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We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated glnA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.

It has previously been shown that phosphorylated nitrogen regulator I (NRI-phosphate) is the activator responsible for increasing the transcription of glnA, the structural gene for glutamine synthetase, and that NRII catalyzes the transfer of the gamma-phosphate of ATP to NRI. We have now shown that the reaction of ATP with NRII results in the reversible transfer of the gamma-phosphate of ATP to a histidine residue of NRII. In turn, NRII-phosphate transfers its phosphate reversibly to an aspartic residue of NRI. NRI-phosphate is hydrolyzed to NRI and inorganic phosphate in a divalent cation-requiring autocatalytic reaction.

Transcription from nitrogen-regulated promoters, such as glnAp2, requires the glnG gene product, NRI, as well as the rpoN(glnF) gene product, sigma60, and is regulated by the glnL gene product, NRII. We find that in a reaction mixture containing NRI, NRII, and ATP, NRII catalyzes the transfer of the gamma phosphate of ATP to NRI. This covalent modification of NRI occurs concurrently with the acquisition of the ability by the reaction mixture to activate transcription from glnAp2. In the presence of PII, the product of glnB, NRII catalyzes the removal of the phosphate from NRI-phosphate. This reaction occurs concurrently with the loss by the reaction mixture of the ability to activate transcription from glnAp2. On the basis of this evidence, we propose that NRI-phosphate activates transcription from nitrogen-regulated promoters and that the role of NRII is control of the formation and breakdown of NRI-phosphate in response to cellular signals of nitrogen availability.

The degradation of agmatine to succinate by Klebsiella aerogenes occurs in five steps. The enzyme catalyzing the first step, agmatinase, is induced by agmatine. The enzymes catalyzing the second and third steps, putrescine aminotransferase and 4-aminobutyraldehyde dehydrogenase, are induced by putrescine and also by their product, 4-aminobutyrate. The enzymes catalyzing the fourth and fifth steps, 4-aminobutyrate aminotransferase and succinate semialdehyde dehydrogenase, are induced by 4-aminobutyrate. This compound also serves as gratuitous inducer of the catabolic acetylornithine aminotransferase. The formation of the enzymes responsible for agmatine degradation is regulated not only by induction, but also by catabolite repression and activation by glutamine synthetase.

We isolated an F' episome of Escherichia coli carrying the glnA+ gene from K. aerogenes and an F' episome of E. coli carrying the glnA4 allele from K. aerogenes responsible for the constitutive synthesis of glutamine synthetase. Complementation tests with these episomes showed that the glnA4 mutation (leading to the constitutive synthesis of active glutamine synthetase) was in the gene identified by mutations glnA20, glnA51, and glnA5 as the structural gene for glutamine synthetase. By using these merodiploid strains we were able to show that the glnA51 mutation lead to the synthesis of a glutamine synthetase that lacked enzymatic activity but fully retained its regulatory properties. Finally, we discuss a model that explains the several phenotypes associated with mutations such as glnA4 located within the structural gene for glutamine synthetase leading to constitutive synthesis of active glutamine synthetase.

Glutamine synthetase could be repressed several hundredfold rather than 6- to 10-fold as previously reported. Ammonia was not the primary repression signal for glutamine synthetase. Repression appeared to be mediated by a high level of glutamine and probably by a high ratio of glutamine to alpha-ketoglutarate. Mutations in glnA (the structural gene for glutamine synthetase) were seen to fall into three phenotypic groups: glutamine auxotrophs that produced no detectable glnA product; glutamine auxotrophs that produced a glnA product lacking enzymatic activity (and hence repressibility by ammonia) but were repressible under appropriate conditions; and glutamine synthetase regulatory mutants, whose glnA product was enzymatically active and not repressible under any conditions.

Urease was purified 24-fold from extracts of Klebsiella aerogenes. The enzyme has a molecular weight of 230,000 as determined by gel filtration, is highly substrate specific, and has a Km for urea of 0.7 mM. A mutant strain lacking urease was isolated; it failed to grow with urea as the sole source of nitrogen but did grow on media containing other nitrogen sources such as ammonia, histidine, or arginine. Urease was present at a high level when the cells were starved for nitrogen; its synthesis was repressed when the external ammonia concentration was high. Formation of urease did not require induction by urea and was not subject to catabolite repression. Its synthesis was controlled by glutamine synthetase. Mutants lacking glutamine synthetase failed to produce urease, and mutants forming glutamine synthetase at a high constitutive level also formed urease constitutively. Thus, the formation of urease is regulated like that of other enzymes of K. aerogenes capable of supplying the cell with ammonia or glutamate.

We have isolated a temperature-sensitive mutant of Klebsiella aerogenes unable to grow aerobically at 42 C in standard glucose minimal medium containing 0.03 M ammonium sulfate as a source of nitrogen. This strain, MK810, will grow at this temperature in significantly lower concentrations of ammonia (1 mM) or when ammonia is replaced by a growth rate-limiting source of nitrogen such as histidine or glutamate. A detailed physiological characterization and preliminary biochemical tests support the contention that the mutant has an altered alpha-ketoglutarate dehydrogenase that at the restrictive condition fails to manufacture sufficient succinyl-coenzyme A. We explain the ammonia sensitivity by the dual role of alpha-ketoglutarate as substrate for the formation of succinyl-coenzyme A and glutamate. A defect in the enzyme necessary for the production of succinyl-coenzyme A makes ammonia an overly effective competitor for alpha-ketoglutarate.

Two super-repressor mutations in the histidine utilization (hut) operons of Salmonella typhimurium are described. Cells bearing either of these mutations have levels of hut enzymes that do not increase above the uninduced levels when growth is in the presence of either histidine or the gratuitous inducer imidazole propionate. Both mutations lie in the region of the gene for the hut repressor, hutC, and reverse mutations of both are to the constitutive (repressor-negative) rather than to the inducible (wild type) phenotype. In hybrid merodiploid strains the super-repressor mutations are dominant over either wild-type (hutC+) or repressor-negative (hutC-) alleles. Whereas both super-repressor mutations cause the uninducible synthesis of hut enzymes, the degree of repression is different. One mutation causes repression of enzyme synthesis in one of the two hut operons to a level below the basal, uninduced level of wild-type cells. The other mutation causes repression to a lesser degree than in wild-type cells, so that the hut enzymes are present at a level above the normal basal level; this partially constitutive synthesis is greater for the enzymes of one of the hut operons than for the enzymes of the other. Thus, both mutations apparently result in repressors with altered operator-binding properties, in addition to altered inducer-binding properties.

Mutations at two sites, glnA and glnB, of the Klebsiella aerogenes chromosome result in the loss of glutamine synthetase. The locations of these sites on the chromosome were established by complementation by episomes of Escherichia coli and by determination of their linkage to other genetic sites by transduction with phage P1. The glnB gene is located at a position corresponding to 48 min on the Taylor map of the E. coli chromosome; it is linked to tryA, nadB, and GUA. The glnA gene is at a position corresponding to 77 min on the Taylor map and is linked to rha and metB; it is also closely linked to rbs, located in E. coli at 74 min, indicating a difference in this chromosomal region between E. coli and K. aerogenes. Mutations in the glnA site can also lead to nonrepressible synthesis of active glutamine synthetase. The examination of the fine genetic structure of glnA revealed that one such mutation is located between two mutations leading to the loss of enzymatic activity. This result, together with evidence that the structural gene for glutamine synthetase is at glnA, suggests that glutamine synthetase controls expression of its own structural gene by repression.

Mutations at two sites of the Klebsiella aerogenes chromosome, unlinked by transduction with phages PW52 and P1, result in the lack of enzymatically active glutamine synthetase. A mutation in the glnB site leads to a marked decrease in the formation of an apparently normal enzyme. Some of the mutations in the glnA site lead to the production of enzymatically inactive material capable of reacting with anti-glutamine synthetase serum. The revertant of a glnA mutant was found to produce a glutamine synthetase with less activity and less stability to heat than the enzyme of the wild type. These results locate the structural gene to the production of enzymatically inactive glutamine synthetase antigen, not subject to repression by exogenously added ammonia. This observation suggests that glutamine synthetase is itself involved in the regulation of the synthesis of glutamine synthetase.

One of the suspected regulators of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] in enteric bacteria is glutamine synthetase itself. We isolated Escherichia coli strains carrying fusions of the beta-galactosidase structural gene to the promoter of the glutamine synthetase gene, with the aid of the Casadaban Mud1 (ApR, lac, cts62) phage. Some aspects of regulation were retained in haploid fusion strains despite the absence of glutamine synthetase, whereas other aspects required glutamine synthetase catalytic or regulatory activity or both. The direction of transcription of the glutamine synthetase gene was also determined.

Mutants of Klebsiella aerogenes containing genetic fusions of glnA to lacZ were isolated by using Mu dl (lac, bla) bacteriophage and a Mu Kmr helper phage with the host range of bacteriophage P1. Synthesis of beta-galactosidase in these strains is regulated in response to nitrogen metabolites and regulatory gln loci and is rendered constitutive by a mutation in the linked glnL gene. Complementation studies indicated that glnL is a separate locus from glnA and glnG and that insertions in glnA are partially polar on glnL expression. These results support the hypothesis that glnA, glnL, and glnG are organized in an operon with multiple promoters.

We isolated mutants of Klebsiella aerogenes with insertions of transposon Tn5 linked to the structural gene for glutamine synthetase, glnA. We found that K. aerogenes, like Escherichia coli and Salmonella typhimurium, contains a regulatory gene, glnG, which is closely linked to but distinct from glnA. The product of glnG is essential for normal regulation of the synthesis of glutamine synthetase and histidase. We analyzed two mutations which affected the regulation of the formation of these enzymes and appeared to be outside glnG. The results of our studies suggest the presence in K. aerogenes of at least two regulatory genes located in the glnA region, namely, glnG and another gene, the transcription of which can be initiated at the promoter of glnA.

The glnG gene product is both a positive regulator and a negative regulator of the expression of glnA, the structural gene for glutamine synthetase, as well as a positive regulator of the expression of a number of genes whose products are involved in the uptake and degradation of nitrogen-containing compounds. The regulation of beta-galactosidase in various strains containing a Mu d1 (lac bla) insertion within glnG leads to the following conclusions regarding the expression of this gene: (i) like the synthesis of glutamine synthetase, the synthesis of the glnG product is regulated in response to the nitrogen source; (ii) high-level expression of glnG under nitrogen-limiting growth conditions depends on transcription initiated at the glnA promoter; and (iii) there is a second, glnA-distal promoter for glnG, whose activity is negatively controlled by the glnG product. Thus, the glnG product regulates the synthesis of the glnG product at two distinct promoters (positively and negatively at the glnA promoter and negatively at the glnA-distal promoter). In addition, a high level of glnG product, corresponding to the level produced by initiation of transcription at the glnA promoter under nitrogen-limiting conditions, is necessary for activation of histidase synthesis. The lower level of glnG product originating from transcription initiated at the glnA-distal promoter is not sufficient to activate histidase synthesis, but is sufficient to activate fully and to repress glnA expression.

DNA was prepared from a strain of Escherichia coli bearing a mutation which confers the GlnC phenotype (inability to reduce the expression of glnA and other nitrogen-regulated operons in response to ammonia in the growth medium). A fragment of this DNA carrying glnA, the structural gene for glutamine synthetase, was cloned on plasmid pBR322. By using recombination in vitro, we mapped the GlnC mutation to a region between glnA and glnG. This region defines a gene, glnL, which codes for a trans-acting product; the GlnC mutant produces an altered product. The glnL product plays a key role in the communication of information concerning the quality and abundance of the nitrogen source in the growth medium to a destination responsible for the regulation of glnA and other genes for enzymes responsible for nitrogen utilization.

Results of studies on proline-nonutilizing (Put-) mutants of the yeast Saccharomyces cerevisiae indicate that proline is an essential intermediate in the degradation of arginine. Put- mutants excreted proline when grown on arginine or ornithine as the sole nitrogen source. Yeast cells contained a single enzyme, delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase, which is essential for the complete degradation of both proline and arginine. The sole inducer of this enzyme was found to be proline. P5C dehydrogenase converted P5C to glutamate, but only when the P5C was derived directly from proline. When the P5C was derived from ornithine, it was first converted to proline by the enzyme P5C reductase. Proline was then converted back to P5C and finally to glutamate by the Put enzymes proline oxidase and P5C dehydrogenase.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.

We sequenced the 274-nucleotide intercistronic glnA-glnL region of Escherichia coli to localize regulatory regions postulated from genetic evidence. The transcriptional start of glnLp, identified by S1 nuclease mapping, preceded the structural gene by 32 bases. NR1, the glnG gene product and a repressor of glnLp, protected from DNase digestion a region of DNA between -12 and +15 from the transcriptional start. A mutation rendering glnLp insensitive to NRI was within the protected region in a-TGCA- sequence found in all nitrogen-regulated operons, providing evidence for involvement of this sequence in interactions with NRI. We also observed in the intercistronic region a potential rho-independent terminator preceding glnLp and a sequence previously found in other intercistronic regions.

Certain mutations at the glaB locus result in the failure to fully derepress glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] and to convert it to the active nonadenylylated form in response to nitrogen limitation. In these mutants the PII regulatory protein is altered such that it cannot be converted by uridylyltransferase to the form stimulating deadenylylation of glutamine synthetase by adenylyltransferase. Additional mutations as well as insertions of transposon Tn5 at the glnB site result in the loss of PII. The loss of PII does not prevent adenylylation and deadenylylation of glutamine synthetase but reduces the rates of these reactions. Cells lacking PII have a high level of glutamine synthetase even when they are grown with an excess of ammonia and the enzyme is highly adenylylated. The results suggest that the PII protein plays a role, independent of its effect on adenylylation, in the regulation of the level of glutamine synthetase.

Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1. The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression. Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis. The response of all four proteins is blocked by gln3 mutations. In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants. Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia. We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.

Production of glutamine synthetase in Saccharomyces cerevisiae is controlled by three regulatory systems. One system responds to glutamine levels and depends on the positively acting GLN3 product. This system mediates derepression of glutamine synthetase in response to pyrimidine limitation as well, but genetic evidence argues that this is an indirect effect of depletion of the glutamine pool. The second system is general amino acid control, which couples derepression of a variety of biosynthetic enzymes to starvation for many single amino acids. This system operates through the positive regulatory element GCN4. Expression of histidinol dehydrogenase, which is under general control, is not stimulated by glutamine limitation. A third system responds to purine limitation. No specific regulatory element has been identified, but depression of glutamine synthetase is observed during purine starvation in gln3 gcn4 double mutants. This demonstrates that a separate purine regulatory element must exist. Pulse-labeling and immunoprecipitation experiments indicate that all three systems control glutamine synthetase at the level of subunit synthesis.

We have examined three mutants of Klebsiella aerogenes whose genetic lesions (glnB, glnD, and glnE) are in loci unlinked to the structural gene for glutamine sythetase (glnA) and in which the control of both the level and state of adenylylation of glutamine synthetase is altered. Each mutation alters a different component of the adenylylation system of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. Inability of the cell to deadenylylate glutamine synthetase (glnB and glnD) greatly decreases its production, while inability to adenylylate glutamine sythetase (glnE) results in its constitutively high production. These results together with our previous results indicate that adenylylated glutamine synthetase inhibits the transcription of glnA.

We have determined that the glnA gene of the complex glnALG operon of Escherichia coli is transcribed from tandem promoters. Expression from the upstream promoter, glnAp1, requires the catabolite activating protein, is repressed by nitrogen regulator I (NRI), the product of glnG, and produces a transcript with an untranslated leader of 187 nucleotides. Expression from the downstream promoter, glnAp2, requires NRI as well as the glnF product; full expression also requires growth in a nitrogen-limited environment. The downstream transcript has an untranslated leader of 73 nucleotides. We also provide evidence that the function of the glnL product is to mediate the interconversion of NRI between a form capable of activating glnAp2 and an inactive form in response to changes in the intracellular concentration of ammonia. The function of the two minor promoters of the glnALG operon, glnAp1 and glnLp, is to maintain the products of glnA, glutamine synthetase, an essential enzyme, and of glnG, NRI, an activator of nitrogen-controlled genes, during carbon-limited growth.

We have shown that the purified glnF (ntrA) product of Escherichia coli binds to core RNA polymerase. Together these proteins initiated transcription at the nitrogen-regulated promoter glnAp2 on a supercoiled template. The initiation of transcription at glnAp2 on a linear template required in addition NRI, the product of glnG (ntrC), and NRII2302, the product of a mutant allele of glnL (ntrB). These results identify the glnF product as a new sigma factor specifically required for the transcription of nitrogen-regulated and of nitrogen-fixation promoters. We propose rpoN as the proper designation for glnF, and sigma 60 for its product. Our results indicate that sigma 60 RNA polymerase recognizes the nitrogen-regulated/nitrogen-fixation promoter consensus sequence C-T-G-G-Y-A-Y-R-N4-T-T-G-C-A. Initiation of transcription in the intact cell appears to require in addition the active form of NRI, the product of glnG. Conversion of NRI to its active form is apparently brought about by NRII, the product of glnL, in response to nitrogen deprivation.

We have cloned and characterized a fragment of the Escherichia coli chromosome spanning glnA, the structural gene for glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The fragment also carriers glnG, whose product is necessary for regulation of glnA expression, and a previously unidentified gene whose function we have not discovered. Transcription of glnA and the newly identified gene occurs divergently from a region between the two genes. Transcription of glnA proceeds toward glnG, which is transcribed in the same direction. A region of DNA between glnA and glnG contains genetic information whose loss may result in the inability to reduce expression of glnA and other operons in response to ammonia (the GlnC phenotype).