Conditions Of Nitrogen Excess Research Articles

Role of Escherichia coli nitrogen regulatory genes in the nitrogen response of the Azotobacter vinelandii NifL-NifA complex.

The redox-sensing flavoprotein NifL inhibits the activity of the nitrogen fixation (nif)-specific transcriptional activator NifA in Azotobacter vinelandii in response to molecular oxygen and fixed nitrogen. Although the mechanism whereby the A. vinelandii NifL-NifA system responds to fixed nitrogen in vivo is unknown, the glnK gene, which encodes a PII-like signal transduction protein, has been implicated in nitrogen control. However, the precise function of A. vinelandii glnK in this response is difficult to establish because of the essential nature of this gene. We have shown previously that A. vinelandii NifL is able to respond to fixed nitrogen to control NifA activity when expressed in Escherichia coli. In this study, we investigated the role of the E. coli PII-like signal transduction proteins in nitrogen control of the A. vinelandii NifL-NifA regulatory system in vivo. In contrast to recent findings with Klebsiella pneumoniae NifL, our results indicate that neither the E. coli PII nor GlnK protein is required to relieve inhibition by A. vinelandii NifL under nitrogen-limiting conditions. Moreover, disruption of both the E. coli glnB and ntrC genes resulted in a complete loss of nitrogen regulation of NifA activity by NifL. We observe that glnB ntrC and glnB glnK ntrC mutant strains accumulate high levels of intracellular 2-oxoglutarate under conditions of nitrogen excess. These findings are in accord with our recent in vitro observations (R. Little, F. Reyes-Ramirez, Y. Zhang, W. Van Heeswijk, and R. Dixon, EMBO J. 19:6041-6050, 2000) and suggest a model in which nitrogen control of the A. vinelandii NifL-NifA system is achieved through the response to the level of 2-oxoglutarate and an interaction with PII-like proteins under conditions of nitrogen excess.

The Rhizobium leguminosarum glnB gene is down-regulated during symbiosis.

Symbiotic nitrogen fixation involves the development, on the legume plant root, of specialised organs called nodules, within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. The glnB gene encodes a signal transduction protein (P(II)) which is a component of the bacterial nitrogen regulation (Ntr) system and an essential regulator of ammonium assimilation. We demonstrate that in Rhizobium leguminosarum the glnB promoter is strongly regulated by nitrogen and NtrC, but still shows a significant level of activity in conditions of nitrogen excess. Expression of genes involved in nitrogen assimilation has been shown to be absent in nitrogen-fixing bacteroids, and, in agreement with this, we find that the glnB promoter is down-regulated during bacteroid differentiation at a time coincident with the arrest of bacterial division in the nodule. This pattern is common to other bacterial genes involved in nitrogen assimilation and it is noteworthy that the zone where the glnB promoter is active is coincident with the region in which NtrC is expressed.

Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.

Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein.

PII-like signal transduction proteins, which respond to the nitrogen status via covalent modification and signal the carbon status through the binding of 2-oxoglutarate, have been implicated in the regulation of nitrogen fixation in several diazotrophs. The NIFL-NIFA two-component regulatory system, which integrates metabolic signals to fine-tune regulation of nitrogenase synthesis in Azotobacter vinelandii, is a potential target for PII-mediated signal transduction. Here we demonstrate that the inhibitory activity of the A.vinelandii NIFL protein is stimulated by interaction with the non-uridylylated form of PII-like regulatory proteins. We also observe that the NIFL-NIFA system is directly responsive to 2-oxoglutarate. We propose that the PII protein signals the nitrogen status by interaction with the NIFL-NIFA system under conditions of nitrogen excess, and that the inhibitory activity of NIFL is relieved by elevated levels of 2-oxoglutarate when PII is uridylylated under conditions of nitrogen limitation. Our observations suggest a model for signal transduction to the NIFL-NIFA system in response to carbon and nitrogen status which is clearly distinct from that suggested from studies on other diazotrophs.

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC∼P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions σ 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of σ 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.

Structural homologues P(II) and P(Z) of Azospirillum brasilense provide intracellular signalling for selective regulation of various nitrogen-dependent functions.

P(II) (glnB) is a signal transduction protein that in Azospirillum brasilense is specifically required for nitrogen fixation. Little is known about whether and how its homologue P(Z) (glnZ) participates in the regulation of cellular functions. In this study, we have shown the regulatory action of the two proteins by analysing the relevant single and double null-mutant strains. The transcription of glnZ is monocistronic, and it starts mainly from a sigma54-dependent promoter, activated by NtrC. glnZ expression is dependent on the ntr system, even under conditions of nitrogen excess, and is greatly enhanced in the presence of aspartate. P(Z) is uridylylated in response to nitrogen limitation, like P(II), although different amounts of the two proteins are synthesized. P(II) is required for the dephosphorylation of NtrC. Thus, in the absence of P(II), the repression of nitrate assimilation is not promoted, which, in turn, leads to a high rate of ammonium excretion. Unexpectedly, P(II) and P(Z) proteins are not essential for the reversible modification of glutamine synthetase. (Methyl)ammonium transport into the cell is negatively regulated by P(Z). The growth of a double-mutant strain (glnB::kan; glnZ::omega) is drastically disabled, although wild-type growth is restored by complementation with either glnB or glnZ. We conclude that P(II) and P(Z), despite their structural similarity, are involved in different regulatory processes, except for that required for cell growth.

The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear. We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation. In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth. Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations. The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway. Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function. In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects. We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.

Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation.

The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P(II) protein, have been determined. glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess. This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain. The increase was not observed in rpoN or ntrC mutants. In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P(II) negatively regulates its own expression. A DNA motif, similar to a sigma54-dependent promoter consensus, was found in the 5' nontranscribed region. Thus, the glnBA operon seems to be transcribed from a sigma54-dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia. Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth. The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type. Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix- nodules on Sesbania rostrata roots. P(II) is the first example in A. caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state.

Effect of nitrogen limitaton on synthesis of enzymes in Saccharomyces cerevisiae during fermentation of high concentration of carbohydrates

The enzyme data presented here support the view that in Saccharomyces cerevisiae the flux of carbon through the glycolytic pathway is greater under nitrogen limiting than under nitrogen excess growth conditions. Enzyme data also suggest that conditions which stimulate the glycolytic pathway depress the flow of carbon through the hexose monophosphate pathway and vice versa.

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.

Bacillus subtilis glnR mutants defective in regulation

The Bacillus subtilis glnR gene (part of the glnRA operon) encodes a 135-amino-acid (aa) repressor, GlnR, that regulates glnRA transcription in response to nitrogen levels in the growth medium. Two glnR mutants unable to repress under nitrogen excess conditions were obtained by mutagenesis. Lesions were found at Leu 77 and Ala 80, aa that lie within a region (between aa 59–83) thought to form the α-helix-turn-α-helix (HTH) motif common among a class of regulatory proteins. Alteration of Gly 72 by site-directed mutagenesis also affected regulation, suggesting that aa within the putative HTH region are critical for GlnR function and may be involved in DNA binding. However, other replacements within the aa 59–83 sequence failed to support the HTH structure proposed for this region. Mutations within the C-terminal region of GlnR were also found to affect regulation. Introduction of an ochre stop codon at aa 110, 116, 123 and 129 resulted in the production of truncated proteins that were constitutively repressed, strongly suggesting that a signal recognition site resides within the last seven aa of GlnR. Substituting Asp 129 with Asn led to loss of repression, indicating that Asp 129 may be directly involved in interacting with either positive or negative effector molecules, or is a target for post-translational modification.

Genetic and biochemical characterization of the two glutamine synthetases GSI and GSII of the phosphinothricyl-alanyl-alanine producer, streptomyces viridochromogenes Tü494.

The 1410 bp DNA region (glnA) encoding glutamine synthetase I (GSI) from Streptomyces viridochromogenes was amplified by PCR, cloned and sequenced. The molecular mass of the deduced GSI protein (469 residues) was determined to be 50 kDa. The DNA region showed 90% nucleotide identity with the Streptomyces coelicolor A3(2) glnA gene, but no significant nucleotide sequence similarity with the glnII (GSII) gene of S. viridochromogenes. The chromosomal glnA and glnII genes of S. viridochromogenes were disrupted by site-specific mutagenesis. Neither glnA nor glnII single mutants required glutamine for growth and both were normal in their sporulation. Measurement of the GS activity in cultures grown with different nitrogen sources revealed that GSI (heat-stable) and GSII (heat-labile) were always expressed together, with GSI as the predominant activity. It could be proposed that GSI, but not GSII is inactivated by adenylylation under conditions of nitrogen excess. GSI and GSII activities are inhibited by amino acids and by nucleotides.

Functional organization of the glnB-glnA cluster of Azospirillum brasilense.

The functional organization of the glnB-A cluster of Azospirillum brasilense, which codes for the PII protein and glutamine synthetase, respectively, was studied with the aid of lacZ fusions, deletion mapping, site-directed mutagenesis, and complementation. It was shown previously by mRNA mapping that the cluster contains two tandemly organized promoters, glnBp1 and glnBp2, of the sigma 70 and sigma 54 types, respectively, upstream of glnB and a third unidentified promoter upstream of glnA. Data obtained with lacZ fusions in the wild-type strain confirmed that cotranscription of glnBA and transcription of glnA alone were oppositely regulated by the cell N status. Quantification of promoter activities showed a high level of transcription from glnBp1p2 and a low level from glnAp under conditions of nitrogen limitation. The opposite situation prevails under conditions of nitrogen excess. As a consequence, PII polypeptide synthesis is increased under conditions of nitrogen fixation, which strongly suggests that PII plays an important role under these conditions. Null mutant strains of glnB, ntrB-ntrC, nifA, and point mutant strains in glnA were analyzed. NtrB and NtrC are not involved in the regulation of glnBA expression, in contrast to PII and glutamine synthetase. Glutamine synthetase probably acts by modulating the intracellular N status, and PII acts by modifying the properties of an unidentified regulator which might be a functional homolog of NtrC. In addition, a Nif- null mutant strain of glnB was characterized further. A Nif+ phenotype was restored to the strain by nifA from Klebsiella pneumoniae but not by nifA from A. brasilense. This mutant strain is not impaired in NifA synthesis, which is relatively independent of the growth conditions in A. brasilense. It is therefore most likely that PII is required for NifA activation under conditions of nitrogen fixation. Deletion mapping and site-directed mutagenesis showed glnAp was located within a 45-bp DNA fragment upstream of the mRNA start site, dissimiar to previously described consensus sites for sigma factors.

Characterization of Escherichia coli glnL mutations affecting nitrogen regulation.

Nitrogen regulator II (NRII), the product of the Escherichia coli glnL (ntrB) gene, regulates the activation of transcription of glnA and the Ntr regulon by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI. Previous results have indicated that under conditions of nitrogen excess, transcriptional activation is prevented by an NRI-phosphate phosphatase activity that is observed when NRII and another signal transduction protein known as PII (the glnB product) interact. The availability of PII for this interaction is controlled by a uridylytransferase/uridylyl-removing enzyme, encoded by glnD, that reversibly modifies PII in response to intracellular signals of nitrogen availability. Here we describe the isolation and characterization of missense mutations in glnL that suppress the Ntr- phenotype resulting from a leaky glnD mutation. The regulation of glnA expression in the pseudorevertants was found to vary from complete insensitivity to ammonia in some strains (GlnC phenotype) to nearly normal regulation by ammonia in other strains. Sequence analysis indicated that in 16 instances suppression was due to point mutations at 14 different sites; 10 different mutations resulting in a variety of phenotypes were identified in a cluster extending from codons 111 to 154 flanking the site of NRII autophosphorylation at His-139. Complementation experiments with multicopy plasmids encoding NRII or PII showed that suppression by GlnC glnL alleles was eliminated upon introduction of the plasmid encoding NRII but was not affected by introduction of the plasmid encoding PII. Conversely, suppression by certain glnL alleles that resulted in regulated expression of glnA was eliminated upon introduction of either the plasmid encoding NRII or that encoding PII. We hypothesize that mutants of the latter type result in a subtle perturbation of the NRII-PII interaction and suggest two possible mechanisms for their effects.

Ammonium transport in Escherichia coli: localization and nucleotide sequence of the amtA gene

Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess. We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation. In this study, a 3.4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3.4 kb fragment were generated by exonuclease III digestion. The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion. This allowed amtA to be localized within a 1.4 kb region which spans the site of the mutation. By application of the Sanger dideoxy method, we sequenced the region containing amtA. The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator. Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system.

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent Km values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 μM and 2.0±1.6 μM respectively, and the Vmax was 47±2.6 nmol min-1 (mg protein)-1 for MNF 2030 and 101±5.1 nmol min-1 (mg protein)-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.

PRODUCTION OF ACETONE AND BUTANOL BY CLOSTRIDIUM ACETOBUTYLICUM IN A PRODUCT LIMITED CHEMOSTAT

Abstract The solvents acetone and butanol are produced by Clostridium acetobutylicum during batch culture only after a considerable lag. The influence of limiting nutrients on the onset of solvent production has been investigated by studying the continuous fermentation under conditions of carbon, nitrogen and phosphate excess over a wide range of dilution rates. A maximum solvent concentration of 12 gl−1 was obtained at the lowest dilution rate investigated, namely 0.05 hr−1, and the average yield of solvents over the range of dilution rates approximated 0.3. These data confirm that a limitation of either of these nutrients is not essential for solvent production.

THE INFLUENCE OF ASSIMILABLE NITROGEN COMPOUNDS IN WORT ON THE ABILITY OF YEAST TO REDUCE DIMETHYL SULPHOXIDE

Dimethyl sulphoxide (DMSO) reductase activity increased at the end of the growth cycle of Saccharomyces cerevisiae NCYC 240 on complex medium. On defined medium, DMSO reductase and thioredoxin reductase activities and thioredoxin were all elevated in cultures limiting in nitrogen, but not in nitrogen-sufficient cultures. In nitrogen limiting cultures it was also shown that the reduction of DMSO (measured as disappearance of [14C] from the medium) was much increased compared with conditions of nitrogen excess. This applied whether ammonium or glutamate was the limiting nitrogen source. When methionine was used as the limiting nitrogen source, DMSO reduction was higher still. Methionine-limited cells accumulated [17C] methionine much more rapidly than did cells from methionine-sufficient cultures, and [14C] methionine sulphoxide could be identified in the cells after 1 min incubation with labelled methionine. It is concluded that DMSO reductase activity is controlled by nitrogen catabolite repression and may be involved in methionine uptake by yeast. The amino nitrogen content of wort will in consequence have an important effect on levels of dimethylsulphide (DMS) produced during fermentation.

Regulation of expression from the glnA promoter of Bacillus subtilis requires the glnA gene product.

Expression of the cloned glnA gene [coding for glutamine synthetase (EC 6.3.1.2)] of Bacillus subtilis was 10-fold higher in an Escherichia coli strain grown under nitrogen-limiting conditions than in the same strain under nitrogen-excess conditions. Mutations in the E. coli glnA, glnB, glnD, glnE, glnF, glnG, and glnL genes had no effect on the observed regulation. To test whether sequences within the B. subtilis DNA (3.2 kilobase pairs) were responsible for the observed regulation, a plasmid carrying a transcriptional fusion of the B. subtilis glnA promoter with E. coli lacZ was constructed. beta-Galactosidase levels coded for by this plasmid were found to be negatively regulated in trans by a plasmid carrying the entire B. subtilis glnA gene. Analysis of various deletion plasmids showed that the 1.4-kilobase-pair region encoding glutamine synthetase was necessary for the observed regulation of beta-galactosidase. Plasmids coding for 67% or more of the glutamine synthetase polypeptide gave at least partial repression, but a plasmid carrying 30% of the structural gene, as well as a plasmid carrying a deletion internal to glnA, gave no repression. DNA downstream from glnA (to within 130 base pairs of the end of the gene) was not required for the observed regulation. These results suggest that the glnA gene of B. subtilis is autoregulated, supporting the model for glnA control proposed by Dean et al. [Dean, D. R., Hoch, J. A. & Aronson, A. I. (1977) J. Bacteriol. 131, 981-987].

The Role of Adenylyltransferase and Uridylyltransferase in the Regulation of Glutamine Synthetase in Escherichia coli

The regulation of GS activity involves two nucleotidylation cycles, the uridylylation cycle of PII and the adenylylation cycle of GS, which are catalyzed by two converter enzymes, uridylyltransferase and adenylyltransferase, respectively. The converter enzymes sense the fluctuation in the availability of nitrogen and accordingly regulate the activity of GS. On the other hand, the posttranslational modification of GS is tightly coupled to the transcriptional regulation of the glnA gene by unmodified PII protein acting as a repressor in the GS synthesis. Therefore, metabolic signals perceived by uridylyltransferase are transmitted through PII to two different levels of the regulation, namely, the posttranslational level and transcriptional level. In order to study the converter enzymes which exist in extremely low concentration, the glnD and glnE genes were cloned into a plasmid vector carrying the strong, regulatable lambda phage promoter. In this way, uridylyltransferase and adenylyltransferase were overproduced to the levels approaching 800- and 500-fold, respectively. The recombinant DNA technology also enabled us to examine the transcriptional regulation of the glnD and glnE genes. The expression of these genes was slightly repressed under nitrogen-excess conditions and the repressions were more pronounced under excess nitrogen plus carbon-limiting conditions. It was found that variations of the concentration of uridylyltransferase and adenylyltransferase also affect the rate of GS synthesis. Studies with strains harboring a multicopy plasmid, pglnD or pglnE, indicate that the elevated synthesis of the converter enzymes causes the enhancement of GS synthesis. In addition, the absence of one of the converter enzymes reduces the expression of the glnA gene. The parallel relationship between the converter enzymes and GS seems to derive from the binding capacity of the converter enzymes for the unbound PII, which is a repressor for the glnA gene. Therefore, it is believed that the metabolic regulation of the glnD and glnE genes is ultimately linked to the expression of the glnA operon.