contribution No. 1649 from the University of Miami, Rosenstiel School of Marine and Atmospheric Science, 10 Rickenbacker Causeway, Miami, Florida 33149, U.S.A.