A previously unrecognized glutamine synthetase expressed in Klebsiella pneumoniae from the glnT locus of Rhizobium leguminosarum

Summary Using glnT DNA of Rhizobium meliloti as a hybridization probe we identified a R. leguminosarum biovar phaseoli (R. l. phaseoli) locus (glnT) expressing a glutamine synthetase activity in Klebsiella pneumoniae. A 2.2 kb DNA fragment from R. l. phaseoli was cloned to give plasmid pMW5a, which shows interspecific complementation of a K. pneumoniae glnA mutant. The cloned sequence did not show cross-hybridization to glnA or glnII, the genes coding for two glutamine synthetase isozymes of Rhizobium spp. While in previous reports on glnT of R. meliloti and Agrobacterium tumefaciens no glutamine synthetase activity was detected, we do find activity with the glnT locus of R. l. phaseoli. The glutamine synthetase (GSIII) activity expressed in a K. pneumoniae glnA strain from pMW5a shows a ratio of biosynthetic to transferase activity 10³-fold higher than that observed for GSI or GSII. GSIII is similar in molecular weight and heat stability to GSI.     

Investigation of the functional properties and regulation of three glutamine synthetase-like genes in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) has three additional glnA-type genes besides the glutamine synthetase genes glnA (encoding GSI) and glnII (encoding GSII). The aim of this work was to characterize their functional properties and regulation. Sequence analyses revealed that GlnA2, GlnA3, and GlnA4 are dissimilar to S. coelicolor GSI and lack highly conserved amino acid residues involved in catalysis. In heterologous expression experiments, glnA2, glnA3, and glnA4, in contrast to glnA and glnII, were not capable of complementing the l-glutamine auxotrophy of an Escherichia coli glnA mutant. The lack of a conserved sequence motif reflecting adenylylation control of enzyme activity suggests that GlnA2, GlnA3, and GlnA4 are not regulated via adenylyltransferase-mediated modification. In DNA-binding assays, the OmpR-like regulator of nitrogen metabolism GlnRII, which interacts with the glnA and glnII promoters, did not bind to the upstream regions of glnA2, glnA3, and glnA4. These findings support the conclusion that glnA2, glnA3, and glnA4 are not directly involved in l-glutamine synthesis and nitrogen assimilation and are not subject to nitrogen control in S. coelicolor. The glnA3 gene product is similar to FluG, which is required for asexual sporulation in Aspergillus nidulans. However, inactivation of glnA3 does not block morphological differentiation in S. coelicolor.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Impaired nitrogen fixation and glutamine synthesis in methionine sulfoximine sensitive (MSs) mutants of Rhizobium phaseoli

Summary Random Tn5 mutagenesis of antibiotic-resistant derivatives of Rhizobium phaseoli CFN42 yielded several independent mutants that were sensitive to methionine sulfoximine (MS^s), a specific inhibitor of glutamine synthetase (GS). These MS^s mutants were analyzed for GSI and GSII activities and for their symbiotic properties. Four classes of MS^s mutants have been distinguished. Class I strains are impaired in their synthesis of glutamine and in their symbiotic properties. Class II strains have wild type levels of GSI and GSII activities but have a reduced capacity to fix nitrogen. Class III strains have lost GSII activity, but their symbiotic properties are wild type. In class IV mutants neither glutamine synthesis nor symbiotic properties are affected. Mutants of classes I, III, and IV all have the Tn5 inserted into the chromosome, whereas in class II mutants the Tn5 is located in plasmid p42e, a plasmid different from the previously identified symbiotic plasmid p42d.     

Physiological characteristics of glutamine synthetases I and II of Frankia sp. strain CpI1

Frankia sp. strain CpI1 has two glutamine synthetases designated GSI and GSII. Biosynthetic activities of both GSI and GSII were strongly inhibited by ADP and AMP. Alanine, aspartate, glycine and serine inhibited both GSI and GSII activities, whereas asparagine and lysine inhibited only slightly. Glutamine inhibited GSII but did not affect GSI. Since GSII is more heat labile than GSI, their relative heat stabilities can be used to determine their contribution to total GS activity. In cells grown on ammonia and on glutamine as sole combined-nitrogen sources most GS activity detected in crude extracts was due to GSI. In cells transferred to glutamate, GSI accounted for all GS activity in the first 15 h and then heat labile GSII was induced and increased to account for 40% of total GS activity within 50 h. Transfer of N₂-fixing cells to ammonia-containing medium led to a rapid decrease of GSII and a slow increase of GSI activity within 24 h. Conversely, when ammonia-grown cells were transferred to combined nitrogen-free medium, GSI activity gradually decreased and GSII increased before total activity leveled off in 50 h. GSII appears to be an ammonia-assimilating enzyme specifically synthesized during perceived N-starvation of Frankia cells.     

The relationship between the state of adenylylation of glutamine synthetase I and the export of ammonium in free-living,N2-fixing Rhizobium

The relationship between ammonium assimilation and ammonium export has been studied in free-living, N₂-fixing Rhizobium sp. 32H1. After 55 to 67 h of microaerobic growth under a gas phase of 0.2% O₂ – 1.0% CO₂ – 98.8% Ar high levels of nitrogenase were observed concomitant with a slightly adenylylated glutamine synthetase (GSI) and some glutamine synthetase (GSII) activity. However, after growth of 89 h, or longer, GSI became adenylylated and the level of GSII had decreased. When the gas phase was shifted to 0.2% O₂ – 1.0% CO₂ – 98.8% N₂, a lag was observed before ammonium export could be detected in the 55 to 67 h cultures. No lag in ammonium export was observed in the cultures previously grown for 89 h. The onset of ammonium export in the 55 to 67 h cultures was found to correlate with the adenylylation state of GSI. There appeared to be no correlation between the level of GSII and the export of ammonium. Neither an increase in the adenylylation level of GSI nor ammonium export was observed when the 55 to 67 h cultures were maintained under the Ar gas mixture.Abbreviations GOGAT Glutamate synthase - GS glutamine synthetase - BES [N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid] - CTAB cetyltrimethylammonium bromide - MES [2-(N-morpholino)-ethane sulfonic acid]     

Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue

The isopentenyl transferase gene was isolated from Agrobacterium tumefaciens AcH5 using polymerase chain reaction and transformed into Petunia and Kalanchoë using both A. tumefaciens and A. rhizogenes transformation systems. Morphological evidence and elevated endogenous cytokinin levels indicated that the PCR product was an active gene. Accurate quantification of the cytokinins was obtained by radioimmunoassay, following purification and separation of the free bases and ribosides by HPLC. Of the six cytokinins quantified, zeatin riboside and its stabilised dihydro-derivative, dihydrozeatin riboside, showed the greatest increases in the transformed Petunia tissue (up to 600-fold). The importance of measuring changes in individual cytokinins is discussed.     

Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation.

We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT). The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation. The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII). The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E. coli strain. The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons. glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+). A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules. glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules. glnII and glnT, but not glnA, expression in R. meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.     

Glutamine synthetase II inRhizobium: Reexamination of the proposed horizontal transfer of DNA from eukaryotes to prokaryotes

Summary We have determined the DNA sequence of aRhizobium meliloti gene that encodes glutamine synthetase II (GSII). The deduced amino acid sequence was compared to that ofBradyrhizobium japonicum GSII and those of various plant and mammalian glutamine synthetases (GS) in order to evaluate a proposal that the gene for this enzyme was recently transferred from plants to their symbiotic bacteria. There is 83.6% identity between theR. meliloti andB. japonicum proteins. The bacterial GSII proteins average 42.5% identity with the plant GS proteins and 41.8% identity with their mammalian counterparts. The plant proteins average 53.7% identity with the mammalian proteins. Thus, the GS proteins are highly conserved and the divergence of these proteins is proportional to the phylogenetic divergence of the organisms from which the sequences were determined. No transfer of genes across large taxonomic gaps is needed to explain the presence of GSII in these bacteria.     

NdgR,an IclR-like regulator involved in amino-acid-dependent growth,quorum sensing,and antibiotic production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

NdgR (regulator for nitrogen source-dependent growth and antibiotic production), an IclR-like regulator, has been initially identified as a binding protein to the promoters of doxorubicin biosynthetic genes in Streptomcyes peucetius by DNA affinity capture assay method. NdgR is well conserved throughout the Streptomcyes species and many other bacteria such as Mycobacteria and Corynebacteria. In Streptomcyes coelicolor, ndgR deletion mutant showed slow cell growth and defects in differentiation and enhances the production of actinorhodin (ACT) in minimal media containing certain amino acids where wild-type strain could not produce ACT. Although deletion mutant of ndgR showed different antibiotic production in minimal media containing Leu or Gln, it only showed reduced mRNA expression levels of the genes involved in leucine metabolism. Neither NdgR-dependent expression of glnA nor direct binding of NdgR protein to glnA, glnII, and glnR promoters was observed. However, ScbR, which is governed by NdgR shown by gel mobility shift assay, binds to promoter of glnR, suggesting indirect regulation of glutamine metabolism by NdgR. NdgR protein binds to intergenic region of ndgR–leuC, and scbR–scbA involved in γ-butyrolactone. Two-dimensional gel analysis has shown a global effect of ndgR deletion in protein expression, including up-regulated proteins involved in ACT synthesis and down-regulation of chaperones such as GroEL, GroES, and DnaK. These results suggest a global regulatory role for NdgR in amino acid metabolisms, quorum sensing, morphological changes, antibiotic production, and expression of chaperonines in S. coelicolor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of the Klebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles

Summary The glnB gene of Klebsiella pneumoniae, which encodes the nitrogen regulation protein P_II has been cloned and sequenced. The gene encodes a 12429 dalton polypeptide and is highly homologous to the Escherichia coli glnB gene. The sequences of a glnB mutation which causes glutamine auxotrophy and of a Tn5 induced Gln⁺ suppressor of this mutation were also determined. The glutamine auxotrophy was deduced to be the result of a modification of the uridylylation site of P_II and the suppression was shown to be caused by Tn5 insertion in glnB. The 3 end of an open reading frame of unknown function was identified upstream of glnB and may be part of an operon containing glnB. Potential homologues of glnB encoding polypeptides extremely similar in sequence to P_II were identified upstream of published sequences of the glutamine synthetase structural gene (glnA) in Rhizobium leguminosarum, Bradyrhizobium japonicum and Azospirillum brasilense.     

The Presence of GSI-Like Genes in Higher Plants: Support for the Paralogous Evolution of GSI and GSII Genes

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution. Received: 4 May 1999 / Accepted: 17 September 1999     

Inhibition of F plasmid replication in htpR mutants of Escherichia coli deficient in sigma 32 protein

Summary In Drosophila melanogaster there are two glutamine synthetase (GS) (EC 6.3.1.2) isozymes. They are called GSI and GSII. The two enzymes have different subunits and different genetic determination. A DNA fragment that comprises 80% of the coding region of the glutamine synthetase gene of Chinese hamster ovary (CHO) cells allowed the identification and cloning of an homologous DNA fragment of Drosophila. This sequence is located at the 10B8-11 region on the X chromosome. Dose variation of a chromosomal segment from 9F3 to 10C1-2, which encompasses the 10B region, leads to proportional variations of GSII without apparently influencing the amount of GSI.     

Close linkage of genes encoding glutamine synthetases I and II in Frankia alni CpI1.

Frankia alni CpI1 has two glutamine synthetases (GSs), GSI and GSII. The GSI gene (glnA) was isolated from a cosmid library of F. alni CpI1 DNA by heterologous probing with glnA from Streptomyces coelicolor. The glnA gene was shown to be located upstream of the GSII gene (glnII) by DNA-DNA hybridization. The nucleotide sequences of the 1,422-bp CpI1 glnA gene and of the 449-bp intervening region between glnA and glnII were determined, and the glnA amino acid sequence was deduced. In common with GSIs from other organisms, CpI1 GSI contains five conserved regions near the active site and a conserved tyrosine at the adenylylation site. F. alni CpI1 glnA complemented the glutamine growth requirement of the Escherichia coli glnA deletion strain YMC11 but only when expressed from an E. coli lac promoter. While the functional significance of maintaining two GSs adjacent to one another remains unclear, this arrangement in F. alni provides support for the recently proposed origin of GSI and GSII as resulting from a gene duplication early in the evolution of life.     

Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups

Abstract: The structure of glutamine synthetase (GS) enzymes from diverse bacterial groups fall into three distinct classes. GSI is the typical bacterial GS, GSII is similar to the eukaryotic GS and is found together with GSI in plant symbionts and Streptomyces , while GSIII has been found in two unrelated anaerobic rumen bacteria. In most cases, the structural gene for GS enzyme is regulated in response to nitrogen. However, different regulatory mechanisms, to ensure optimal utilization of nitrogen substrates, control the GS enzyme in each class.