Glutamate catabolism in Rhizobium meliloti

The pathway by which glutamate is degraded as a carbon source has not previously been elucidated, but enzymatic analysis of Rhizobium meliloti CMF1 indicated that both glutamate dehydrogenase (GDH) and gamma-aminobutyrate (GABA) bypass activities were present in free living cells. However, when similar studies were performed on R. meliloti CMF1 bacteroids, isolated from alfalfa nodules, only GABA bypass activities were detectable. Both GDH and GABA bypass activities were influenced by the carbon source provided, with maximum activities being detected when glutamate was present as sole carbon and nitrogen source. Addition of a second carbon source, such as succinate, to the growth medium did not influence GDH activity but substantially decreased levels of the first enzyme of the GABA bypass, glutamate decarboxylase (GDC). Cyclic adenosine 3′5′-monophosphate (cAMP) failed to increase GDC activities in R. meliloti CMF1 cells grown in the presence of an additional carbon source. It is proposed that the GABA bypass is a major mechanism of glutamate carbon degradation in R. meliloti CMF1, a system whose enzymatic activities are influenced by the nature of the carbon source present in the growth environment.     

Control of the Pathway of γ-Aminobutyrate Breakdown in Escherichia coli K-12

Mutants of Escherichia coli K-12 isolated for their ability to utilize gamma-aminobutyrate (GABA) as the sole source of nitrogen exhibit a concomitant several-fold increase in the activities of gamma-aminobutyrate-alpha-ketoglutarate transaminase (GSST, EC 2.6.1.19) and succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16). The increase in rate of enzymatic activity is not accompanied by any changes in the affinities of the mutant enzymes for their respective substrates. The synthesis of the two enzymes is highly coordinate under a great variety of conditions, in spite of the wide range of activities observed. In cultures grown in minimal media with ammonium salts as the source of nitrogen, both GSST and SSDH are severely repressed by glucose. Substitution of ammonia with GABA, glutamate, or aspartate greatly reduces the effect of glucose on the synthesis of the GABA utilization enzymes. This escape from catabolite repression is specific for GSST and SSDH and does not involve other enzymes sensitive to catabolite repression (e.g., beta-galactosidase, EC 3.2.1.23, and aspartase, EC 4.3.1.1).     

Cycling of amino compounds in symbiotic lupin

The composition of amino acids was determined in the xylem andphloem sap of symbiotic lupins grown under a variety of treatmentsdesigned to alter the rate of nitrogen fixation. Asparaginewas the major amino acid in both xylem and phloem with glutamine,glutamate and aspartate also major components. GABA had a highconcentration in the xylem while valine was a major componentin the phloem. Exposure to combined nitrogen in the form ofeither ammonium or nitrate caused a reduction in specific nitrogenaseactivity and was associated with subsequent changes in bothof the translocated saps. Inhibiting nitrogen fixation by exposingnodules to oxygen produced a lower amide to amine ratio in thexylem sap (1.3:1) compared with control and nitrate ratios (2.6:1)and ammonium ratios (7.1:1). Similar ratios for amide aminewere also observed in the phloem sap. Labelling studies using¹⁵N₂ to follow nitrogen fixation, ammonium assimilation andamino acid transport have shown rapid accumulation of labelinto glutamine with subsequent enrichment in glutamate, aspartate,alanine, and GABA. Asparagine was found in high concentrationsin nodules and became slowly enriched. Labelled nitrogen fixedand assimilated in nodules was detected 40 min later in stemxylem extracts, largely as the amides glutamine and asparagine.These experiments provide evidence that large amounts of nitrogenouscompounds are cycled through the root nodules of symbiotic plants(contributing approximately 50% of xylem N) and that differencesin the composition of the phloem sap may influence nodule growthand activity. Key words: Nitrogen fixation, nitrogen translocation, isotope labelling, legumes, GC-MS     

Rhizobium meliloti glutamate synthase: cloning and initial characterization of the glt locus.

The genetic locus glt, encoding glutamate synthase from Rhizobium meliloti 1021, was selected from a pLAFR1 clone bank by complementation of the R. meliloti 41 Glt- mutant AK330. A fragment of cloned DNA complementing this mutant also served to complement the Escherichia coli glt null mutant PA340. Complementation studies using these mutants suggested that glutamate synthase expression requires two complementation groups present at this locus. Genomic Southern analysis using a probe of the R. meliloti 1021 glt region showed a close resemblance between R. meliloti 1021, 41, and 102f34 at glt, whereas R. meliloti 104A14 showed many differences in restriction fragment length polymorphism patterns at this locus. R. meliloti 102f34, but not the other strains, showed an additional region with sequence similarity to glt. Insertion alleles containing transposable kanamycin resistance elements were constructed and used to derive Glt- mutants of R. meliloti 1021 and 102f34. These mutants were unable to assimilate ammonia and were Nod+ Fix+ on alfalfa seedlings. The mutants also showed poor or no growth on nitrogen sources such as glutamate, aspartate, arginine, and histidine, which are utilized by the wild-type parental strains. Strains that remained auxotrophic but grew nearly as well as the wild type on these nitrogen sources were readily isolated from populations of glt insertion mutants, indicating that degradation of these amino acids is negatively regulated in R. meliloti as a result of disruptions of glt.     

Specificity and regulation of gamma-aminobutyrate transport in Escherichia coli.

A specific gamma-aminobutyrate (GABA) transport system in Escherichia coli K-12 cells with a K(m) of 12 muM and a V(max) of 278 nmol/ml of intracellular water per min is described. Membrane vesicles contained d-lactate-dependent activity of the system. Mutants defective in GABA transport were isolated; they lost the ability to utilize GABA as a nitrogen source, although the activities of glutamate-succinylsemialdehyde transaminase (GSST) (EC 2.6.1.19) and succinylsemialdehyde dehydrogenase (SSDH) (EC 1.2.1.16), the enzymes that catalyze GABA utilization, remained as high as in the parental CS101B strain. The ability to utilize l-ornithine, l-arginine, putrescine, l-proline, and glycine as a nitrogen source was preserved in the mutants. The genetic lesions resulting in the loss of GABA transport, gabP5 and gabP9, mapped in the gab gene cluster in close linkage to gabT and gabD, the structural genes of GSST and SSDH, and to gabC, a gene controlling the utilization of GABA, arginine, putrescine, and ornithine. The synthesis of the GABA transport carrier is subject to dual physiological control by (i) catabolite repression and (ii) nitrogen availability. Experiments with glutamine synthetase (EC 6.3.1.2)-negative and with glutamine synthetase-constitutive strains strongly indicate that this enzyme is the effector in the regulation of GABA carrier synthesis by route (ii).     

Nitrogen fixation by Rhizobium in pure cultures.

Strains of Rhizobium trifolii and Rhizobium meliloti were tested for their asymbiotic nitrogen fixation ability. From among ten tested strains two R. trifolii and one R. meliloti expressed nitrogenase activity within the range of 1.3--9.3 nM C2H4/h/mg protein. Asymbiotic nitrogen fixation was affected by the composition of the medium.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Mutations affecting the regulation of γ-aminobutyrate utilization inEscherichia coli K-12

Four genes,gabCPDT, are involved in the utilization of γ-aminobutyrate (GABA) byEscherichia coli K-12. Thegab gene cluster maps nearrecA andsrl, at 57.5 min.gabP, gabD andgabT specify the synthesis of GABA transport carrier, succinic semialdehyde dehydrogenase (SSDH), and glutamate-succinic semialdehyde transaminase (GSST), respectively;gabC controls the synthesis of all three proteins. GABA-nonutilizing mutants carrying deletions insrl extended into thegab cluster have been isolated. The mutants completely lost the capacity for GABA transport, while preserving full activity of GSST and SSDH, suggesting thatgabC is not a promoter-operator locus or a gene coding for an activator protein. A mutation ingabD (M-16) that abolished SSDH activity had the following additional properties: It exerted a bipolar effect on the neighboring genes, greatly reducing the activities of GSST and SSDH; the polar effect ongabP but not ongabT was fully suppressed by the knownrho mutation suA78; at least three classes of GABA-utilizing revertants of M-16 were obtained: (i) revertants with allgab activities restored to the parental levels; (ii) revertants with SSDH activity still missing, but with the other activities fully repaired; (iii) revertants with no SSDH activity, with GSST partly recovered, but with transport fully repaired. It is suggested that thegab cluster is transcribed bidirectionally from a promoter in thegabD region and that the mutation in strain M-16 may be due to DNA insertion in that region.     

Rhizobium meliloti mutants altered in ammonium utilization.

Derivatives of Rhizobium meliloti 2011 required trace amounts of glutamate to use ammonium as the nitrogen source for growth, although they could use serine as the sole nitrogen source. Specific activities of ammonium assimilatory enzymes were similar to those in strain Rm2011. The mutants were deficient in nitrogen fixation.     

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.     

Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.)

Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.     

Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions.

We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R. meliloti ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation. DNA sequence analysis showed that R. meliloti NtrA is 38% homologous with Klebsiella pneumoniae NtrA. Subcloning and complementation analysis suggested that the R. meliloti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.     

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

The effect of combined and separate inoculation of alfalfa plants with Azospirillum lipoferum and Sinorhizobium meliloti on denitrification and nitrogen-fixing activities

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N2O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N2O (11 micromol N2O/pot x 24 h) with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C2H4 nmol/flask x 24 h) and low denitrification activity (1.8 x micromol N2O/flask x 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.     

Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.     

Glutamate, a neurotransmitter--and so much more. A synopsis of Wierzba III

It appears almost incredible that the first indications that glutamate excites brain tissue were obtained during the second half of the 20th century, that vesicles containing glutamate were demonstrated in glutamatergic neurons less than 25 years ago, and that glutamate was not accepted as the major excitatory transmitter until about the same time. During this span of time it has also become realized that glutamate is so much more than a conventional neurotransmitter: (1) astrocytes express vesicles accumulating glutamate by vesicular transporters akin to the vesicular glutamate transporters in glutamatergic neurons, and they release glutamate by exocytosis; (2) a series of metabolic processes in astrocytes (glutamate uptake, glutamine synthetase activity, glutamine release) are involved in neuronal reutilization of transmitter glutamate; (3) glutamine may also be utilized for synthesis of GABA, the major inhibitory transmitter; (4) de novo synthesis of glutamate accounts for 20% of cerebral glucose metabolism, all of which initially occurs in astrocytes, and at steady state a corresponding amount of glutamate is oxidatively degraded, mainly or exclusively in astrocytes; (5) tissue contents of glutamate/glutamine increase during enhanced glutamatergic activity, i.e., astrocytic de novo synthesis exceeds astrocytic metabolic degradation of glutamate.     

Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity.

The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia. Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species. These are encoded by the glnA and glnII genes, respectively. Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source. For two auxotrophs that lacked any detectable GS activity, R. meliloti DNA of the mutated region was cloned and partially characterized. Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity. One of the cloned regions was identified as glnII. An R. meliloti glnII mutant and an R. meliloti glnA glnII double mutant were constructed. Both formed effective nodules on alfalfa. This is unlike the B. japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop. However, the R. meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.