Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria

In this work, we report the detection of aromatic amino acid aminotransferase (AAT) activity from cell-free crude extracts of nine strains of N(2)-fixing bacteria from three genera. Using tyrosine as substrate, AAT activity ranged in specific activity from 0.084 to 0.404 micromol min(-1)mg(-1). When analyzed under non-denaturating PAGE conditions; and using tryptophan, phenylalanine, tyrosine, and histidine as substrates Pseudomonas stutzeri A15 showed three isoforms with molecular mass of 46, 68 and 86 kDa, respectively; Azospirillum strains displayed two isoforms which molecular mass ranged from 44 to 66 kDa and Gluconacetobacter strains revealed one enzyme, which molecular mass was estimated to be much more higher than those of Azospirillum and P. stutzeri strains. After SDS-PAGE, some AAT activity was lost, indicating a differential stability of proteins. All the strains tested produced IAA, especially with tryptophan as precursor. Azospirillum strains produced the highest concentrations of IAA (16.5-38 microg IAA/mg protein), whereas Gluconacetobacter and P. stutzeri strains produced lower concentrations of IAA ranging from 1 to 2.9 microg/mg protein in culture medium supplemented with tryptophan. The IAA production may enable bacteria promote a growth-promoting effect in plants, in addition to their nitrogen fixing ability.     

Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti

Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied. Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant. The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation. Histology of the nodules induced by trpE and aro mutants exhibited striking similarities. Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone. Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency. A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation. The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted. These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S. meliloti. The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.     

Rhizobium meliloti mutants unable to synthesize anthranilate display a novel symbiotic phenotype.

Analyses of Rhizobium meliloti trp auxotrophs suggest that anthranilate biosynthesis by the R. meliloti trpE(G) gene product is necessary during nodule development for establishment of an effective symbiosis. trpE(G) mutants, as well as mutants blocked earlier along this pathway in aromatic amino acid biosynthesis, form nodules on alfalfa that have novel defects. In contrast, R. meliloti trp mutants blocked later in the tryptophan-biosynthetic pathway form normal, pink, nitrogen-fixing nodules. trpE(G) mutants form two types of elongated, defective nodules containing unusually extended invasion zones on alfalfa. One type contains bacteroids in its base and is capable of nitrogen fixation, while the other lacks bacteroids and cannot fix nitrogen. The trpE(G) gene is expressed in normal nodules. Models are discussed to account for these observations, including one in which anthranilate is postulated to act as an in planta siderophore.     

Gas chromatography-mass spectrometry analysis of indoleacetic acid and tryptophan following aqueous chloroformate derivatisation of Rhizobium exudates.

A new method for preparing alkyl esters of indole-3-acetic acid (IAA) in aqueous solution is adapted from the chloroformate method originally described by Husek for the analysis of amino acids. This method has the significant advantage of avoiding the generation and use of diazomethane, and is done in aqueous solution without the need to dry the sample with concomitant non-specific losses of IAA. The effectiveness of this method is demonstrated by its use in an isotope dilution gas chromatography-mass spectrometry (GC-MS) assay of IAA and L-tryptophan (Trp) in the culture supernatant of a series of Sinorhizobium meliloti and Rhizobium leguminosarum bv. trifolii strains that can interact with rice to either enhance or inhibit rice plant growth. We were testing the hypothesis that the rice growth inhibition was related to the biosynthesis of IAA. It was found that S. meliloti and Rhizobium strains produced high amounts of IAA in Trp supplemented BIII minimal medium compared to BIII media. All the strains produced more than the minimum amount of IAA required to inhibit rice growth and thus IAA is not the major inhibitory factor of rice seedling growth from S. meliloti and Rhizobium strains.     

Aspartate aminotransferase activity is required for aspartate catabolism and symbiotic nitrogen fixation in Rhizobium meliloti.

A mutant of Rhizobium meliloti, 4R3, which is unable to grow on aspartate has been isolated. The defect is specific to aspartate utilization, since 4R3 is not an auxotroph and grows as well as its parent strain on other carbon and nitrogen sources. The defect was correlated with an inability to fix nitrogen within nodules formed on alfalfa. Transport of aspartate into the mutant cells was found to be normal. Analysis of enzymes involved in aspartate catabolism showed a significantly lower level of aspartate aminotransferase activity in cell extracts of 4R3 than in the wild type. Two unrelated regions identified from a genomic cosmid bank each complemented the aspartate catabolism and symbiotic defects in 4R3. One of the cosmids was found to encode an aspartate aminotransferase enzyme and resulted in restoration of aspartate aminotransferase activity in the mutant. Analysis of the region cloned in this cosmid by transposon mutagenesis showed that mutations within this region generate the original mutant phenotypes. The second type of cosmid was found to encode an aromatic aminotransferase enzyme and resulted in highly elevated levels of aromatic aminotransferase activity. This enzyme apparently compensated for the mutation by its ability to partially utilize aspartate as a substrate. These findings demonstrate that R. meliloti contains an aspartate aminotransferase activity required for symbiotic nitrogen fixation and implicate aspartate as an essential substrate for bacteria in the nodule.     

Genomic structure,expression and evolution of the alfalfa aspartate aminotransferase genes

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.     

Tryptophan metabolism in Klebsiella aerogenes: regulation of the utilization of aromatic amino acids as sources of nitrogen.

Klebsiella aerogenes utilized aromatic amino acids as sole sources of nitrogen but not as sole sources of carbon. K. aerogenes abstracted the alpha-amino group of these compounds by transamination and excreted the arylpyruvate portions into the medium. When tryptophan was utilized as the sole source of nitrogen by K. aerogenes, indolepyruvate was excreted into the medium, where it polymerized non-enzymatically to form a brick red pigment. At least four separate aromatic aminotransferase activities were found in K. aerogenes. One activity (aromatic aminotransferase I) appeared to be solely responsible for the aminotransferase reaction necessary for the growth of K. aerogenes when tryptophan was the source of nitrogen; the loss of this activity by mutation (tut) prevented the growth of cells on media containing this and other aromatic amino acids. None of the other aminotransferase activities in the cells could substitute for aromatic aminotransferase in this regard. Tryptophan-dependent pigment formation in K. aerogenes was positively controlled by the intracellular level of glutamine synthetase. Nevertheless, the aromatic aminotransferase activity in cells varied less than 2-fold in response to 10-fold or greater changes in the levels of glutamine synthetase. Glutamine synthetase affected the ability of the cells to take up tryptophan from the medium.     

Production of indole-3-acetic acid, aromatic amino acid aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere isolates

The production of auxins, such as indole-3-acetic acid (IAA), by rhizobacteria has been associated with plant growth promotion, especially root initiation and elongation. Six indole-producing bacteria isolated from the rhizosphere of legumes grown in Saskatchewan soils and identified as Pantoea agglomerans spp. were examined for their ability to promote the growth of canola, lentil and pea under gnotobiotic conditions and for tryptophan (Trp)-dependent IAA production. Five of the isolates enhanced root length, root weight or shoot weight by 15–37% in at least one of the plant species, but isolates 3–117 and 5–51 were most consistent in enhancing plant growth across the three species. Indole concentrations in the rhizosphere of plants grown under gnotobiotic conditions increased in the presence of the rhizosphere isolates and when Trp was added 3 days prior to plant harvest. Isolates 3–117, 5–51 and 5–105 were most effective in increasing rhizosphere indole concentrations. Colony hybridization confirmed that all of the isolates possessed the ipdC gene which codes for a key enzyme in the Trp-dependent IAA synthetic pathway. The activity of amino acid aminotransferase (AAT), catalyzing the first step in the Trp-dependent synthetic pathway, was examined in the presence of Trp and other aromatic amino acids. All of the isolates accumulated Trp internally and released different amounts of IAA. The production of IAA from the isolates was greatest in the presence of Trp, ranging from 2.78 to 16.34 μg mg protein⁻¹ in the presence of 250 μg of Trp ml⁻¹. The specific activity of AAT was correlated with the concentration of IAA produced in the presence of Trp but not when tyrosine (Tyr), phenylalanine (Phe) or aspartate (Asp) was used as a sole nitrogen source. Isolate 3–117, which produced significant concentrations of IAA in the presence and absence of Trp, was able to use aromatic amino acids as sole sources of nitrogen and was most consistent in enhancing the growth of canola, lentil and pea may have potential for development as a plant growth-promoting inoculant. Responsible Editor: Peter A. H. Bakker.     

Characterization of an aromatic amino acid aminotransferase from Rhizobium leguminosarum biovar trifolii.

The most abundant aromatic amino acid aminotransferase of Rhizobium leguminosarum biovar trifolii was partially purified. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration. The enzyme transaminated aromatic amino acids and histidine. It used aromatic keto acids and alpha-ketoglutaric and oxalacetic acids as amino-group acceptors. The optimum temperature was 35 degrees C. Using phenylalanine and alpha-ketoglutaric acid as substrates the activation energy was 46.2 kJ.mol-1 and for the couple tryptophan:alpha-ketoglutaric acid it was 70.3 kJ.mol-1. The optimum pH was different for each substrate: 7.3 for phenylalanine, 7.9 for histidine and 8.7 for tryptophan.     

Partial purification and characterisation of an aromatic amino acid aminotransferase from mung bean (Vigna radiata L. Wilczek)

An aromatic amino acid aminotransferase (aromAT) was purified over 33 000-fold from the shoots and primary leaves of mung beans (Vigna radiata L. Wilczek). The enzyme was purified by ammonium sulfate precipitation, gel filtration and anion exchange followed by fast protein liquid chromatography using Mono Q and Phenylsuperose. The relative amino transferase activities using the most active amino acid substrates were: tryptophan 100, tyrosine 83 and phenylalanine 75, withK _m values of 0.095, 0.08 and 0.07 mM, respectively. The enzyme was able to use 2-oxoglutarate, oxaloacetate and pyruvate as oxo acid substrates at relative activities of 100, 128 and 116 andK _m values of 0.65, 0.25 and 0.24 mM, respectively. In addition to the aromatic amino acids the enzyme was able to transaminate alanine, arginine, aspartate, leucine and lysine to a lesser extent. The reverse reactions between glutamate and the oxo acids indolepyruvate and hydroxyphenylpyruvate occurred at 30 and 40% of the forward reactions of tryptophan and tyrosine, withK _m, values of 0.1 and 0.8 mM, respectively. The enzyme was not inhibited by indoleacetic acid, although -naphthaleneacetic acid did inhibit slightly. Addition of the cofactor pyridoxal phosphate only slightly increased the activity of the purified enzyme. The aromAT had a molecular weight of 55–59 kDa. The possible role of the aromAT in the biosynthesis of indoleacetic acid is discussed.Abbreviations AAT aspartate aminotransferase - aromAT aromatic amino acid aminotransferase - FPLC fast protein liquid chromatography - IPyA indolepyruvate - OHPhPy hydroxyphenylpyruvate - PLP pyridoxal phosphate - TAT tryptophan aminotransferase     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Functional significance of aromatic amino acid: aromatic keto acid aminotransferases in rat brain and liver: competition for tryptophan between aminotransferases and tryptophan hydroxylase in vitro.

Using low concentrations of substrates and cofactors, a comparison was made of the relative rates by which aminotransferases catalysed transaminations between aromatic amino acids and aromatic or aliphatic keto acids. Tryptophan aminotransferase in homogenates of rat midbrain and liver transaminated phenylpyruvate at a rate 70 to 150-fold greater than the rate with α-ketoglutarate at low concentrations of substrates. Phenylalanine aminotransferase in liver and midbrain also was more active with aromatic keto acids than with aliphatic keto acids. However, tyrosine aminotransferase in dialysed homogenates of midbrain transaminated α-ketoglutarate and phenylpyruvate at approximately equal rates. Fresh homogenates of midbrain contained an inhibitor which markedly decreased tyrosine aminotransferase activity with α-ketoglutarate but not with phenylpyruvate. Tyrosine aminotransferase in homogenates of rat liver transaminated α-ketoglutarate and phenylpyruvate at equal rates below 10 μM keto acid, but above 10 μM, transamination of α-ketoglutarate was favoured. With homogenates of liver, transamination of α-ketoglutarate, but not phenylpyruvate, by tyrosine was increased 650% by exogenous pyridoxal phosphate. Since tryptophan aminotransferase in the brain may compete with tryptophan hydroxylase for available tryptophan, a comparison was made of the relative activities of tryptophan hydroxylase and tryptophan aminotransferase. At concentrations above 7.5 μM phenylpyruvate, transamination was 8 to 17-fold greater than the rate of hydroxylation of 50 μM tryptophan.     

Purification and properties of aromatic amino acid aminotransferase from Klebsiella aerogenes.

We describe the complete purification of aromatic aminotransferase I, the enzyme responsible for the ability of Klebsiella aerogenes to use tryptophan and phenylalanine as sole sources of nitrogen, as well as the partial purification of aromatic aminotransferase IV. An examination of the properties of these enzymes revealed that aminotransferase I had much greater affinity for the aromatic amino acids than aminotransferase IV, explaining the essential role of aminotransferase I in the utilization of exogenously supplied aromatic amino acids. The properties of aminotransferase IV suggest that this enzyme is actually an aspartate aminotransferase (EC 2.6.1.1), corresponding to the product of the aspC gene of Escherichia coli.     

Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.     

Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.     

Enzymatic Basis for Overproduction of Tryptophan and Its Metabolites in Hansenula polymorpha Mutants

3-Deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase and anthranilate synthetase are key regulatory enzymes in the aromatic amino acid biosynthetic pathway. The DAHP synthetase activity of Hansenula polymorpha was subject to additive feedback inhibition by phenylalanine and tyrosine but not by tryptophan. The synthesis of DAHP synthetase in this yeast was not repressed by exogenous aromatic amino acids, singly or in combinations. The activity of anthranilate synthetase was sensitive to feedback inhibition by tryptophan, but exogenous tryptophan did not repress the synthesis of this enzyme. Nevertheless, internal repression of anthranilate synthetase probably exists, since the content of this enzyme in H. polymorpha strain 3-136 was double that in the wild-type and less sensitive 5-fluorotryptophan-resistant strains. The biochemical mechanism for the overproduction of indoles by the 5-fluorotryptophan-resistant mutants was due primarily to a partial desensitization of the anthranilate synthetase of these strains to feedback inhibition by tryptophan. These results support the concept that inhibition of enzyme activities rather than enzyme repression is more important in the regulation of aromatic amino acid biosynthesis in H. polymorpha.     

Modular structure of the Rhizobium meliloti DctB protein

Abstract To investigate the modular structure of the Rhizobium meliloti dicarboxylic acid sensor protein, DctB, three truncated DctB proteins (DctB4, DctB5 and DctB4G) were constructed, overproduced in Escherichia coli and purified. The DctB4G protein was composed of 446 amino acids of the DctB C-terminus and displayed strong autophosphorylation activity in vitro. This activity was sustained when a further 120 amino acids at the N-terminus of the polypeptide were deleted (DctB5). This protein which has an intact transmitter domain exhibits specific but inefficient phospho-transfer capabilities. Removal of 58 amino acids from the DctB4G C-terminus which included blocks F and G2 of the transmitter domain, rendered the resultant protein (DctB4) incompetent in autophosphorylation. Phosphorylation activity was restored to DctB4 through intramolecular complementation with DctB. Therefore, it would appear that the R. meliloti DctB protein is active as a dimer (or higher order oligomer). Furthermore, the intramolecular complementation experiments indicate that the amino acids 171–291, a predicted periplasmic stretch, play an important role in the dimerization process.     

Alfalfa Root Flavonoid Production Is Nitrogen Regulated

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.     

The disruption of a gene encoding a putative arylesterase impairs pyruvate dehydrogenase complex activity and nitrogen fixation in Sinorhizobium meliloti

Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the trichloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3' end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.