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.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.     

Atomic structure of plant glutamine synthetase: a key enzyme for plant productivity

Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to generate glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Elucidation of the atomic structure of higher plant GS is important to understand its detailed reaction mechanism and to obtain further insight into plant productivity and agronomical utility. Here we report the first crystal structures of maize (Zea mays L.) GS. The structure reveals a unique decameric structure that differs significantly from the bacterial GS structure. Higher plants have several isoenzymes of GS differing in heat stability and catalytic properties for efficient responses to variation in the environment and nutrition. A key residue responsible for the heat stability was found to be Ile-161 in GS1a. The three structures in complex with substrate analogues, including phosphinothricin, a widely used herbicide, lead us to propose a mechanism for the transfer of phosphate from ATP to glutamate and to interpret the inhibitory action of phosphinothricin as a guide for the development of new potential herbicides.     

Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum

Here, we describe two nearly identical expressed genes for cytosolic glutamine synthetase (GS3A and GS3B) in Pisum sativum L. RFLP mapping data indicates that the GS3A and GS3B genes are separate loci located on different chromosomes. DNA sequencing of the GS3A and GS3B genes revealed that the coding regions are 99% identical with only simple nucleotide substitutions resulting in three amino acid differences. Surprisingly, the non-coding regions (5 non-coding leader, the 11 introns, and 3 non-coding tail) all showed a high degree of identity (96%). In these non-coding regions, 25% of the observed differences between the GS3A and GS3B genes were deletions or duplications. The single difference in the 3 non-coding regions of the GS3A and GS3B genes was a 25 bp duplication of an AU-rich element in the GS3B gene. As the GS3B mRNA accumulates to lower levels than the GS3A gene, we tested whether this sequence which resembles an mRNA instability determinant functioned as such in the context of the GS mRNA. Using the GS3B 3 tail as part of a chimeric gene in transgenic plants, we showed that this AU-rich sequence has little effect on transgene mRNA levels. To determine whether the GS3A/GS3B genes represent a recent duplication, we examined GS3-like genes in genomic DNA of ancient relatives of P. sativum. We observed that several members of the Viceae each contain two genomic DNA fragments homologous to the GS3B gene, suggesting that this is an ancient duplication event. Gene conversion has been invoked as a possible mechanism for maintaining the high level of nucleotide similarity found between the GS3A and GS3B genes. Possible evolutionary reasons for the maintenance of these twin GS genes in pea, and the general duplication of genes for cytosolic GS in all plant species are discussed.     

Molecular basis of glutathione synthetase deficiency and a rare gene permutation event.

Glutathione synthetase (GS) catalyses the production of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Malfunctioning of GS results in disorders including metabolic acidosis, 5-oxoprolinuria, neurological dysfunction, haemolytic anaemia and in some cases is probably lethal. Here we report the crystal structure of human GS (hGS) at 2.1 A resolution in complex with ADP, two magnesium ions, a sulfate ion and glutathione. The structure indicates that hGS belongs to the recently identified ATP-grasp superfamily, although it displays no detectable sequence identity with other family members including its bacterial counterpart, Escherichia coli GS. The difficulty in identifying hGS as a member of the family is due in part to a rare gene permutation which has resulted in a circular shift of the conserved secondary structure elements in hGS with respect to the other known ATP-grasp proteins. Nevertheless, it appears likely that the enzyme shares the same general catalytic mechanism as other ligases. The possibility of cyclic permutations provides an insight into the evolution of this family and will probably lead to the identification of new members. Mutations that lead to GS deficiency have been mapped onto the structure, providing a molecular basis for understanding their effects.     

Concerted gene recruitment in early plant evolution

Background  

Horizontal gene transfer occurs frequently in prokaryotes and unicellular eukaryotes. Anciently acquired genes, if retained among descendants, might significantly affect the long-term evolution of the recipient lineage. However, no systematic studies on the scope of anciently acquired genes and their impact on macroevolution are currently available in eukaryotes.     

植物LTR-反转座子中Orf1基因的分子进化

LTR-反转座子是植物基因组的主要组成部分。它们在结构上非常保守, 通常含有gag和pol两个基因, 是完成其转座过程所必需的。在前期研究中, 本项目组对大豆基因组SARE转座子家族进行了详细的分析。结果表明, 该家族的拷贝中还存在第3个基因——Orf1。文章借助生物信息学的研究方法, 对33个已测序的基因组进行了全基因组注释。结果发现, 在7个植物基因组(桉树、杨树、棉花、大豆、百脉根、亚麻和苜蓿)中, 部分LTR-反转座子元件在gag基因的上游存在约1~2 kb未知的Orf1基因或基因片段。这类转座子多数在0~3百万年内插入到其所在的寄主基因组中, 但它们在不同物种中的分子结构、发生的频率、扩增的强度和活跃的时期等方面差异较大。系统进化树分析表明, 这类具有特殊结构的转座子较整齐的聚类到双子叶植物的一个进化分支上, 表明它们可能是部分双子叶植物在进化过程中所产生的。不同物种间的相对保守性、大量拷贝的转录活性以及可能存在的多个功能结构域, 提示Orf1基因可能具有一定的生物学功能。     

Phylogenetic relationships among Passiflora species based on the glutamine synthetase nuclear gene expressed in chloroplast (ncpGS)

This paper presents the first molecular phylogeny of the genus Passiflora encompassing almost all sections of this large genus. The nuclear-encoded chloroplast-expressed glutamine synthetase gene (ncpGS) was used to examine the relationships among Passiflora species (passionflowers), which was then compared with the new classification proposed by Feuillet and MacDougal. The resulting Bayesian, likelihood, and parsimony trees are congruent and well supported. The 90 Passiflora species examined apparently split into eight main subgenera: Plectostemma, Granadilla, Astrophea, Deidamioides, Polyanthea, Dysosmia, Tetrapathea, and Tryphostemmatoides. These results are in overall agreement with the Feuillet and MacDougal's classification but here we propose that three additional subgenera, Polyanthea, Dysosmia, and Tetrapathea, should be maintained. We observe a striking overall correlation between the phylogenetic position of the different species and their chromosome number. The first clade contains the arborescent species of the subgenus Astrophea, with n=12. The second clade, subgenus Plectostemma, includes species from four subgenera of Killip's classification with n=6 chromosomes. The last clade, subgenus Granadilla, includes species of seven old subgenera with n=9. Subgenus Dysosmia, with a variable chromosome number of n=9-11, is considered here as a separate subgenus closely related to the subgenus Granadilla.     

Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS₂) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS₁). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS₂ polypeptide (NH₂-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS₂ subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS₂-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS₂ mRNA, and an anti-GS antiserum to assay for GS₂ protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS₂ mRNA; class II, which had normal or increased levels of GS₂ mRNA but very little GS₂ protein; and class III, which had significant amounts of GS₂ protein but little or no GS₂ activity.     

Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase

An improved method was developed to align related protein sequences and search for homology. A glutamine amide transfer domain was identified in an NH2-terminal segment of GMP synthetase from Escherichia coli. Amino acid residues 1-198 in GMP synthetase are homologous with the glutamine amide transfer domain in trpG X D-encoded anthranilate synthase component II-anthranilate phosphoribosyltransferase and the related pabA-encoded p-aminobenzoate synthase component II. This result supports a model for gene fusion in which a trpG-related glutamine amide transfer domain was recruited to augment the function of a primitive NH3-dependent GMP synthetase. Sequence analyses emphasize that glutamine amide transfer domains are thus far found only at the NH2 terminus of fused proteins. Two rules are formulated to explain trpG and trpG-related fusions. (i) trpG and trpG-related genes must have translocated immediately up-stream of genes destined for fusion in order to position a glutamine amide transfer domain at the NH2 terminus after fusion. (ii) trpG and trpG-related genes could not translocate adjacent to a regulatory region at the 5' end of an operon. These rules explain known trpG-like fusions and explain why trpG and pabA are not fused to trpE and pabB, respectively. Alignment searches of GMP synthetase with two other enzymes that bind GMP, E. coli amidophosphoribosyltransferase and human hypoxanthine-guanine phosphoribosyltransferase, suggest a structurally homologous segment which may constitute a GMP binding site.