PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates.

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

The Mastigamoeba balamuthi Genome and the Nature of the Free-Living Ancestor of Entamoeba.

The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

The Origin and Diversification of Mitochondria.

Mitochondria are best known for their role in the generation of ATP by aerobic respiration. Yet, research in the past half century has shown that they perform a much larger suite of functions and that these functions can vary substantially among diverse eukaryotic lineages. Despite this diversity, all mitochondria derive from a common ancestral organelle that originated from the integration of an endosymbiotic alphaproteobacterium into a host cell related to Asgard Archaea. The transition from endosymbiotic bacterium to permanent organelle entailed a massive number of evolutionary changes including the origins of hundreds of new genes and a protein import system, insertion of membrane transporters, integration of metabolism and reproduction, genome reduction, endosymbiotic gene transfer, lateral gene transfer and the retargeting of proteins. These changes occurred incrementally as the endosymbiont and the host became integrated. Although many insights into this transition have been gained, controversy persists regarding the nature of the original endosymbiont, its initial interactions with the host and the timing of its integration relative to the origin of other features of eukaryote cells. Since the establishment of the organelle, proteins have been gained, lost, transferred and retargeted as mitochondria have specialized into the spectrum of functional types seen across the eukaryotic tree of life.

Minimal cytosolic iron-sulfur cluster assembly machinery of Giardia intestinalis is partially associated with mitosomes.

Iron-sulfur (Fe-S) clusters are essential cofactors that enable proteins to transport electrons, sense signals, or catalyze chemical reactions. The maturation of dozens of Fe-S proteins in various compartments of every eukaryotic cell is driven by several assembly pathways. The ubiquitous cytosolic Fe-S cluster assembly (CIA) pathway, typically composed of eight highly conserved proteins, depends on mitochondrial Fe-S cluster assembly (ISC) machinery. Giardia intestinalis contains one of the smallest eukaryotic genomes and the mitosome, an extremely reduced mitochondrion. Because the only pathway known to be retained within this organelle is the synthesis of Fe-S clusters mediated by ISC machinery, a likely function of the mitosome is to cooperate with the CIA pathway. We investigated the cellular localization of CIA components in G. intestinalis and the origin and distribution of CIA-related components and Tah18-like proteins in other Metamonada. We show that orthologs of Tah18 and Dre2 are missing in these eukaryotes. In Giardia, all CIA components are exclusively cytosolic, with the important exception of Cia2 and two Nbp35 paralogs, which are present in the mitosomes. We propose that the dual localization of Cia2 and Nbp35 proteins in Giardia might represent a novel connection between the ISC and the CIA pathways.

An ancestral bacterial division system is widespread in eukaryotic mitochondria.

Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such as Saccharomyces cerevisiae, mammals, and Arabidopsis thaliana seem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid Andalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.

A large number of nuclear genes in the human parasite blastocystis require mRNA polyadenylation to create functional termination codons.

Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3'-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3'-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left.

Evolution of the cytosolic iron-sulfur cluster assembly machinery in Blastocystis species and other microbial eukaryotes.

The cytosolic iron/sulfur cluster assembly (CIA) machinery is responsible for the assembly of cytosolic and nuclear iron/sulfur clusters, cofactors that are vital for all living cells. This machinery is uniquely found in eukaryotes and consists of at least eight proteins in opisthokont lineages, such as animals and fungi. We sought to identify and characterize homologues of the CIA system proteins in the anaerobic stramenopile parasite Blastocystis sp. strain NandII. We identified transcripts encoding six of the components-Cia1, Cia2, MMS19, Nbp35, Nar1, and a putative Tah18-and showed using immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation that the last three of them localized to the cytoplasm of the cell. We then used comparative genomic and phylogenetic approaches to investigate the evolutionary history of these proteins. While most Blastocystis homologues branch with their eukaryotic counterparts, the putative Blastocystis Tah18 seems to have a separate evolutionary origin and therefore possibly a different function. Furthermore, our phylogenomic analyses revealed that all eight CIA components described in opisthokonts originated before the diversification of extant eukaryotic lineages and were likely already present in the last eukaryotic common ancestor (LECA). The Nbp35, Nar1 Cia1, and Cia2 proteins have been conserved during the subsequent evolutionary diversification of eukaryotes and are present in virtually all extant lineages, whereas the other CIA proteins have patchy phylogenetic distributions. Cia2 appears to be homologous to SufT, a component of the prokaryotic sulfur utilization factors (SUF) system, making this the first reported evolutionary link between the CIA and any other Fe/S biogenesis pathway. All of our results suggest that the CIA machinery is an ubiquitous biosynthetic pathway in eukaryotes, but its apparent plasticity in composition raises questions regarding how it functions in nonmodel organisms and how it interfaces with various iron/sulfur cluster systems (i.e., the iron/sulfur cluster, nitrogen fixation, and/or SUF system) found in eukaryotic cells.

Evidence for a hydrogenosomal-type anaerobic ATP generation pathway in Acanthamoeba castellanii.

Diverse, distantly-related eukaryotic lineages have adapted to low-oxygen environments, and possess mitochondrion-related organelles that have lost the capacity to generate adenosine triphosphate (ATP) through oxidative phosphorylation. A subset of these organelles, hydrogenosomes, has acquired a set of characteristic ATP generation enzymes commonly found in anaerobic bacteria. The recipient of these enzymes could not have survived prior to their acquisition had it not still possessed the electron transport chain present in the ancestral mitochondrion. In the divergence of modern hydrogenosomes from mitochondria, a transitional organelle must therefore have existed that possessed both an electron transport chain and an anaerobic ATP generation pathway. Here, we report a modern analog of this organelle in the habitually aerobic opportunistic pathogen, Acanthamoeba castellanii. This organism possesses a complete set of enzymes comprising a hydrogenosome-like ATP generation pathway, each of which is predicted to be targeted to mitochondria. We have experimentally confirmed the mitochondrial localizations of key components of this pathway using tandem mass spectrometry. This evidence is the first supported by localization and proteome data of a mitochondrion possessing both an electron transport chain and hydrogenosome-like energy metabolism enzymes. Our work provides insight into the first steps that might have occurred in the course of the emergence of modern hydrogenosomes.

The site-wise log-likelihood score is a good predictor of genes under positive selection.

The strength and direction of selection on the identity of an amino acid residue in a protein is typically measured by the ratio of the rate of non-synonymous substitutions to the rate of synonymous substitutions. In attempting to predict positively selected sites from amino acid alignments, we made the unexpected observation that the site likelihood of an alignment column for a given tree tends to be negatively correlated with the posterior probability that site is in the positive selection class under widely-used codon models. This is likely because positively selected sites tend to be more variable and display more "radical" amino acid changes; both of these features are expected to result in low site log-likelihoods. We explored the efficacy of using the site log-likelihood (SLL) score as a predictor for positive selection. Through simulation we show that a SLL-based test has a low false positive rate and comparable power as the codon models. In one case where the simulated data violated the assumption that synonymous substitution rates were constant across the sites, the codon models were not able to detect positive selection in the data while the SLL test did. We applied the new method to ten empirical datasets and found that it made similar predictions as the codon models in eight of them. For the tax gene dataset the SLL test seemed to produce more reasonable results. The SLL methods are a valuable complement to codon models, especially for some cases where the assumptions of codon models are likely violated.

Amoeba stages in the deepest branching heteroloboseans, including Pharyngomonas: evolutionary and systematic implications.

The taxon Heterolobosea (Excavata) is a major group of protists well known for its diversity of life stages. Most are amoebae capable of transforming into flagellates (amoeboflagellates), while others are known solely as flagellates or solely as amoebae. The deepest-branching heterolobosean taxon confirmed previously, Pharyngomonas, was generally assumed to be a pure flagellate, suggesting that the amoeba form arose later in the evolution of Heterolobosea sensu lato. Here we report that multiple isolates of Pharyngomonas are actually amoeboflagellates that also have cyst stages, with only amoebae transforming into cysts. The amoeba form of Pharyngomonas showed heterolobosean characteristics (e. g. eruptive movement), but also possessed unusual morphological features like slow-flowing crenulated hyaline crescents with conical subpseudopodia, finger-like projections and branching posterior extensions. Furthermore, phylogenetic analyses of 18S ribosomal RNA gene sequences that included two undescribed species of amoebae showed that Pharyngomonas is not the only deep-branching heterolobosean to possess an amoeba stage. These results suggest that possession of an amoeba stage was ancestral for Heterolobosea, unifying this taxon as a group of species with amoeba stages in their lifecycle or derived from organisms with such stages.

Complete local elimination of infectious trachoma from severely affected communities after six biannual mass azithromycin distributions.

OBJECTIVE: To determine whether infectious trachoma can be completely eliminated from severely affected villages. DESIGN: Cross-sectional survey of 2 villages previously enrolled and monitored over 42 months as part of a larger, group-randomized clinical trial. PARTICIPANTS: A total of 758 individuals residing in 2 villages with high baseline trachoma prevalence, of a total population of 768 (98.7%). METHODS: All members of the 2 villages were offered 6 biannual mass treatments with oral azithromycin. At 42 months, each current village member was examined. The right upper tarsal conjunctiva was everted and swabbed. Samples were processed for evidence of Chlamydia trachomatis RNA. MAIN OUTCOME MEASURES: Clinical activity by World Health Organization simplified grading scale for trachoma and laboratory evidence of chlamydial RNA. RESULTS: Average antibiotic coverage over the study period was 90% and 94% in the 2 villages. Clinical trachoma activity in children aged 1 to 5 years decreased from 78% and 83% in the 2 villages before treatment to 17% and 24% at 42 months. Polymerase chain reaction (PCR) evidence of infection in the same age group decreased from 48% to 0% in both villages at 42 months. When all age groups were examined, there were zero cases with evidence of chlamydial RNA among 758 total villagers tested. CONCLUSIONS: Biannual mass distribution of azithromycin can locally eliminate ocular chlamydial infection from severely affected communities.

Expanded phylogenies of canonical and non-canonical types of methionine adenosyltransferase reveal a complex history of these gene families in eukaryotes.

Most eukaryotes possess the highly-conserved enzyme methionine adenosyltransferase (MAT) that produces S-adenosyl-l-methionine, a molecule essential to a variety of cellular processes. However, a recent study revealed that genomes of a very few eukaryote lineages encode a highly divergent type of MAT (called MATX), instead of the canonical MAT enzyme. Since MATX-containing eukaryotes are phylogenetically interspersed with MAT-containing organisms, it is likely that the MATX gene was spread into the MAT-containing groups via multiple eukaryote-to-eukaryote lateral gene transfer events. Here, we further investigate the evolutionary history of these gene families by vastly increasing the sampling of species containing MAT (22 new taxa) and MATX (8 new taxa). Our expanded analyses reveal the first example of lateral transfer of a MAT gene between the pelagophycean alga Aureococcusanophagefferens and a cryptomonad. The increased MATX sampling also provided new insights into the evolution of MATX. Specifically, our MATX phylogeny robustly grouped the haptophyte homologues with the Aureococcus homologue to the exclusion of the diatom homologues, suggesting a transfer of the MATX gene between haptophytes and pelagophytes. Various scenarios of MAT and MATX gene family evolution in diatoms are re-evaluated in light of the new data.

A new divergent type of eukaryotic methionine adenosyltransferase is present in multiple distantly related secondary algal lineages.

S-adenosylmethionine is one of the most important metabolites in living cells and is synthesized in a single reaction catalyzed by methionine adenosyltransferase (MAT). At the sequence and structural level, this enzyme is one of the most conserved proteins known. Here we show that some representatives of three distantly related eukaryotic lineages--dinoflagellates, haptophytes, and euglenids--possess a highly divergent type of MAT, which we call MATX. Even though MATX contains all the sites known to be involved in catalysis and the association of monomers, it also has four insertions throughout the protein that are not observed in other MAT homologs. The phylogenetic distribution and affinities of MATX suggest that it originated in a single eukaryotic lineage and was spread via multiple events of eukaryote-to-eukaryote lateral gene transfer. We suggest a tentative model in which the origin of MATX is connected with the progression of secondary endosymbiosis.

A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution.

BACKGROUND: Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus). RESULTS: The analyses revealed a compact genome with few, if any, introns and very short 3' untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes--mostly encoding metabolic proteins--that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. CONCLUSION: Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution.

Rapid evolution in conformational space: a study of loop regions in a ubiquitous GTP binding domain.

The rapidly evolving subsets of a protein are often evident in multiple sequence alignments as poorly defined, gap-containing regions. We investigated the 3D context of these regions observed in 28 protein structures containing a GTP-binding domain assumed to be homologous to the transforming factor p21-RAS. The phylogenetic depth of this data set is such that it is possible to observe lineages sharing a common protein core that diverged early in the eukaryotic cell history. The sequence variability among these homolog proteins is directly linked to the structural variability of surface loops. We demonstrate that these regions are self-contained and thus mostly free of the evolutionary constraints imposed by the conserved core of the domain. These intraloop interactions have the property to create stem-like structures. Interestingly, these stem-like structures can be observed in loops of varying size, up to the size of small protein domains. We propose a model under which the diversity of protein topologies observed in these loops can be the product of a stochastic sampling of sequence and conformational space in a near-neutral fashion, while the proximity of the functional features of the domain core allows novel beneficial traits to be fixed. Our comparative observations, limited here to the proteins containing the RAS-like GTP-binding domain, suggest that a stochastic process of insertion/deletion analogous to "budding" of loops is a likely mechanism of structural innovation. Such a framework could be experimentally exploited to investigate the folding of increasingly complex model inserts.