Complex distribution of EFL and EF-1α proteins in the green algal lineage

Background  

EFL (or elongation factor-like) is a member of the translation superfamily of GTPase proteins. It is restricted to eukaryotes, where it is found in a punctate distribution that is almost mutually exclusive with elongation factor-1 alpha (EF-1α). EF-1α is a core translation factor previously thought to be essential in eukaryotes, so its relationship to EFL has prompted the suggestion that EFL has spread by horizontal or lateral gene transfer (HGT or LGT) and replaced EF-1α multiple times. Among green algae, trebouxiophyceans and chlorophyceans have EFL, but the ulvophycean Acetabularia and the sister group to green algae, land plants, have EF-1α. This distribution singles out green algae as a particularly promising group to understand the origin of EFL and the effects of its presence on EF-1α.     

What Is Characteristic of Fungal Lysine Synthesis Through the α-Aminoadipate Pathway?

Recent finding that a prokaryote synthesizes lysine through the α-aminoadipate pathway demonstrates that the lysine synthesis through the α-aminoadipate pathway is not typical of fungi. However, the fungal lysine biosynthesis is not completely the same as the prokaryotic one. We point out that α-aminoadipate reductase is a key enzyme to the evolution of fungal lysine synthesis. In addition, fungi have two different saccharopine dehydrogenases, which is also characteristic of fungi. Received: 18 February 2000 / Accepted: 19 June 2000     

Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase

Candida albicans LYS1-encoded saccharopine dehydrogenase (CaLys1p, SDH) catalyzes the final biosynthetic step (saccharopine to lysine + α-ketoglutarate) of the novel α-aminoadipate pathway for lysine synthesis in fungi. The reverse reaction catalyzed by lysine-α-ketoglutarate reductase (LKR) is used exclusively in animals and plants for the catabolism of excess lysine. The 1,146 bp C. albicans LYS1 ORF encodes a 382 amino acid SDH. In the present investigation, we have used E. coli-expressed recombinant C. albicans Lys1p for the determination of both forward and reverse SDH activities in vitro, compared the sequence identity of C. albicans Lys1p with other known SDHs and LKRs, performed extensive site-directed mutational analyses of conserved amino acid residues and analyzed the phylogenetic relationship of C. albicans Lys1p to other known SDHs and LKRs. We have identified 14 of the 68 amino acid substitutions as essential for C. albicans Lys1p SDH activity, including two highly conserved functional motifs, H93XXF96XH98 and G138XXXG142XXG145. These results provided new insight into the functional and phylogenetic characteristics of the distinct biosynthetic SDH in fungi and catabolic LKR in higher eukaryotes.     

A unique fungal lysine biosynthesis enzyme shares a common ancestor with tricarboxylic acid cycle and leucine biosynthetic enzymes found in diverse organisms

Fungi have evolved a unique α-aminoadipate pathway for lysine biosynthesis. The fungal-specific enzyme homoaconitate hydratase from this pathway is moderately similar to the aconitase-family proteins from a diverse array of taxonomic groups, which have varying modes of obtaining lysine. We have used the similarity of homoaconitate hydratase to isopropylmalate isomerase (serving in leucine biosynthesis), aconitase (from the tricarboxylic acid cycle), and iron-responsive element binding proteins (cytosolic aconitase) from fungi and other eukaryotes, eubacteria, and archaea to evaluate possible evolutionary scenarios for the origin of this pathway. Refined sequence alignments show that aconitase active site residues are highly conserved in each of the enzymes, and intervening sequence sites are quite dissimilar. This pattern suggests strong purifying selection has acted to preserve the aconitase active site residues for a common catalytic mechanism; numerous other substitutions occur due to adaptive evolution or simply lack of functional constraint. We hypothesize that the similarities are the remnants of an ancestral gene duplication, which may not have occurred within the fungal lineage. Maximum likelihood, neighbor joining, and maximum parsimony phylogenetic comparisons show that the α-aminoadipate pathway enzyme is an outgroup to all aconitase family proteins for which sequence is currently available. Received: 7 October 1997     

Old genes in new places: A taxon-rich analysis of interdomain lateral gene transfer events

Vertical inheritance is foundational to Darwinian evolution, but fails to explain major innovations such as the rapid spread of antibiotic resistance among bacteria and the origin of photosynthesis in eukaryotes. While lateral gene transfer (LGT) is recognized as an evolutionary force in prokaryotes, the role of LGT in eukaryotic evolution is less clear. With the exception of the transfer of genes from organelles to the nucleus, a process termed endosymbiotic gene transfer (EGT), the extent of interdomain transfer from prokaryotes to eukaryotes is highly debated. A common critique of studies of interdomain LGT is the reliance on the topology of single-gene trees that attempt to estimate more than one billion years of evolution. We take a more conservative approach by identifying cases in which a single clade of eukaryotes is found in an otherwise prokaryotic gene tree (i.e. exclusive presence). Starting with a taxon-rich dataset of over 13,600 gene families and passing data through several rounds of curation, we identify and categorize the function of 306 interdomain LGT events into diverse eukaryotes, including 189 putative EGTs, 52 LGTs into Opisthokonta (i.e. animals, fungi and their microbial relatives), and 42 LGTs nearly exclusive to anaerobic eukaryotes. To assess differential gene loss as an explanation for exclusive presence, we compare branch lengths within each LGT tree to a set of vertically-inherited genes subsampled to mimic gene loss (i.e. with the same taxonomic sampling) and consistently find shorter relative distance between eukaryotes and prokaryotes in LGT trees, a pattern inconsistent with gene loss. Our methods provide a framework for future studies of interdomain LGT and move the field closer to an understanding of how best to model the evolutionary history of eukaryotes.     

Aminoadipate reductase gene: a new fungal-specific gene for comparative evolutionary analyses

Background  

In fungi, aminoadipate reductase converts 2-aminoadipate to 2-aminoadipate 6-semialdehyde. However, other organisms have no homologue to the aminoadipate reductase gene and this pathway appears to be restricted to fungi. In this study, we designed degenerate primers for polymerase chain reaction (PCR) amplification of a large fragment of the aminoadipate reductase gene for divergent fungi.     

Phylogenomic Analysis of the α Proteasome Gene Family from Early-Diverging Eukaryotes

We employed a phylogenomic approach to study the evolution of α subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven α proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple α subunit sequences generated single α proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct α proteasome genes each. The presence of seven distinct α proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the α proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the α proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented. Received: 14 February 2000 / Accepted: 14 August 2000     

Presence of a Bacterial-Like Citrate Synthase Gene in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>: Recent Lateral Gene Transfers (LGT) or Multiple Gene Losses Subsequent to a Single Ancient LGT?

Citrate synthase is the initial enzyme in the tricarboxylic acid cycle of mitochondria. In plants and fungi, it is the second isozyme in the glyoxylate cycle of peroxisomes (or glyoxysomes), and it is also present in bacteria. Some of the biochemical reactions in the glyoxylate cycle of the ciliated protozoan Tetrahymena pyriformis depend upon mitochondrial enzymes, as T. pyriformis lacks some glyoxysome-specific enzymes. Here we demonstrate a new citrate synthase gene from Tetrahymena thermophila that is different from the mitochondrial counterpart. A potential peroxysome-targeted signal was detected in the N-terminus, suggesting the localization of the enzyme in peroxysomes. Phylogenetic analysis placed the Tetrahymena sequence in a clade consisting of a few sequences from eukaryotes such as cellular slime molds and two land plants, near a green sulfur bacterium and many proteobacteria as a sister group but not in a mitochondrial clade. Southern blot analysis revealed that this type of gene was absent from distantly related ciliates and other species of Tetrahymena except for the closest species, T. mallaccensis. The scattered presence of the bacterial-like genes among distantly related eukaryotes suggests three alternative interpretations of acquisition of the novel glyoxysomal citrate synthase gene via lateral gene transfer (LGT). (1) Some eukaryotes independently acquired the gene from a common bacterium or closely related bacteria via LGT. (2) A hypothetical eukaryote once acquired the gene, which was thereafter independently transferred from the eukaryote to other eukaryotes. (3) A single event of LGT (or duplication) occurred in a certain common ancestor of eukaryotes, followed by multiple losses in many eukaryotic lineages during the subsequent evolution. Considering the monophyly of the bacterial-like eukaryotic citrate synthase genes, the first model is somewhat unlikely, even though it is not impossible. The second and third models can rationally explain the present observation, so these models are discused in some detail.     

Evolution of the proteasome components

 A phylogenetic analysis of proteasome subunits revealed two major families (α and β) which originated by an ancient gene duplication prior to the divergence of archaebacteria and eukaryotes. Numerous gene duplications have subsequently occurred in eukaryotes; at least nine of these duplications were shown to have occurred prior to the divergence of animals and fungi. In mammals, two genes encoding proteasome subunits (LMP2 and LMP7) are located in the major histocompatibility complex (MHC) region and play a specific role in generation of peptides for presentation by class I MHC molecules. Phylogenetic analysis of LMP7 and related sequences from mammals and lower vertebrates indicated that this locus arose by gene duplication prior to the divergence of jawed and jawless vertebrates; the time of this duplication was estimated to have been about 600 million years ago. The evolutionary history of the proteasome subunits provides support for a model of the evolution of new gene function postulating that, after gene duplication, the proteins encoded by daughter loci can adapt to specialized functions previously performed by the product of a single generalized ancestral locus. Received: 19 August 1996 / Revised: 24 December 1996     

Characterization of the lys2 gene of Penicillium chrysogenum encoding α -aminoadipic acid reductase

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr^ 154 859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomycespombe (51.3% identity) and Candida albicans (48.12% identity) α-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the α-aminoadipate-activating domain of the α-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5′-region and other in the 3′-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region. Received: 26 January 1998 / Accepted: 4 May 1998     

Evolution of four gene families with patchy phylogenetic distributions: influx of genes into protist genomes

Background  

Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject in need of further study. Here we present gene distribution and phylogenetic analyses of the genes encoding the hybrid-cluster protein, A-type flavoprotein, glucosamine-6-phosphate isomerase, and alcohol dehydrogenase E. These four genes have a limited distribution among sequenced prokaryotic and eukaryotic genomes and were previously implicated in gene transfer events affecting eukaryotes. If our previous contention that these genes were introduced by LGT independently into the diplomonad and Entamoeba lineages were true, we expect that the number of putative transfers and the phylogenetic signal supporting LGT should be stable or increase, rather than decrease, when novel eukaryotic and prokaryotic homologs are added to the analyses.     

Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9, affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, α-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known α-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium. Received: 21 October 1996 / Accepted: 23 September 1997     

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae

Summary The lysA gene of Escherichia coli has been cloned from a transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.     

Replacement of the Arginine Biosynthesis Operon in Xanthomonadales by Lateral Gene Transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the γ-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.     

Gain and loss of elongation factor genes in green algae

Background  

Two key genes of the translational apparatus, elongation factor-1 alpha (EF-1α) and elongation factor-like (EFL) have an almost mutually exclusive distribution in eukaryotes. In the green plant lineage, the Chlorophyta encode EFL except Acetabularia where EF-1α is found, and the Streptophyta possess EF-1α except Mesostigma, which has EFL. These results raise questions about evolutionary patterns of gain and loss of EF-1α and EFL. A previous study launched the hypothesis that EF-1α was the primitive state and that EFL was gained once in the ancestor of the green plants, followed by differential loss of EF-1α or EFL in the principal clades of the Viridiplantae. In order to gain more insight in the distribution of EF-1α and EFL in green plants and test this hypothesis we screened the presence of the genes in a large sample of green algae and analyzed their gain-loss dynamics in a maximum likelihood framework using continuous-time Markov models.     

Targeted disruption of a G protein α subunit gene results in reduced growth and pathogenicity in <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Cloning and disruption of Rga1, the gene encoding the G protein α subunit in the rice sheath blight fungus Rhizoctonia solani, was investigated. The deduced primary structure of the Rga1-encoded protein showed high identity to those of Gα subunits from other filamentous fungi. Disruption of Rga1 led to decreased vegetative growth and pathogenicity. The Rga1 disruptant showed altered colony morphology. In addition, the sclerotia formation ability of the disruptant was completely lost. These results suggest that the Gα subunit encoded by Rga1 is involved in a signal transduction pathway in R. solani that controls growth, development and pathogenicity.     

The Frequency of Eubacterium-to-Eukaryote Lateral Gene Transfers Shows Significant Cross-Taxa Variation Within Amoebozoa

Single-celled bacterivorous eukaryotes offer excellent test cases for evaluation of the frequency of prey-to-predator lateral gene transfer (LGT). Here we use analysis of expressed sequence tag (EST) data sets to quantify the extent of LGT from eubacteria to two amoebae, Acanthamoeba castellanii and Hartmannella vermiformis. Stringent screening for LGT proceeded in several steps intended to enrich for authentic events while at the same time minimizing the incidence of false positives due to factors such as limitations in database coverage and ancient paralogy. The results were compared with data obtained when the same methodology was applied to EST libraries from a number of other eukaryotic taxa. Significant differences in the extent of apparent eubacterium-to-eukaryote LGT were found between taxa. Our results indicate that there may be substantial inter-taxon variation in the number of LGT events that become fixed even between amoebozoan species that have similar feeding modalities. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Martin Kreitman]     

Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships

Background  

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (NPS) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.     

Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

Background  

Capping protein (CP), a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform.