A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli.

Pahel, G; Tyler, Bonnie

doi:10.1073/pnas.76.9.4544

Cited by 134 publications

(131 citation statements)

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“…These insertion strains are found in classes 1 and 2 and thus represent at least two different genes and perhaps more. One of these may be gltB, the gene coding for glutamate synthase, since strains with mutations in gltB were originally described as being unable to use L-arginine, proline, or glycine as a nitrogen source (19).…”

Section: Resultsmentioning

confidence: 99%

Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine

1992

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The leucine regulon coordinates the expression of several Escherichia coli genes according to the presence of exogenous leucine, which interacts with the bp gene product, Lrp. We isolated and characterized 22 strains with k placMu insertions in Lrp-regulated genes. Lrp and leucine influenced gene expression in a surprising variety of ways. We identified two genes that are regulated by Lrp and not affected by L-leucine. We therefore rename this the leucine-fp regulon. Genes coding for glycine cleavage and leucine biosynthesis enzymes have been identified as members of the leucine4rp regulon. We suggest that the brp gene product activates genes needed for growth in minimal medium, and we show that the gene is repressed by its own product and is highly repressed during growth in rich medium.We recently showed that in Escherichia coli the branchedchain amino acid L-leucine is the effector of a global response that governs a group of genes known as the leucine regulon (11). Regulation of the synthesis of a number of enzymes from different metabolic pathways is affected by the presence of L-leucine in the growth medium (11,16). This response to exogenous L-leucine was altered in an E. coli mutant carrying an insertion in a gene at 20 min (11). This gene, referred to originally as rbl, was renamed lip by consensus among interested investigators (11,20).The lip gene codes for a 38-kDa dimer, the leucineresponsive regulatory protein (Lrp), which has been purified extensively (31). Under the name of the ilvIH binding protein, it was shown to bind to two sites upstream of ilvIH (22), for which it is a positive regulator. A mutation in lrp made the strain constitutive for ilvIH expression (20) and changed only one nucleotide, resulting in a substitution of Glu for Asp. The lip gene was shown to be identical to oppI (2) and is also thought to be identical to livR (12,20).Until now, the genes of the leucine regulon have been identified by guesswork or by the accident of a previously known regulatory gene proving to be identical to lip. As a result of a somewhat different accident, lysU has now been added to the regulon. We present here the results of a screening among random X placMu9 insertions for leucineand Lrp-regulated expression of lacZ. We used a system based on that used for the isolation of din genes (8). We also show that some genes are regulated by Lrp without leucine, and therefore we rename the regulon as the leucine-Lrp regulon. MATERIALS AND METHODSCultures. The strains used in this study-all derivatives of E. coli K-12-are described in Table 1. The plasmids used are also listed in Table 1. Cultures were grown as described previously (11) with L-isoleucine and L-valine (each at 50 ,g/ml) added to the minimal medium used to grow strain CU1008 and all its derivatives to compensate for the ilvA mutation carried by these strains. Other genetic techniques. Plasmid isolations, DNA manipulations, transductions, and transformations were performed as described previously (13, 14).Enzyme assays. L-Serine deaminase was as...

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Section: Resultsmentioning

confidence: 99%

Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine

1992

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“…2 shows that the loss of PL drastically lowers the rate of deadenylylation, and somewhat impairs the rate of adenylylation. It has been shown that the product of the glnF gene is required for the synthesis of glutamine synthetase (14,17,18). One model suggests that this product converts the product of another gene, ginG, from a repressor to an activator of glutamine synthetase formation (14,18,19).…”

Section: Resultsmentioning

confidence: 99%

“…It has been shown that the product of the glnF gene is required for the synthesis of glutamine synthetase (14,17,18). One model suggests that this product converts the product of another gene, ginG, from a repressor to an activator of glutamine synthetase formation (14,18,19). The fact that glnF mutants lacking PII due to a mutation in glnB fail to produce glutamine synthetase indicates that the product of the ginG gene, even in the absence of PIIA, is fully capable of repressing glutamine synthetase (14).…”

Section: Resultsmentioning

confidence: 99%

Regulation of the synthesis of glutamine synthetase by the PII protein in Klebsiella aerogenes.

Foor¹,

Reuveny²,

Magasanik³

1980

Proc. Natl. Acad. Sci. U.S.A.

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Certain mutations at the glnB 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 P11 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 gInB site result in the loss of P11. The loss of PI, 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. The biosynthetic activity of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] in enteric bacteria is progressively decreased by the specific covalent attachment of adenylyl groups to each of its 12 subunits (1). This reversible modification is catalyzed by the enzyme adenylyltransferase (ATase). Adenylylation is stimulated by the small (44,000 dalton) regulatory protein PITA (2). Deadenylylation is stimulated by PIID, a uridylylated form of PIIA. The interconversion of PIIA and PIID is catalyzed by uridylyltransferase (UTase) and uridylyl-removing enzyme(s).Glutamine stimulates the adenylylation reaction as well as the conversion of PID to PITA; 2-ketoglutarate inhibits adenylylation but stimulates the deadenylylation reaction and the conversion of PITA to PIID. Glutamine inhibits the latter two reactions. Thus, the relative amounts of PITA and PIID and the adenylylation state of glutamine synthetase are determined in a reciprocal manner by the concentrations of these two metabolites; these in turn are determined by the availability of nitrogen in the cell. When growth is limited by the nitrogen source, the level of 2-ketoglutarate is high, the level of glutamine is low, and the adenylylation state of glutamine synthetase is low. When growth is limited by some other component, such as carbon, glutamine is high, 2-ketoglutarate is low, and the adenylylation state of glutamine synthetase is high (3). In addition, the synthesis of glutamine synthetase is regulated by the availability of nitrogen. During nitrogen-limited growth the level of glutamine synthetase is high, whereas during carbonlimited growth the level of glutamine synthetase is low (3,4).We have studied the regulation of the level and the adenyl- MATERIALS AND METHODS Bacterial Strains. All strains are phage Pl-sensitive derivatives (5) of K. aerogenes MK-1 (6). Media have been described (7).Genetic Techniques. Generalized transduction was carried out as described by Goldberg et al. (5) with phage P1clrLO0CM (obtained from B. Tyler). Lysates of donor st...

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“…Bacterial strains and plasmids used in this work are listed in Table 1. Defined W-salts-based media and rich media were as described previously (32). Transformation of CaCl2-treated cells with plasmid DNA and transduction using Plvir were as described previously (23,38).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Escherichia coli glnL mutations affecting nitrogen regulation

Atkinson

Ninfa

1992

J Bacteriol

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A signal transduction pathway consisting of at least four gene products is responsible for the regulation of g1nA, encoding glutamine synthetase (GS), and the Ntr regulon in Escherichia coli and similar bacteria ( Fig. 1) (reviewed in references 21, 22, and 41). The activation of transcription of glnA and the Ntr regulon in response to nitrogen starvation requires the phosphorylated form of the ginG (ntrC) gene product, nitrogen regulator I (NRI), and this phosphorylation is in turn catalyzed by the protein kinase activity of the ginL (ntrB) gene product, NRI, (17,28,44). NRI-phosphate stimulates the initiation of transcription from specialized promoters by RNA polymerase containing the alternative sigma factor r54 (15,16,28). The sensitivity of any given Ntr promoter to activation by NR,-phosphate is determined at least in part by the number, sequence, and position of NRI-P-binding sites, which are functionally analogous to enhancer sequences (27,30,33,35). The glnAp2 promoter, endowed with two high-affinity NR1-binding sites ideally positioned 110 and 140 bp upstream from the site of transcript initiation, is exquisitely sensitive to activation by NR,-P, while other Ntr promoters, containing only a single NRI-P-binding site or multiple sites whose position is less than ideal, are less sensitive to activation by NRI-P (8,14,34,45; reviewed in references 18 and 41). These differences in promoter architecture probably contribute to the temporal staging of the cellular response to a shift from nitrogen excess to nitrogen-limiting conditions (discussed in reference 41).Under conditions of nitrogen excess, the transcriptional activation of glnA and the Ntr regulon is prevented by a phosphatase activity that results from the interaction of NR11 * Corresponding author. and the product of the glnB gene, known as PI1 (6,17,28).The activity of PII is in turn regulated by its reversible uridylylation catalyzed by the product of ginD, a uridylyltransferase/uridylyl-removing enzyme (UT/UR) (1, 5, 9, 12, 13). Under conditions of nitrogen excess, PI, is kept in the unmodified form, and upon nitrogen starvation PI, is converted to the uridylylated form by the UT/UR. The most important small molecules sensed by the UT/UR seem to be glutamine, which stimulates the UR activity, and 2-ketoglutarate, which stimulates the UT activity (9, 12, 13). Thus, the UT/UR enzyme serves to continuously monitor the intracellular ratio of glutamine to 2-ketoglutarate, a measure of nitrogen status, and communicates this information to PI,.PI, is a central element of nitrogen regulation in bacteria:it interacts with the kinase-phosphatase NRI to elicit the phosphatase activity and thus control transcription of nitrogen-regulated genes, and it also interacts with an adenylyltransferase enzyme (ATase)

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A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli.

Abstract: Mutations in the ginA region of the Escherichia coli chromosome due to Mu prophage insertion result in two phenotypic classes. One class is G1n and does not synthesize glutamine synthetase IL-glutamate:ammonia ligase (ADPforming), EC 6.3

Cited by 134 publications

References 13 publications

Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine

Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine

Regulation of the synthesis of glutamine synthetase by the PII protein in Klebsiella aerogenes.

Characterization of Escherichia coli glnL mutations affecting nitrogen regulation

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