Meteora sporadica, a protist with incredible cell architecture, is related to Hemimastigophora.

"Kingdom-level" branches are being added to the tree of eukaryotes at a rate approaching one per year, with no signs of slowing down.1,2,3,4 Some are completely new discoveries, whereas others are morphologically unusual protists that were previously described but lacked molecular data. For example, Hemimastigophora are predatory protists with two rows of flagella that were known since the 19th century but proved to represent a new deep-branching eukaryote lineage when phylogenomic analyses were conducted.2Meteora sporadica5 is a protist with a unique morphology; cells glide over substrates along a long axis of anterior and posterior projections while a pair of lateral "arms" swing back and forth, a motility system without any obvious parallels. Originally, Meteora was described by light microscopy only, from a short-term enrichment of deep-sea sediment. A small subunit ribosomal RNA (SSU rRNA) sequence was reported recently, but the phylogenetic placement of Meteora remained unresolved.6 Here, we investigated two cultivated Meteora sporadica isolates in detail. Transmission electron microscopy showed that both the anterior-posterior projections and the arms are supported by microtubules originating from a cluster of subnuclear microtubule organizing centers (MTOCs). Neither have a flagellar axoneme-like structure. Sequencing the mitochondrial genome showed this to be among the most gene-rich known, outside jakobids. Remarkably, phylogenomic analyses of 254 nuclear protein-coding genes robustly support a close relationship with Hemimastigophora. Our study suggests that Meteora and Hemimastigophora together represent a morphologically diverse "supergroup" and thus are important for resolving the tree of eukaryote life and early eukaryote evolution.

Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

Inteins are self-splicing protein elements found in viruses and all three domains of life. How the DNA encoding these selfish elements spreads within and between genomes is poorly understood, particularly in eukaryotes where inteins are scarce. Here, we show that the nuclear genomes of three strains of Anaeramoeba encode between 45 and 103 inteins, in stark contrast to four found in the most intein-rich eukaryotic genome described previously. The Anaeramoeba inteins reside in a wide range of proteins, only some of which correspond to intein-containing proteins in other eukaryotes, prokaryotes, and viruses. Our data also suggest that viruses have contributed to the spread of inteins in Anaeramoeba and the colonization of new alleles. The persistence of Anaeramoeba inteins might be partly explained by intragenomic movement of intein-encoding regions from gene to gene. Our intein dataset greatly expands the spectrum of intein-containing proteins and provides insights into the evolution of inteins in eukaryotes.

RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins.

RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.

Rescue of Escherichia coli auxotrophy by de novo small proteins.

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (≤50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

Ancient and pervasive expansion of adaptin-related vesicle coat machinery across Parabasalia.

The eukaryotic phylum Parabasalia is composed primarily of anaerobic, endobiotic organisms such as the veterinary parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis, the latter causing the most prevalent, non-viral, sexually transmitted disease world-wide. Although a parasitic lifestyle is generally associated with a reduction in cell biology, T. vaginalis provides a striking counter-example. The 2007 T. vaginalis genome paper reported a massive and selective expansion of encoded proteins involved in vesicle trafficking, particularly those implicated in the late secretory and endocytic systems. Chief amongst these were the hetero-tetrameric adaptor proteins or 'adaptins', with T. vaginalis encoding ∼3.5 times more such proteins than do humans. The provenance of such a complement, and how it relates to the transition from a free-living or endobiotic state to parasitism, remains unclear. In this study, we performed a comprehensive bioinformatic and molecular evolutionary investigation of the heterotetrameric cargo adaptor-derived coats, comparing the molecular complement and evolution of these proteins between T. vaginalis, T. foetus and the available diversity of endobiotic parabasalids. Notably, with the recent discovery of Anaeramoeba spp. as the free-living sister lineage to all parabasalids, we were able to delve back to time points earlier in the lineage's history than ever before. We found that, although T. vaginalis still encodes the most HTAC subunits amongst parabasalids, the duplications giving rise to the complement took place more deeply and at various stages across the lineage. While some duplications appear to have convergently shaped the parasitic lineages, the largest jump is in the transition from free-living to endobiotic lifestyle with both gains and losses shaping the encoded complement. This work details the evolution of a cellular system across an important lineage of parasites and provides insight into the evolutionary dynamics of an example of expansion of protein machinery, counter to the more common trends observed in many parasitic systems.

Draft genomes of Blastocystis subtypes from human samples of Colombia.

BACKGROUND: Blastocystis is one of the most common eukaryotic microorganisms colonizing the intestines of both humans and animals, but the conditions under which it may be a pathogen are unclear. METHODS: To study the genomic characteristics of circulating subtypes (ST) in Colombia, we established nine xenic cultures from Blastocystis isolated from human fecal samples, we identified 10 different subtypes, since one sample had a mixed infection. Thus, the genomes of the subtypes ST1 (n = 3), ST2 (n = 1), ST3 (n = 2), ST6 (n = 1), ST7 (n = 1), and ST8 (n = 2) were sequenced using Illumina and Oxford Nanopore Technologies (ONT). RESULTS: Analyses of these draft nuclear genomes indicated remarkable diversity in terms of genome size and guanine-cytosine (GC) content among the compared STs. Illumina sequencing-only draft genomes contained 824 to 2077 scaffolds, with total genome size ranging from 12 to 13.2 Mb and N50 values ranging from 10,585 to 29,404 base pairs (bp). The genome of one ST1 isolate was sequenced using ONT. This assembly was more contiguous, with a size of 20 million base pairs (Mb) spread over 116 scaffolds, and an N50 of 248,997 bp. CONCLUSION: This work represents one of the few large-scale comparative genomic analyses of Blastocystis isolates, providing an additional glimpse into its genomic diversity.

Comprehensive characterization of Cysteine-rich protein-coding genes of Giardia lamblia and their role during antigenic variation.

Giardia lamblia encodes several families of cysteine-rich proteins, including the Variant-specific Surface Proteins (VSPs) involved in the process of antigenic variation. Their characteristics, definition and relationships are still controversial. An exhaustive analysis of the Cys-rich families including organization, features, evolution and levels of expression was performed, by combining pattern searches and predictions with massive sequencing techniques. Thus, a new classification for Cys-rich proteins, genes and pseudogenes that better describes their involvement in Giardia's biology is presented. Moreover, three novel characteristics exclusive to the VSP genes, comprising an Initiator element/Kozak-like sequence, an extended polyadenylation signal and a unique pattern of mutually exclusive transcript accumulation are presented, as well as the finding that High Cysteine Membrane Proteins, upregulated under stress, may protect the parasite during VSP switching. These results allow better interpretation of previous reports providing the basis for further studies of the biology of this early-branching eukaryote.

A Detailed Gene Expression Map of Giardia Encystation.

Giardia intestinalis is an intestinal protozoan parasite that causes diarrheal infections worldwide. A key process to sustain its chain of transmission is the formation of infectious cysts in the encystation process. We combined deep RNAseq of a broad range of encystation timepoints to produce a high-resolution gene expression map of Giardia encystation. This detailed transcriptomic map of encystation confirmed a gradual change of gene expression along the time course of encystation, showing the most significant gene expression changes during late encystation. Few genes are differentially expressed early in encystation, but the major cyst wall proteins CWP-1 and -2 are highly up-regulated already after 3.5 h encystation. Several transcription factors are sequentially up-regulated throughout the process, but many up-regulated genes at 7, 10, and 14 h post-induction of encystation have binding sites in the upstream regions for the Myb2 transcription factor, suggesting that Myb2 is a master regulator of encystation. We observed major changes in gene expression of several meiotic-related genes from 10.5 h of encystation to the cyst stage, and at 17.5 h encystation, there are changes in many different metabolic pathways and protein synthesis. Late encystation, 21 h to cysts, show extensive gene expression changes, most of all in VSP and HCMP genes, which are involved in antigenic variation, and genes involved in chromatin modifications. This high-resolution gene expression map of Giardia encystation will be an important tool in further studies of this important differentiation process.

Anaeramoebae are a divergent lineage of eukaryotes that shed light on the transition from anaerobic mitochondria to hydrogenosomes.

Discoveries of diverse microbial eukaryotes and their inclusion in comprehensive phylogenomic analyses have crucially re-shaped the eukaryotic tree of life in the 21st century.1 At the deepest level, eukaryotic diversity comprises 9-10 "supergroups." One of these supergroups, the Metamonada, is particularly important to our understanding of the evolutionary dynamics of eukaryotic cells, including the remodeling of mitochondrial function. All metamonads thrive in low-oxygen environments and lack classical aerobic mitochondria, instead possessing mitochondrion-related organelles (MROs) with metabolisms that are adapted to low-oxygen conditions. These MROs lack an organellar genome, do not participate in the Krebs cycle and oxidative phosphorylation,2 and often synthesize ATP by substrate-level phosphorylation coupled to hydrogen production.3,4 The events that occurred during the transition from an oxygen-respiring mitochondrion to a functionally streamlined MRO early in metamonad evolution remain largely unknown. Here, we report transcriptomes of two recently described, enigmatic, anaerobic protists from the genus Anaeramoeba.5 Using phylogenomic analysis, we show that these species represent a divergent, phylum-level lineage in the tree of metamonads, emerging as a sister group of the Parabasalia and reordering the deep branching order of the metamonad tree. Metabolic reconstructions of the Anaeramoeba MROs reveal many "classical" mitochondrial features previously not seen in metamonads, including a disulfide relay import system, propionate production, and amino acid metabolism. Our findings suggest that the cenancestor of Metamonada likely had MROs with more classical mitochondrial features than previously anticipated and demonstrate how discoveries of novel lineages of high taxonomic rank continue to transform our understanding of early eukaryote evolution.

Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist.

Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family.

The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5'-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha-beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.

Bacteria can be infected by viruses known as bacteriophages. These viruses inject their genetic material into bacterial cells and use the bacteria's own machinery to build the proteins they need to survive and infect other cells. To protect themselves, bacteria produce a molecule called S-adenosyl methionine, or SAM for short, which deposits marks on the bacteria's DNA. These marks help the bacteria distinguish their own genetic material from the genetic material of foreign invaders: any DNA not bearing the mark from SAM will be immediately broken down by the bacterial cell. This system helps to block many types of bacteriophage infections, but not all. Some bacteriophages carry genes that code for enzymes called SAMases, which can break down SAM, switching off the bacteria's defenses. The most well-known SAMase was first discovered in the 1960s in a bacteriophage called T3. Chemical studies of this SAMase suggested that it works as a 'hydrolase', meaning that it uses water to break SAM apart. New SAMases have since been discovered in bacteriophages from environmental water samples, which, despite being able to degrade SAM, are genetically dissimilar to one another and the SAMase in T3. This brings into question whether these enzymes all use the same mechanism to break SAM down. To gain a better understanding of how these SAMases work, Guo, Söderholm, Kanchugal, Isaksen et al. solved the crystal structure of one of the newly discovered enzymes called Svi3-3. This revealed three copies of the Svi3-3 enzyme join together to form a unit that SAM binds to at the border between two of the enzymes. Computer simulations of this structure suggested that Svi3-3 holds SAM in a position where it cannot interact with water, and that once in the grip of the SAMase, SAM instead reacts with itself and splits into two. Experiments confirmed these predictions for Svi3-3 and the other tested SAMases. Furthermore, the SAMase from bacteriophage T3 was also found to degrade SAM using the same mechanism. This shows that this group of SAMases are not hydrolases as originally thought, but in fact 'lyases': enzymes that break molecules apart without using water. These findings form a starting point for further investigations into how SAM lyases help bacteriophages evade detection. SAM has various different functions in other living organisms, and these lyases could be used to modulate the levels of SAM in future studies investigating its role.

The compact genome of Giardia muris reveals important steps in the evolution of intestinal protozoan parasites.

Diplomonad parasites of the genus Giardia have adapted to colonizing different hosts, most notably the intestinal tract of mammals. The human-pathogenic Giardia species, Giardia intestinalis, has been extensively studied at the genome and gene expression level, but no such information is available for other Giardia species. Comparative data would be particularly valuable for Giardia muris, which colonizes mice and is commonly used as a prototypic in vivo model for investigating host responses to intestinal parasitic infection. Here we report the draft-genome of G. muris. We discovered a highly streamlined genome, amongst the most densely encoded ever described for a nuclear eukaryotic genome. G. muris and G. intestinalis share many known or predicted virulence factors, including cysteine proteases and a large repertoire of cysteine-rich surface proteins involved in antigenic variation. Different to G. intestinalis, G. muris maintains tandem arrays of pseudogenized surface antigens at the telomeres, whereas intact surface antigens are present centrally in the chromosomes. The two classes of surface antigens engage in genetic exchange. Reconstruction of metabolic pathways from the G. muris genome suggest significant metabolic differences to G. intestinalis. Additionally, G. muris encodes proteins that might be used to modulate the prokaryotic microbiota. The responsible genes have been introduced in the Giardia genus via lateral gene transfer from prokaryotic sources. Our findings point to important evolutionary steps in the Giardia genus as it adapted to different hosts and it provides a powerful foundation for mechanistic exploration of host-pathogen interaction in the G. muris-mouse pathosystem.

Evolution of a New Function by Fusion between Phage DNA and a Bacterial Gene.

Mobile genetic elements, such as plasmids, phages, and transposons, are important sources for evolution of novel functions. In this study, we performed a large-scale screening of metagenomic phage libraries for their ability to suppress temperature-sensitivity in Salmonella enterica serovar Typhimurium strain LT2 mutants to examine how phage DNA could confer evolutionary novelty to bacteria. We identified an insert encoding 23 amino acids from a phage that when fused with a bacterial DNA-binding repressor protein (LacI) resulted in the formation of a chimeric protein that localized to the outer membrane. This relocalization of the chimeric protein resulted in increased membrane vesicle formation and an associated suppression of the temperature sensitivity of the bacterium. Both the host LacI protein and the extracellular 23-amino acid stretch are necessary for the generation of the novel phenotype. Furthermore, mutational analysis of the chimeric protein showed that although the native repressor function of the LacI protein is maintained in this chimeric structure, it is not necessary for the new function. Thus, our study demonstrates how a gene fusion between foreign DNA and bacterial DNA can generate novelty without compromising the native function of a given gene.

Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida.

BACKGROUND: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive. RESULTS: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis. CONCLUSION: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

Proximity Staining Using Enzymatic Protein Tagging in Diplomonads.

The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3'-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalisIMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida This protocol was also applicable to the human pathogen Giardia intestinalis Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function.

One key concept in the evolution of new functions is the ability of enzymes to perform promiscuous side-reactions that serve as a source of novelty that may become beneficial under certain conditions. Here, we identify a mechanism where a bacteriophage-encoded enzyme introduces novelty by inducing expression of a promiscuous bacterial enzyme. By screening for bacteriophage DNA that rescued an auxotrophic Escherichia coli mutant carrying a deletion of the ilvA gene, we show that bacteriophage-encoded S-adenosylmethionine (SAM) hydrolases reduce SAM levels. Through this perturbation of bacterial metabolism, expression of the promiscuous bacterial enzyme MetB is increased, which in turn complements the absence of IlvA. These results demonstrate how foreign DNA can increase the metabolic capacity of bacteria, not only by transfer of bona fide new genes, but also by bringing cryptic bacterial functions to light via perturbations of cellular physiology.

On the reversibility of parasitism: adaptation to a free-living lifestyle via gene acquisitions in the diplomonad Trepomonas sp. PC1.

BACKGROUND: It is generally thought that the evolutionary transition to parasitism is irreversible because it is associated with the loss of functions needed for a free-living lifestyle. Nevertheless, free-living taxa are sometimes nested within parasite clades in phylogenetic trees, which could indicate that they are secondarily free-living. Herein, we test this hypothesis by studying the genomic basis for evolutionary transitions between lifestyles in diplomonads, a group of anaerobic eukaryotes. Most described diplomonads are intestinal parasites or commensals of various animals, but there are also free-living diplomonads found in oxygen-poor environments such as marine and freshwater sediments. All these nest well within groups of parasitic diplomonads in phylogenetic trees, suggesting that they could be secondarily free-living. RESULTS: We present a transcriptome study of Trepomonas sp. PC1, a diplomonad isolated from marine sediment. Analysis of the metabolic genes revealed a number of proteins involved in degradation of the bacterial membrane and cell wall, as well as an extended set of enzymes involved in carbohydrate degradation and nucleotide metabolism. Phylogenetic analyses showed that most of the differences in metabolic capacity between free-living Trepomonas and the parasitic diplomonads are due to recent acquisitions of bacterial genes via gene transfer. Interestingly, one of the acquired genes encodes a ribonucleotide reductase, which frees Trepomonas from the need to scavenge deoxyribonucleosides. The transcriptome included a gene encoding squalene-tetrahymanol cyclase. This enzyme synthesizes the sterol substitute tetrahymanol in the absence of oxygen, potentially allowing Trepomonas to thrive under anaerobic conditions as a free-living bacterivore, without depending on sterols from other eukaryotes. CONCLUSIONS: Our findings are consistent with the phylogenetic evidence that the last common ancestor of diplomonads was dependent on a host and that Trepomonas has adapted secondarily to a free-living lifestyle. We believe that similar studies of other groups where free-living taxa are nested within parasites could reveal more examples of secondarily free-living eukaryotes.

Comparative Cell Biology and Evolution of Annexins in Diplomonads.

Annexins are multifunctional, calcium-binding proteins found in organisms across all kingdoms. Most studies of annexins from single-celled eukaryotes have focused on the alpha-giardins, proteins assigned to the group E annexins, expressed by the diplomonad Giardia intestinalis. We have characterized the annexin gene family in another diplomonad parasite, Spironucleus salmonicida, by phylogenetic and experimental approaches. We constructed a comprehensive phylogeny of the diplomonad group E annexins and found that they are abundant across the group with frequent gene duplications and losses. The annexins of S. salmonicida were found to be related to alpha-giardins but with better-preserved type II Ca(2+) coordination sites. Two annexins were confirmed to bind phospholipids in a Ca(2+)-dependent fashion but with different specificities. Superresolution and confocal microscopy of epitope-tagged S. salmonicida annexins revealed localization to distinct parts of the cytoskeleton and membrane. The ultrastructural details of the localization of several annexins were determined by proximity labeling and transmission electron microscopy. Two annexins localize to a novel cytoskeletal structure in the anterior of the cell. Our results show that the annexin gene family is expanded in diplomonads and that these group E annexins are associated mostly with cytoskeletal and membrane structures. IMPORTANCE Annexins are proteins that associate with phospholipids in a Ca(2+)-dependent fashion. These proteins have been intensely studied in animals and plants because of their importance in diverse cellular processes, yet very little is known about annexins in single-celled eukaryotes, which represent the largest diversity of organisms. The human intestinal parasite Giardia intestinalis is known to have more annexins than humans, and they contribute to its pathogenic potential. In this study, we investigated the annexin complement in the salmon pathogen Spironucleus salmonicida, a relative of G. intestinalis. We found that S. salmonicida has a large repertoire of annexins and that the gene family has expanded separately across diplomonads, with members showing sequence diversity similar to that seen across kingdom-level groups such as plants and animals. S. salmonicida annexins are prominent components of the cytoskeleton and membrane. Two annexins are associated with a previously unrecognized structure in the anterior of the cell.