Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium.

Glutamic acid is synthesized in enteric bacteria by either glutamate dehydrogenase or by the coupled activities of glutamate synthase and glutamine synthetase. A hybrid plasmid containing a fragment of the Salmonella typhimurium chromosome cloned into pBR328 restores growth of glutamate auxotrophs of S. typhimurium and Escherichia coli strains which have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 2.2-kilobase pair region was shown by complementation analysis, enzyme activity measurements, and the maxicell protein synthesizing system to carry the entire glutamate dehydrogenase structural gene, gdhA. Glutamate dehydrogenase encoded by gdhA carried on recombinant plasmids was elevated 5- to over 100-fold in S. typhimurium or E. coli cells and was regulated in both organisms. The gdhA promoter was located by recombination studies and by the in vitro fusion to, and activation of, a promoter-deficient galK gene. Additionally, S. typhimurium gdhA DNA was shown to hybridize to single restriction fragments of chromosomes from other enteric bacteria and from Saccharomyces cerevisiae.     

Genetic characterization of the glutamate dehydrogenase gene (gdhA) of Salmonella typhimurium.

Salmonella typhimurium mutants, either devoid or glutamate dehydrogenase activity or having a thermolabile glutamate dehydrogenase protein, were used to identify the structural gene (gdhA) for this enzyme. Transductions showed that the mutations producing these phenotypes were linked to both the pncA and nit genes, placing the gdhA locus between 23 and 30 U on the S. typhimurium chromosome. Additional transductions with several Tn10 insertions established the gene order as pncA-gdhA-nit. Since few genetic markers exist in this region of the chromosome, Hfr strains were constructed to orient the pncA-gdhA-nit cluster with outside genes. Conjugation experiments provided evidence for the gene order pyrD-pncA-gdhA-nit-trp. To further characterize gdhA, we used Mu cts d1 (Apr lac) insertions in this gene to select numerous strains containing deletions with various endpoints. Transductions of these deletions with strains containing different gdh mutations and with a mutant having a thermolabile glutamate dehydrogenase protein permitted us to construct a deletion map of the gdhA region.     

Gene expression analysis of methylotrophic oxidoreductases involved in the oligotrophic growth of Rhodococcus erythropolis N9T-4

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO? without any additional carbon or energy source. We performed a gene expression analysis of the oxidoreductases, which are involved in methanol metabolism, under various growth and induction conditions in N9T-4. A real-time PCR analysis revealed that the genes encoding NAD-dependent formaldehyde dehydrogenase (nFADH) and N,N'-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase (MDH) were strongly expressed under the oligotrophic conditions at levels of 20-100 fold those under heterotrophic conditions, in which n-tetradecane was used as the sole carbon source, while glucose did not affect the gene expression pattern in a minimum medium. The genes encoding mycothiol-dependent formaldehyde dehydrogenase (mFADH) and formate dehydrogenase were not induced under oligotrophic conditions, although mFADH expression was observed when formaldehyde was added to the induction medium. These results suggest that N9T-4 had three distinct formaldehyde oxidation systems, and that MDH and nFADH were the key enzymes in its oligotrophic growth.     

Deletion of a repetitive extragenic palindromic (REP) sequence downstream from the structural gene of Escherichia coli glutamate dehydrogenase affects the stability of its mRNA

A deletion that removes one repetitive extragenic palindromic sequence downstream of the structural gene of Escherichia coli glutamate dehydrogenase, reduces twofold the half-life of gdhA mRNA.     

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.     

The gdhA gene is located at 38.6 minutes on the Escherichia coli map.

We report here that the gdhA gene of Escherichia coli, which encodes the NADP-specific glutamate dehydrogenase, is located at 38.6 min on the map. We have confirmed this location by showing linkage with three Tn10 insertions that are linked to the aroD, pheS, and ansA loci, by complementation by a restriction-mapped lambda clone, and by showing correspondence between the restriction maps of the chromosome and the cloned and sequenced gdhA gene.     

Growth inhibition caused by overexpression of the structural gene for glutamate dehydrogenase (gdhA) from Klebsiella aerogenes

Two linked mutations affecting glutamate dehydrogenase (GDH) formation (gdh-1 and rev-2) had been isolated at a locus near the trp cluster in Klebsiella aerogenes. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. The gdhA gene from K. aerogenes was cloned and sequenced, and an insertion mutation was generated and shown to be linked to trp. A region of gdhA from a strain bearing gdh-1 was sequenced and shown to have a single-base-pair change, confirming that the locus defined by gdh-1 is the structural gene for GDH. Mutants with the same phenotype as rev-2 were isolated, and their sequences showed that the mutations were located in the promoter region of the gdhA gene. The linkage of gdhA to trp in K. aerogenes was explained by postulating an inversion of the genetic map relative to other enteric bacteria. Strains that bore high-copy-number clones of gdhA displayed an auxotrophy that was interpreted as a limitation for alpha-ketoglutarate and consequently for succinyl-coenzyme A (CoA). Three lines of evidence supported this interpretation: high-copy-number clones of the enzymatically inactive gdhA1 allele showed no auxotrophy, repression of GDH expression by the nitrogen assimilation control protein (NAC) relieved the auxotrophy, and addition of compounds that could increase the alpha-ketoglutarate supply or reduce the succinyl-CoA requirement relieved the auxotrophy.     

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.     

Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen.     

Changes in glutamate dehydrogenase activity of Chlamydomonas reinhardtii under different trophic and stress conditions

Abstract. Under stress conditions (darkness, nitrogen starvation, high ammonium concentrations, glutamine synthetase and glutamate synthase inhibition) glutamate dehydrogenase animating activity levels of Chlamydomonas cells varied inversely to those of glutamine synthetase. Nitrogen and carbon sources also influenced glutamate dehydrogenase levels in Chlamydomonas , the highest values being found in cells cultured mixotrophically with ammonium, under which conditions glutamate dehydrogenase and glutamine synthetase levels were likewise inversely related. These facts, together with the analysis of internal fluctuations of ammonium, 2-oxoglutarate, and the amino acid pool as well as the variations of certain enzymes involved in carbon metabolism indicate that glutamate dehydrogenase animating activity is adaptative, being involved in the maintenance of intracellular levels of L-glutamate when they cannot be maintained by the GS-GOGAT cycle, and probably more connected with carbon than nitrogen metabolism.     

Temperature-sensitive glutamate dehydrogenase mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in glutamate dehydrogenase activity were isolated in parent strains lacking glutamate synthase activity by localizcd mutagenesis or by a general mutagenesis combined with a cycloserine enrichment for glutamate auxotrophs. Two mutants with temperature-sensitive phenotypes had glutamate dehydrogenase activities that were more thermolabile than that of an isogenic control strain. Eight other mutants had less than 10% of the wild-type glutamate dehydrogenase activity. All the mutations were cotransducible with a Tn10 element (zed-2:Tn10) located at approximately 23 U on the S. typhimurium linkage map. These data strongly indicate that this region contains the structural gene (gdhA) for glutamate dehydrogenase.     

Mutations affecting the synthesis of NADP-dependent glutamate dehydrogenase in Pseudomonas aeruginosa

NADP-dependent glutamate dehydrogenase (NADP-GDH) was purified to homogeneity from Pseudomonas aeruginosa strain 8602 (PAC 1). The Mr determined by Sephadex gel filtration was 280,000; the subunit Mr determined by SDS-PAGE was 45,000. Mutant strains lacking NADP-GDH and glutamate synthase (Gdh-Glt-) required glutamate for growth. Transductants that lacked only NADP-GDH were indistinguishable from the wild-type strain in growth properties. It was concluded that NADP-GDH is not essential for growth of the wild-type organism and that glutamate formation via NAD-dependent glutamate dehydrogenase does not occur to a significant extent. A mutant strain, 39, producing high NADP-GDH activity, synthesized normal NADP-GDH and had the same intracellular glutamate concentrations as its parent. The mutation responsible for the synthesis of high levels of NADP-GDH was shown, by transduction, to be closely linked to the NADP-GDH structural gene (gdhA).