The occurrence of eukaryotic type III glutamine synthetase in the marine diatom Chaetoceros compressum

Glutamine synthetase (GS) has been described as one of the oldest functioning genes and thus a good molecular clock protein. GS is diverged into three distinct forms, type I (GSI), type II (GSII) and type III (GSIII), the last type of which is a member of the most recently discovered family among GSs and thus has been reported from a limited number of prokaryotes. In the present study, we determined the full-length sequence of GSIII from the marine diatom Chaetoceros compressum. The 3′ untranslated region of the diatom GSIII gene was composed of a polyadenylation signal followed by a poly (A)⁺ tail, clearly demonstrating that its mRNA is transcribed from the eukaryotic genome. We also screened available genome databases and identified full-length GSIII sequences from 5 eukaryotic species. These eukaryotic GSIIIs specifically contained regions A–D and a long additional sequence flanking region V toward the C-terminal site, both being specific to GSIII. Phylogenic analysis revealed that eukaryotic GSIIIs are not within a monophyletic relationship with the possible occurrence of lateral gene transfer in GSIII during evolution.     

Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

Background  

Glutamine synthetase (GS) is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII) are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria) to the Chloroplastida.     

MOLECULAR EVOLUTION OF GLUTAMINE SYNTHETASE II AND III IN THE CHROMALVEOLATES1

Glutamine synthetase (GS) is encoded by three distinct gene families (GSI, GSII, and GSIII) that are broadly distributed among the three domains of life. Previous studies established that GSII and GSIII isoenzymes were expressed in diatoms; however, less is known about the distribution and evolution of the gene families in other chromalveolate lineages. Thus, GSII cDNA sequences were isolated from three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyrenomonas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were obtained from Bangia atropurpurea (Mertens ex Roth) C. Agardh; Compsopogon caeruleus (Balbis ex C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and Porphyridium aerugineum Geitler; Rhodella violacea (Kornmann) Wehrmeyer and Dixoniella grisea (Geitler) J. L. Scott, S. T. Broadwater, B. D. Saunders, J. P. Thomas et P. W. Gabrielson; and Stylonema alsidii (Zanardini) K. M. Drew. In Bayesian inference and maximum‐likelihood (ML) phylogenetic analyses, chromalveolate GSII sequences formed a weakly supported clade that nested among sequences from glaucophytes, red algae, green algae, and plants. Red algal GSII sequences formed two distinct clades. The largest clade contained representatives from the Cyanidiophytina and Rhodophytina and grouped with plants and green algae. The smaller clade (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested within the chromalveolates, although its placement was unresolved. Chromalveolate GSIII sequences formed a well‐supported clade in Bayesian and ML phylogenies, and mitochondrial transit peptides were identified in many of the sequences. There was strong support for a stramenopile‐haptophyte‐cryptophyte GSIII clade in which the cryptophyte sequence diverged from the deepest node. Overall, the evolutionary history of the GS gene families within the algae is complex with evidence for the presence of orthologous and paralogous sequences, ancient and recent gene duplications, gene losses and replacements, and the potential for both endosymbiotic and lateral gene transfers.     

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.     

GLUTAMINE SYNTHETASE IN MARINE ALGAE: NEW SURPRISES FROM AN OLD ENZYME

Glutamine synthetase (GS), which catalyzes the formation of glutamine from ammonium and glutamate in the presence of ATP, is encoded by three distinct gene families: GSI, GSII, and GSIII. Genes encoding GSI are found in the Bacteria and Archaea, whereas GSII genes are found in eukaryotes and a few species of Bacteria. Members of the third family, GSIII, have been described from a limited number of bacteria; however, recent biochemical and molecular data suggest that this type of enzyme is broadly distributed among the algae. Peptide fragments obtained from GS purified from the marine diatom Skeletonema costatum (Greville) Cleve are 77% identical to a partial sequence of GSIII from Chaetoceros compressum Lauder, which permits the unambiguous assignment of the biochemically characterized enzyme to the GSIII gene family. The N-terminal sequence was 43% identical to the GSIII-like enzyme purified from the haptophyte Emiliania huxleyi (Lohm.) Hay et Miller and several residues were conserved among bacterial and eukaryotic GSIII enzymes. The presence of genes encoding GSIII in diatoms and haptophytes indicates that this enzyme family is more broadly distributed in eukaryotes than previously suspected.     

Modulation of different forms of glutamine synthetases inRhizobium phaseoli and their possible role in nitrogen fixation

The effect of a number of compounds structurally related to glutamic acid and other nitrogenous compounds on the composition of three forms of glutamine synthetase (GS) inRhizobium phaseoli has been examined in detail. Amino acids like glutamic acid, glutamine, and a fixed source of nitrogen like ammonium chloride did not alter the relative glutamine synthetase composition.l-Methioninedl-sulfoximine (MSX), a glutamate analogue, significantly repressed the synthesis of GSIII to a greater extent.,N-oxalyl,-diaminopropionic acid (ODAP), another glutamate analogue, selectively stimulated the synthesis of GSII, and the effect of ODAP on GSII synthesis was greatly enhanced in the presence of ethylenediamine or ammonium chloride. Ethylenediamine itself caused a predominant synthesis of GSIII.-Cyanoalanine-grownR. phaseoli did not synthesize GSI. The synthesis of the three different glutamine synthetases can thus be differentially modulated.     

Evolutionary relationships of bacterial and archaeal glutamine synthetase genes

Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII. Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria. GSIII genes have been found in only a few bacterial species. Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred. In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii. Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-a and GSI-. GSIa-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles). GSI--type genes occur in all other bacteria. GSI-- and GSI--type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter. Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it. The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision. The S. solfataricus GSI gene has a shorter GSI--type insertion, but like GSI-a-type genes, lacks conserved sequences about the adenylylation site. We suspect that the similarity of GSI- genes from Euryarchaeota and several bacterial species does not reflect a common phylogeny but rather lateral transmission between archaea and bacteria.Correspondence to: J.R. Brown 1073     

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.     

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.     

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.     

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     

The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications

Glutamine synthetase exists in at least two related forms, GSI and GSII, the sequences of which have been used in evolutionary molecular clock studies. GSI has so far been found exclusively in bacteria, and GSII has been found predominantly in eukaryotes. To date, only a minority of bacteria, including rhizobia, have been shown to express both forms of GS. The sequences of equivalent internal fragments of the GSI and GSII genes for the type strains of 16 species of rhizobia have been determined and analyzed. The GSI and GSII data sets do not produce congruent phylogenies with either neighbor-joining or maximum-likelihood analyses. The GSI phylogeny is broadly congruent with the 16S rDNA phylogeny for the same bacteria; the GSII phylogeny is not. There are three striking rearrangements in the GSII phylograms, all of which might be explained by horizontal gene transfer to Bradyrhizobium (probably from Mesorhizobium), to Rhizobium galegae (from Rhizobium), and to Mesorhizobium huakuii (perhaps from Rhizobium). There is also evidence suggesting intrageneric DNA transfer within Mesorhizobium. Meta-analysis of both GS genes from the different genera of rhizobia and other reference organisms suggests that the divergence times of the different rhizobium genera predate the existence of legumes, their host plants.     

Identification and characterization of a heat-labile type I glutamine synthetase fromStreptomyces cinnamonensis

Streptomycetes have two distinct glutamine synthetases (GS): a heat-stable dodecameric GSI and a heat-labile octameric GSII. A heat-inactivated GS activity was detected in crude extracts ofStreptomyces cinnamonensis cells grown with nitrate or glutamate as the nitrogen source. The purified enzyme obtained from crude extracts of the nitrate-grown cells after affinity and anion-exchange chromatography was also heat-labile; it was inactivated by 80 % when incubated at 50 °C for 1 h. However, the enzyme has properties typical of GSI and similar with those of the heat-stable GSI purified fromS. aureofaciens: It is composed of twelve subunits, each ofM 55 kDa, and has a native molar mass of 625 kDa and an isoelectric point at pH 4.2. In addition, its activity is regulated by reversible adenylylation. Mg²⁺ and NaCl but not Mn²⁺ protected the purified enzyme from thermal inactivation, and both NaCl and Mn²⁺ or Mg²⁺ stabilized its activity at 4–8 °C. As compared with GSI fromS. aureofaciens, theS. cinnamonensis enzyme was cleaved more extensively during SDS-PAGE, was less sensitive to feedback inhibitors, and similarly affected by divalent cations. TheK _m values were 12.5 mmol/L forl-glutamate, 0.1 for NH ₄ ⁺ , 1.25 for ATP, 18.5 forl-glutamine, 3.3 for hydroxylamine and 0.087 for ADP. To our best knowledge, this is the first report of a heatlabile GSI from any source.     

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.     

Cellular and clinical report of new Griscelli syndrome type III cases

The RAB27A/Melanophilin/Myosin-5a tripartite protein complex is required for capturing mature melanosomes in the peripheral actin network of melanocytes for subsequent transfer to keratinocytes. Mutations in any one member of this tripartite complex cause three forms of Griscelli syndrome (GS), each with distinct clinical features but with a similar cellular phenotype. To date, only one case of GS type III (GSIII), caused by mutations in the Melanophilin (MLPH) gene, has been reported. Here, we report seven new cases of GSIII in three distinct Arab pedigrees. All affected individuals carried a homozygous missense mutation (c.102C>T; p.R35W), located in the conserved Slp homology domain of MLPH, and had hypomelanosis of the skin and hair. We report the first cellular studies on GSIII melanocytes, which demonstrated that MLPH(R35W) causes perinuclear aggregation of melanosomes in melanocytes, typical for GS. Additionally, co-immunoprecipitation assays showed that MLPH(R35W) lost its interaction with RAB27A, indicating pathogenicity of the R35W mutation.     

Cloning and analysis of Agrobacterium tumefaciens C58 loci involved in glutamine biosynthesis: Neither the glnA (GSI) nor the glnII (GSII) gene plays a special role in virulence

Summary Using heterologous complementation of a glutamine synthetase deficient (glnA; GS^-) Escherichia coli mutant strain and heterologous DNA hybridization probes from Rhizobium meliloti and Bradyrhizobium japonicum, three distinct Agrobacterium tumefaciens loci involved in glutamine biosynthesis were identified. These loci correspond to the glnA (GSI), glnII (GSII) and a third previously unidentified locus, which is capable of complementing an E. coli glnA mutant, but may be cryptic in A. tumefaciens. The gene products encoded by the cloned glnA and glnII loci were identified using maxicells. Single insertion mutations in the glnA (GSI) and glnII (GSII) genes and a glnA glnII double mutant were constructed using gene replacement techniques. These mutant strains were examined for GSI and II activities, for growth on a variety of nitrogen (N) sources and for virulence properties on Kalanchoë plants. Neither glnA (GSI) nor glnII (GSII) were found to be essential for tumour induction on Kalanchoë nor for opine catabolism.     

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.     

Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway.

Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway. No glutamate dehydrogenase activity was detected. R. phaseoli has two GS enzymes, as do other rhizobia. The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation. When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated. When GSI is inactivated, GSII is induced. However, induction of GSII activity varied depending on the rate of change of this ratio. GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly. Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII. GSII inactivation was not relieved by shifting of the cultures to glutamate. After GSII inactivation, ammonium was excreted into the medium. Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.     

Evolution of glutamine synthetase in heterokonts: evidence for endosymbiotic gene transfer and the early evolution of photosynthesis

Although the endosymbiotic evolution of chloroplasts through primary and secondary associations is well established, the evolutionary timing and stability of the secondary endosymbiotic events is less well resolved. Heterokonts include both photosynthetic and nonphotosynthetic members and the nonphotosynthetic lineages branch basally in phylogenetic reconstructions. Molecular and morphological data indicate that heterokont chloroplasts evolved via a secondary endosymbiosis, involving a heterotrophic host cell and a photosynthetic ancestor of the red algae and this endosymbiotic event may have preceded the divergence of heterokonts and alveolates. If photosynthesis evolved early in this lineage, nuclear genomes of the nonphotosynthetic groups may contain genes that are not essential to photosynthesis but were derived from the endosymbiont genome through gene transfer. These genes offer the potential to trace the evolutionary history of chloroplast gains and losses within these lineages. Glutamine synthetase (GS) is essential for ammonium assimilation and glutamine biosynthesis in all organisms. Three paralogous gene families (GSI, GSII, and GSIII) have been identified and are broadly distributed among prokaryotic and eukaryotic lineages. In diatoms (Heterokonta), the nuclear-encoded chloroplast and cytosolic-localized GS isoforms are encoded by members of the GSII and GSIII family, respectively. Here, we explore the evolutionary history of GSII in both photosynthetic and nonphotosynthetic heterokonts, red algae, and other eukaryotes. GSII cDNA sequences were obtained from two species of oomycetes by polymerase chain reaction amplification. Additional GSII sequences from eukaryotes and bacteria were obtained from publicly available databases and genome projects. Bayesian inference and maximum likelihood phylogenetic analyses of GSII provided strong support for the monophyly of heterokonts, rhodophytes, chlorophytes, and plants and strong to moderate support for the Opisthokonts. Although the phylogeny is reflective of the unikont/bikont division of eukaryotes, we propose based on the robustness of the phylogenetic analyses that the heterokont GSII gene evolved via endosymbiotic gene transfer from the nucleus of the red-algal endosymbiont to the nucleus of the host. The lack of GSIII sequences in the oomycetes examined here further suggests that the GSIII gene that functions in the cytosol of photosynthetic heterokonts was replaced by the endosymbiont-derived GSII gene.