An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli

The P_II protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that P_II, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB , which encodes a homologue of P_II and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as P_II, reacted with polyclonal P_II antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli P_II. Like P_II, purified GlnK can activate the adenylylation of glutamine synthetase in vitro , and, in vivo , the GlnK protein is uridylylated in a glnD -dependent fashion. Unlike P_II, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a σ^N (σ⁵⁴) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on P_II. Possible regulatory implications of this conditional redundancy of P_II are discussed.     

Modification of the pII protein in response to carbon and nitrogen availability in filamentous heterocystous cyanobacteria

Abstract In the filamentous cyanobacterium Calothrix PCC 7504, which fixes N₂ aerobically, the modification state of the regulatory P_II protein (GlnB) was shown to depend on nitrogen and carbon availability, as observed in the unicellular non-fixing strain Synechococcus PCC 7942. However, the conditions for modifications, the time dependence of the process and the electrophoretic behavior of the native P_II isoforms differed somewhat between the two strains. In another strain, Calothrix PCC 7601, which has lost the capability to fix N₂, P_II was modified only if ammonia plus an inhibitor of glutamine synthetase were present. It is proposed that: (i) the behavior of the P_II proteins depends upon the physiological properties of the strains; and (ii) the modification system of P_II per se may differ between the two cyanobacterial genera.     

Dephosphorylation of the phosphoprotein PII in Synechococcus PCC 7942: identification of an ATP and 2-oxoglutarate-regulated phosphatase activity

The phosphorylation state of the putative signal transduction protein P_II from the cyanobacterium Synechococcus sp. strain PCC 7942 depends on the cellular state of nitrogen and carbon assimilation. In this study, dephosphorylation of phosphorylated P_II protein (P_II-P) was investigated both in vivo and in vitro . The in vivo studies implied that P_II-P dephosphorylation is regulated by inhibitory metabolites involved in the glutamine synthetase–glutamate synthase pathway of ammonium assimilation. An in vitro assay for P_II-P dephosphorylation was established that revealed a Mg²⁺-dependent P_II-P phosphatase activity. P_II-P phosphatase and P_II kinase activities could be separated biochemically. A partially purified P_II-P phosphatase preparation also catalysed the dephosphorylation of phosphoserine/phosphothreonine residues on other proteins in a Mg²⁺-dependent manner. However, only dephosphorylation of P_II-P was regulated by synergistic inhibition by ATP and 2-oxoglutarate. As the same metabolites stimulate the P_II kinase activity, it appears that the phosphorylation state of P_II is determined by ATP and 2-oxoglutarate-dependent reciprocal reactivity of P_II towards its phosphatase and kinase.     

Cloning, heterologous expression, and sequencing of the Proteus vulgaris glnAntrBC operon and implications of nitrogen control on heterologous urease expression

Abstract The glnAntrBC operon of Proteus vulgaris was cloned and heterologously expressed in Escherichia coli . The nucleotide sequence was determined. An open reading frame of 1407 bp was identified as the glnA gene and the deduced amino acid sequence showed 82% identity with the E. coli glutamine synthetase protein. Heterologous expression of the glnA gene in E. coli restored glutamine synthetase (GS) activity in a GS-negative mutant and a 52 kDa protein was detected and addressed as the GS subunit of P. vulgaris . Adjacent to the glnA gene the regulatory genes ntrB and ntrC were identified. Their coding regions comprised 1053 and 1452 bp, respectively, and the deduced gene products NR_II (NtrB) and NR_I (NtrC) shared 72% identity with the corresponding E. coli proteins. Heterologous expression in E. coli revealed only a 54 kDa protein which was shown to be NR_I. NR_II was not detectable using the methods employed.     

The glnB gene of Rhizobium leguminosarum biovar viceae

Abstract An open-reading frame (ORF111) upstream of the glutamine synthetase I structural gene ( glnA ) in Rhizobium leguminosarum biovar viceae encodes a protein which is highly homologous to the P_II protein (encoded by glnB ) of enteric bacteria. ORF111 was cloned in a number of different plasmid vectors and shown to complement a K. pneumoniae glnB mutant. We propose that ORF111 encodes the P_II protein of R. leguminosarum and that it should be designated glnB .     

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge

This communication presents a short outline of the current knowledge on the molecular basis of P_II signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P_II signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P_II signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P_II phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P_II (P_II-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P_II-P dephosphorylation, novel insights into the signals, to which P_II-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P_II-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P_II-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P_II-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum.

Rhizobium leguminosarum, biovar viceae, strain RCC1001 contains two glutamine synthetase activities, GSI and GSII. We report here the identification of glnA, the structural gene for GSI. A 2 kb fragment of DNA was shown to complement the Gln- phenotype of Klebsiella pneumoniae glnA mutant strains. DNA sequence analysis revealed an open reading frame (ORF) of 469 codons specifying a polypeptide of 52,040 daltons. Its deduced amino acid sequence was found to be highly homologous to other glutamine synthetase sequences. This ORF was expressed in Escherichia coli minicells and the corresponding polypeptide reacted with an antiserum raised against GSI. Upstream of glnA we found an ORF of 111 codons (ORF111) preceded by the consensus sequence for an ntrA-dependent promoter. Minicells experiments showed a protein band, with a molecular weight in good agreement with that (10,469) deduced from the nucleotide sequence. On the basis of homology studies we discuss the possibility that the product of ORF111 is equivalent to the PII protein of E. coli and plays a similar role in regulation of nitrogen metabolism.     

The nucleotide sequence of the promoter region of hisS, the structural gene for histidyl-tRNA synthetase

A plasmid has been constructed which carries hisS, the structural gene for histidyl-RNA synthetase of E. coli, on a 1.6-kb fragment bounded by PvuII and BstEII sites. The DNA sequence of both ends of this fragment was determined. The amino-terminal sequence of histidyl-tRNA synthetase was also determined to locate the promoter proximal coding region and the frame in which it is read. Three promoters were identified by consensus criteria. The region surrounding these promoters contains extensive twofold symmetry.     

Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence

Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia. A 1922-base pair fragment from a cDNA containing the glnA structural gene for E. coli glutamine synthetase has been sequenced. An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814. With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously. The secondary structure predicted for the E. coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data. Comparison of the amino acid sequences of glutamine synthetase from E. coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N. E., Robinson, S. T., and Haselkorn, R. (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology. Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.     

Regulation of nitrogenase activity in Anabaena variabilis by modification of the Fe protein

The glutamine synthetase (GS) gene from Bacillus subtilis PCI 219 was cloned in Escherichia coli using the vector pBR329. A plasmid, pSGS2, was isolated from a glnA+ transformant and the cloned GS gene was found to be located in a 3.6 kb DNA fragment. The nucleotide sequence of a 1.8 kb segment encoding the GS was determined. This segment showed an open reading frame which would encode a polypeptide of 444 amino acids. The amino acid sequence of this GS gene product has higher homology with that of the Clostridium acetobutylicum GS than that of the E. coli GS.     

Glutamine synthetase isoforms in leaves of a C4 plant: Sorghum vulgare

In Sorghum vulgare L. (cv. INRA 450) leaves, two isoforms of glutamine synthetase GS₁ and GS₂ were identified by DEAE Sephacel chromatography and polyac-rylamide gel electrophoresis. GS₁ was present both in etiolated and green leaves and its activity remained constant during the greening process. In green leaves another isoform GS₂ was identified that was responsible for a 33% increase in total glutamine synthetase activity after light exposure. The two isoforms differed in their heat stability but exhibited similar pH optima and Km values for L-glutamate. The total glutamine synthetase activity was equally distributed between mesophyll and bundle sheath cells; moreover in the two types of cells both GS₁ and GS₂ were present in the proportions of 67% and 33% of the total respectively. In mesophyll cells the two isoforms had a different subcellular distribution: GS₂ was localized in the chloroplast and GS₁ in the cytosol.     

The ntr genes of Escherichia coli activate the hut and nif operons of Klebsiella pneumoniae

Regulation of the synthesis of glutamine synthetase and of the arginine and glutamine transport systems (Ntr phenotype) in Salmonella have been shown to require two regulatory genes on the C-terminal side of the glnA gene (McFarland et al., 1981). We have cloned a HindIII-EcoRI DNA fragment from Escherichia coli coding for analogous properties with respect to the Ntr phenotype in E. coli. A plasmid containing this E. coli DNA fragment joined to another fragment carrying a cyanobacterial glnA gene (but no functional regulatory genes) was introduced into a Klebsiella pneumoniae mutant with a Gln-Ntr- phenotype, i.e., which could not derepress nitrogenase. The cyanobacterial gene made the Klebsiella strain Gln+ and the E. coli DNA fragment made the strain Ntr+, including the ability to derepress nitrogenase fully. Thus the products of the glnA-linked ntr genes of E. coli can regulate expression of the Ntr-dependent genes of Klebsiella.     

Thioredoxin and related proteins in procaryotes

Abstract Thioredoxin is a small ( M _r 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S₂) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)₂] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)₂ serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)₂ is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S₂ by light and ferrodoxin; Trx-(SH)₂ is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control.
Thioredoxin-negative mutants ( trxA ) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.     

Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli.

The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene.     

The activation of glutamine synthetase from the cyanobacterium Anabaena cylindrica by thioredoxin

Abstract The present communication defines the conditions under which thioredoxin activates glutamine synthetase from Anabaena cylindrica . Effects are obtained at pH values around neutrality, and the activation is affected by Mg²⁺ in the assays. The thioredoxin systems from A. cylindrica and spinach are functionally interchangeable in the activation of glutamine synthetase. The enzyme is efficiently activated by thioredoxin_m and also by thioredoxin_f, but at much higher concentrations. Thioredoxin_m has previously been shown to activate NADPH-dependent malate dehydrogenase and isocitrate dehydrogenase from cyanobacteria. It is speculated that thioredoxin_m plays a role in the differentiation of vegetative cells to heterocysts.     

Cloning and expression in Escherichia coli of a species-specific Chlamydia trachomatis outer membrane antigen

Abstract A gene library of Chlamydia trachomatis serovar L₂ (strain 434) was constructed in Escherichia coli using plasmid pBR322. Amongst 200 recombinants we have identified and characterized a recombinant E. coli that expresses a protein antigen of M _r 74 000 similar in size to an outer membrane antigen produced by elementary bodies of C. trachomatis . Immunologically, the molecule synthesised by E. coli has the same specificity as the protein encoded by serovar L₂. A 1.8 kb DNA fragment from the recombinant insert, used as a hybridization probe, confirmed the species specificity of this clone at the gene level.