Lophomonas as a respiratory pathogen-jumping the gun.

Human infections with the protozoan Lophomonas have been increasingly reported in the medical literature over the past three decades. Initial reports were based on microscopic identification of the purported pathogen in respiratory specimens. Later, a polymerase chain reaction (PCR) was developed to detect Lophomonas blattarum, following which there has been a significant increase in reports. In this minireview, we thoroughly examine the published reports of Lophomonas infection to evaluate its potential role as a human pathogen. We examined the published images and videos of purported Lophomonas, compared its morphology and motility characteristics with host bronchial ciliated epithelial cells and true L. blattarum derived from cockroaches, analyzed the published PCR that is being used for its diagnosis, and reviewed the clinical data of patients reported in the English and Chinese literature. From our analysis, we conclude that the images and videos from human specimens do not represent true Lophomonas and are predominantly misidentified ciliated epithelial cells. Additionally, we note that there is insufficient clinical evidence to attribute the cases to Lophomonas infection, as the clinical manifestations are non-specific, possibly caused by other infections and comorbidities, and there is no associated tissue pathology attributable to Lophomonas. Finally, our analysis reveals that the published PCR is not specific to Lophomonas and can amplify DNA from commensal trichomonads. Based on this thorough review, we emphasize the need for rigorous scientific scrutiny before a microorganism is acknowledged as a novel human pathogen and discuss the potential harms of misdiagnoses for patient care and scientific literature.

Molecular phylogeny of Spirotrichonymphea (Parabasalia) with emphasis on Spironympha, Spirotrichonympha, and three new genera Pseudospironympha, Nanospironympha, and Brugerollina.

Spirotrichonymphea, one of the six classes of phylum Parabasalia, are characterized by bearing many flagella in spiral rows, and they occur exclusively in the guts of termites. Phylogenetic relationships among the 13 described genera are not well understood due to complex morphological evolution and a paucity of molecular data. One such understudied genus is Spironympha. It has been variously considered a valid genus, a subgenus of Spirotrichonympha, or an "immature" life cycle stage of Spirotrichonympha. To clarify this, we sequenced the small subunit rRNA gene sequences of Spironympha and Spirotrichonympha cells isolated from the hindguts of Reticulitermes species and Hodotermopsis sjostedti and confirmed the molecular identity of H. sjostedti symbionts using fluorescence in situ hybridization. Spironympha as currently circumscribed is polyphyletic, with both H. sjostedti symbiont species branching separately from the "true" Spironympha from Reticulitermes. Similarly, the Spirotrichonympha symbiont of H. sjostedti branches separately from the "true" Spirotrichonympha found in Reticulitermes. Our data support Spironympha from Reticulitermes as a valid genus most closely related to Spirotrichonympha, though its monophyly and interspecific relationships are not resolved in our molecular phylogenetic analysis. We propose three new genera to accommodate the H. sjostedti symbionts and two new species of Spirotrichonympha from Reticulitermes.

True molecular phylogenetic position of the cockroach gut commensal Lophomonas blattarum (Lophomonadida, Parabasalia).

Lophomonas blattarum is a facultative commensal gut dweller of common pest cockroaches. Its cells are roughly spherical in shape with an apical tuft of ~50 flagella. Controversially, it has been implicated in human respiratory infections based on light microscopic observations of similarly shaped cells in sputum or bronchoalveolar lavage fluid. Here, we have sequenced the 18S rRNA gene of L. blattarum and its sole congener, Lophomonas striata, isolated from cockroaches. Both species branch in a fully supported clade with Trichonymphida, consistent with a previous study of L. striata, but not consistent with sequences from human samples attributed to L. blattarum.

Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes.

The division of life into producers and consumers is blurred by evolution. For example, eukaryotic phototrophs can lose the capacity to photosynthesize, although they may retain vestigial plastids that perform other essential cellular functions. Chrysophyte algae have undergone a particularly large number of photosynthesis losses. Here, we present a plastid genome sequence from a nonphotosynthetic chrysophyte, "Spumella" sp. NIES-1846, and show that it has retained a nearly identical set of plastid-encoded functions as apicomplexan parasites. Our transcriptomic analysis of 12 different photosynthetic and nonphotosynthetic chrysophyte lineages reveals remarkable convergence in the functions of these nonphotosynthetic plastids, along with informative lineage-specific retentions and losses. At one extreme, Cornospumella fuschlensis retains many photosynthesis-associated proteins, although it appears to have lost the reductive pentose phosphate pathway and most plastid amino acid metabolism pathways. At the other extreme, Paraphysomonas lacks plastid-targeted proteins associated with gene expression and all metabolic pathways that require plastid-encoded partners, indicating a complete loss of plastid DNA in this genus. Intriguingly, some of the nucleus-encoded proteins that once functioned in the expression of the Paraphysomonas plastid genome have been retained. These proteins were likely to have been dual targeted to the plastid and mitochondria of the chrysophyte ancestor, and are uniquely targeted to the mitochondria in Paraphysomonas Our comparative analyses provide insights into the process of functional reduction in nonphotosynthetic plastids.

Molecular Phylogenetic Position of Hoplonympha natator (Trichonymphea, Parabasalia): Horizontal Symbiont Transfer or Differential Loss?

Hoplonympha natator is an obligate symbiont of Paraneotermes simplicicornis (Kalotermitidae), from southwestern North America. Another Hoplonympha species inhabits Hodotermopsis sjostedti (Archotermopsidae), from montane Southeast Asia. The large phylogenetic and geographical distance between the hosts makes the distribution of Hoplonympha puzzling. Here, we report the phylogenetic position of H. natator from P. simplicicornis through maximum likelihood and Bayesian analysis of 18S rRNA genes. The two Hoplonympha species form a clade with a deep node, making a recent symbiont transfer unlikely. The distribution of Hoplonympha may be due to an ancient transfer or strict vertical inheritance with differential loss from other hosts.

The Complete Protist Symbiont Communities of Coptotermes formosanus and Coptotermes gestroi: Morphological and Molecular Characterization of Five New Species.

Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann) (Blattoidea: Rhinotermitidae) are invasive subterranean termite pest species with a major global economic impact. However, the descriptions of the mutualistic protist communities harbored in their respective hindguts remain fragmentary. The C. formosanus hindgut has long been considered to harbor three protist species, Pseudotrichonympha grassii (Trichonymphida), Holomastigotoides hartmanni, and Cononympha (Spirotrichonympha) leidyi (Spirotrichonymphida), but molecular data have suggested that the diversity may be higher. Meanwhile, the C. gestroi community remains undescribed except for Pseudotrichonympha leei. To complete the characterization of these communities, hindguts of workers from both termite species were investigated using single-cell PCR, microscopy, cell counts, and 18S rRNA amplicon sequencing. The two hosts were found to harbor intriguingly parallel protist communities, each consisting of one Pseudotrichonympha species, two Holomastigotoides species, and two Cononympha species. All protist species were unique to their respective hosts, which last shared a common ancestor ~18 MYA. The relative abundances of protist species in each hindgut differed remarkably between cell count data and 18S rRNA profiles, calling for caution in interpreting species abundances from amplicon data. This study will enable future research in C. formosanus and C. gestroi hybrids, which provide a unique opportunity to study protist community inheritance, compatibility, and potential contribution to hybrid vigor.

Molecular Identity of Holomastigotes (Spirotrichonymphea, Parabasalia) with Descriptions of Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp.

Holomastigotes is a protist genus (Parabasalia: Spirotrichonymphea) that resides in the hindguts of "lower" termites. It can be distinguished from other parabasalids by spiral flagellar bands that run along the entire length of the cell, an anterior nucleus, a reduced or absent axostyle, the presence of spherical vesicles inside the cells, and the absence of ingested wood particles. Eight species have been described based on their morphology so far, although no molecular data were available prior to this study. We determined the 18S rRNA gene sequences of Holomastigotes from the hindguts of Hodotermopsis sjostedti, Reticulitermes flavipes, Reticulitermes lucifugus, and Reticulitermes tibialis. Phylogenetic analyses placed all sequences in an exclusive and well-supported clade with the type species, Holomastigotes elongatum from R. lucifugus. However, the phylogenetic position of Holomastigotes within the Spirotrichonymphea was not resolved. We describe two new species, Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp., inhabiting the hindguts of R. flavipes and R. tibialis, respectively.

Effects of surface treatments on trapping with DC insulator-based dielectrophoresis.

A central challenge in measuring the biophysical properties of cells with electrokinetic approaches is the assignment of these biophysical properties to specific biological characteristics. Changes in the electrokinetic behavior of cells may come from mutations, altered gene expression levels, post-translation modifications, or environmental effects. Here we assess the electrokinetic behavior of chemically surface-modified bacterial cells in order to gain insight into the biophysical properties that are specifically affected by changes in surface chemistry. Using E. coli as a scaffold, an amine coupling reaction was used to covalently attach glycine, spermine, bovine serum albumin (protein), or 7-amino-4-methyl-3-coumarinylacetic acid (fluorescent dye) to the free carboxylic acid groups on the surface of the cells. These populations, along with unlabeled control cells, were subject to electrokinetic and dielectrophoretic measurements to quantify any changes in the biophysical properties upon alteration. The properties associated with each electrokinetic force are discussed relative to the specific reactant used. We conclude that relatively modest and superficial changes to cell surfaces can cause measurable changes in their biophysical properties.

New Species of Spirotrichonympha from Reticulitermes and the Relationships Among Genera in Spirotrichonymphea (Parabasalia).

Spirotrichonymphea is a class of hypermastigote parabasalids defined by their spiral rows of many flagella. They are obligate hindgut symbionts of lower termites. Despite more than 100 yr of morphological and ultrastructural study, the group remains poorly characterised by molecular data and the phylogenetic positions and taxonomic validity of most genera remain in question. The genus Spirotrichonympha has been reported to inhabit several termite genera, including Reticulitermes, Coptotermes, and Hodotermopsis. The type species for this genus, Spirotrichonympha flagellata, was described from Reticulitermes lucifugus but no molecular data are yet available for this species. In this study, three new Spirotrichonympha species are described from three species of Reticulitermes. Their molecular phylogenetic position indicates that the genus is not monophyletic, as Spirotrichonympha species from Coptotermes, Paraneotermes, and Hodotermopsis branch separately. In contrast, the genus Holomastigotoides is monophyletic, as demonstrated using new sequences from Holomastigotoides species. The presence of Holomastigotoides in Prorhinotermes and the distinct phylogenetic positions of Spirotrichonympha from Reticulitermes and Coptotermes are consistent with a previously proposed symbiont fauna replacement in the ancestor of Reticulitermes.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Evolutionary Origins of Rhizarian Parasites.

The SAR group (Stramenopila, Alveolata, Rhizaria) is one of the largest clades in the tree of eukaryotes and includes a great number of parasitic lineages. Rhizarian parasites are obligate and have devastating effects on commercially important plants and animals but despite this fact, our knowledge of their biology and evolution is limited. Here, we present rhizarian transcriptomes from all major parasitic lineages in order to elucidate their evolutionary relationships using a phylogenomic approach. Our results suggest that Ascetosporea, parasites of marine invertebrates, are sister to the novel clade Apofilosa. The phytomyxean plant parasites branch sister to the vampyrellid algal ectoparasites in the novel clade Phytorhiza. They also show that Ascetosporea + Apofilosa + Retaria + Filosa + Phytorhiza form a monophyletic clade, although the branching pattern within this clade is difficult to resolve and appears to be model-dependent. Our study does not support the monophyly of the rhizarian parasitic lineages (Endomyxa), suggesting independent origins for rhizarian animal and plant parasites.

Molecular Evidence for the Polyphyly of Macrotrichomonas (Parabasalia: Cristamonadea) and a Proposal for Macrotrichomonoides n. gen.

Macrotrichomonas (Cristamonadea: Parabasalia) is an anaerobic, amitochondriate flagellate symbiont of termite hindguts. It is noteworthy for being large but not structurally complex compared with other large parabasalians, and for retaining a structure similar in appearance to the undulating membrane (UM) of small flagellates closely related to cristamonads, e.g. Tritrichomonas. Here, we have characterised the SSU rDNA from two species described as Macrotrichomonas: M. restis Kirby 1942 from Neotermes jouteli and M. lighti Connell 1932 from Paraneotermes simplicicornis. These species do not form a clade: M. lighti branches with previously characterised Macrotrichomonas sequences from Glyptotermes, while M. restis branches with the genus Metadevescovina. We examined the M. restis UM by light microscopy, scanning electron microscopy, and transmission electron microscopy, and we find common characteristics with the proximal portion of the robust recurrent flagellum of devescovinids. Altogether, we show the genus Macrotrichomonas to be polyphyletic and propose transferring M. restis to a new genus, Macrotrichomonoides. We also hypothesise that the macrotrichomonad body plan represents the ancestral state of cristamonads, from which other major forms evolved.

The phylogenetic position of Kofoidia loriculata (Parabasalia) and its implications for the evolution of the Cristamonadea.

Kofoidia loriculata is a parabasalid symbiont inhabiting the hindgut of the lower termite Paraneotermes simplicicornis. It was initially described as a lophomonad due to its apical tuft of multiple flagella that disintegrate during cell division, but its phylogenetic relationships have not been investigated using molecular evidence. From single cell isolations, we sequenced the small subunit rRNA gene and determined that K. loriculata falls within the Cristamonadea, but is unrelated to other lophomonads. This analysis further demonstrates the polyphyly of the lophomonads and the necessity to re-assess the morphological and cellular evolution of the Cristamonadea.

Protist symbionts of termites: diversity, distribution, and coevolution.

The symbiosis between termites and their hindgut protists is mutually obligate and vertically inherited. It was established by the late Jurassic in the cockroach ancestors of termites as they transitioned to wood feeding. Since then, protist symbionts have been transmitted from host generation to host generation by proctodeal trophallaxis (anal feeding). The protists belong to multiple lineages within the eukaryotic superphylum Metamonada. Most of these lineages have evolved large cells with complex morphology, unlike the non-termite-associated Metamonada. The species richness and taxonomic composition of symbiotic protist communities varies widely across termite lineages, especially within the deep-branching clade Teletisoptera. In general, closely related termites tend to harbour closely related protists, and deep-branching termites tend to harbour deep-branching protists, reflecting their broad-scale co-diversification. A closer view, however, reveals a complex distribution of protist lineages across hosts. Some protist taxa are common, some are rare, some are widespread, and some are restricted to a single host family or genus. Some protist taxa can be found in only a few, distantly related, host species. Thus, the long history of co-diversification in this symbiosis has been complicated by lineage-specific loss of symbionts, transfer of symbionts from one host lineage to another, and by independent diversification of the symbionts relative to their hosts. This review aims to introduce the biology of this important symbiosis and serve as a gateway to the diversity and systematics literature for both termites and protists. A searchable database with all termite-protist occurrence records and taxonomic references is provided as a supplementary file to encourage and facilitate new research in this field.

Morphology and molecular phylogeny of Staurojoenina mulleri sp. nov. (Trichonymphida, Parabasalia) from the hindgut of the kalotermitid Neotermes jouteli.

Staurojoenina is a large and structurally complex genus of hypermastigont parabasalians found in the hindgut of lower termites. Although several species of Staurojoenina have been described worldwide, all Staurojoenina observed to date in different species of North American termites have been treated as the same species, S. assimilis. Here, we characterize Staurojoenina from the North American termite Neotermes jouteli using light microscopy, scanning electron microscopy, and phylogenetic analysis of small subunit ribosomal RNA, and compare it with S. assimilis from its type host, Incisitermes minor. The basic morphological characteristics of the N. jouteli symbiont, including its abundant bacterial epibionts, are similar as far as they may be compared with existing data from S. assimilis, although not consistently identical. In contrast, we find that they are extremely distantly related at the molecular level, sharing a pairwise similarity of SSU rRNA genes comparable to that seen between different genera or even families of other parabasalians. Based on their evolutionary distance and habitat in different termite genera, we consider the N. jouteli Staurojoenina to be distinct from S. assimilis, and describe a new species, Staurojoenina mulleri, in honor of the pioneering parabasalian researcher, Miklos Muller.

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.

An examination of protist diversity in serpentinization-hosted ecosystems of the Samail Ophiolite of Oman.

In the Samail Ophiolite of Oman, the geological process of serpentinization produces reduced, hydrogen rich, hyperalkaline (pH > 11) fluids. These fluids are generated through water reacting with ultramafic rock from the upper mantle in the subsurface. On Earth's continents, serpentinized fluids can be expressed at the surface where they can mix with circumneutral surface water and subsequently generate a pH gradient (∼pH 8 to pH > 11) in addition to variations in other chemical parameters such as dissolved CO2, O2, and H2. Globally, archaeal and bacterial community diversity has been shown to reflect geochemical gradients established by the process of serpentinization. It is unknown if the same is true for microorganisms of the domain Eukarya (eukaryotes). In this study, using 18S rRNA gene amplicon sequencing, we explore the diversity of microbial eukaryotes called protists in sediments of serpentinized fluids in Oman. We demonstrate that protist community composition and diversity correlate significantly with variations in pH, with protist richness being significantly lower in sediments of hyperalkaline fluids. In addition to pH, the availability of CO2 to phototrophic protists, the composition of potential food sources (prokaryotes) for heterotrophic protists and the concentration of O2 for anaerobic protists are factors that likely shape overall protist community composition and diversity along the geochemical gradient. The taxonomy of the protist 18S rRNA gene sequences indicates the presence of protists that are involved in carbon cycling in serpentinized fluids of Oman. Therefore, as we evaluate the applicability of serpentinization for carbon sequestration, the presence and diversity of protists should be considered.

Characterization of microbiomic and geochemical compositions across the photosynthetic fringe.

Hot spring outflow channels provide geochemical gradients that are reflected in microbial community compositions. In many hot spring outflows, there is a distinct visual demarcation as the community transitions from predominantly chemotrophs to having visible pigments from phototrophs. It has been hypothesized that this transition to phototrophy, known as the photosynthetic fringe, is a result of the pH, temperature, and/or sulfide concentration gradients in the hot spring outflows. Here, we explicitly evaluated the predictive capability of geochemistry in determining the location of the photosynthetic fringe in hot spring outflows. A total of 46 samples were taken from 12 hot spring outflows in Yellowstone National Park that spanned pH values from 1.9 to 9.0 and temperatures from 28.9 to 92.2°C. Sampling locations were selected to be equidistant in geochemical space above and below the photosynthetic fringe based on linear discriminant analysis. Although pH, temperature, and total sulfide concentrations have all previously been cited as determining factors for microbial community composition, total sulfide did not correlate with microbial community composition with statistical significance in non-metric multidimensional scaling. In contrast, pH, temperature, ammonia, dissolved organic carbon, dissolved inorganic carbon, and dissolved oxygen did correlate with the microbial community composition with statistical significance. Additionally, there was observed statistical significance between beta diversity and the relative position to the photosynthetic fringe with sites above the photosynthetic fringe being significantly different from those at or below the photosynthetic fringe according to canonical correspondence analysis. However, in combination, the geochemical parameters considered in this study only accounted for 35% of the variation in microbial community composition determined by redundancy analysis. In co-occurrence network analyses, each clique correlated with either pH and/or temperature, whereas sulfide concentrations only correlated with individual nodes. These results indicate that there is a complex interplay between geochemical variables and the position of the photosynthetic fringe that cannot be fully explained by statistical correlations with the individual geochemical variables included in this study.

Single-Cell Genomics Reveals the Divergent Mitochondrial Genomes of Retaria (Foraminifera and Radiolaria).

Mitochondria originated from an ancient bacterial endosymbiont that underwent reductive evolution by gene loss and endosymbiont gene transfer to the nuclear genome. The diversity of mitochondrial genomes published to date has revealed that gene loss and transfer processes are ongoing in many lineages. Most well-studied eukaryotic lineages are represented in mitochondrial genome databases, except for the superphylum Retaria-the lineage comprising Foraminifera and Radiolaria. Using single-cell approaches, we determined two complete mitochondrial genomes of Foraminifera and two nearly complete mitochondrial genomes of radiolarians. We report the complete coding content of an additional 14 foram species. We show that foraminiferan and radiolarian mitochondrial genomes contain a nearly fully overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. In contrast to animals and fungi, many protists encode a diverse set of proteins on their mitochondrial genomes, including several ribosomal genes; however, some aerobic eukaryotic lineages (euglenids, myzozoans, and chlamydomonas-like algae) have reduced mitochondrial gene content and lack all ribosomal genes. Similar to these reduced outliers, we show that retarian mitochondrial genomes lack ribosomal protein and tRNA genes, contain truncated and divergent small and large rRNA genes, and contain only 14 or 15 protein-coding genes, including nad1, -3, -4, -4L, -5, and -7, cob, cox1, -2, and -3, and atp1, -6, and -9, with forams and radiolarians additionally carrying nad2 and nad6, respectively. In radiolarian mitogenomes, a noncanonical genetic code was identified in which all three stop codons encode amino acids. Collectively, these results add to our understanding of mitochondrial genome evolution and fill in one of the last major gaps in mitochondrial sequence databases. IMPORTANCE We present the reduced mitochondrial genomes of Retaria, the rhizarian lineage comprising the phyla Foraminifera and Radiolaria. By applying single-cell genomic approaches, we found that foraminiferan and radiolarian mitochondrial genomes contain an overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. An alternative genetic code was identified in radiolarian mitogenomes in which all three stop codons encode amino acids. Collectively, these results shed light on the divergent nature of the mitochondrial genomes from an ecologically important group, warranting further questions into the biological underpinnings of gene content variability and genetic code variation between mitochondrial genomes.

Characterization of periplastidal compartment-targeting signals in chlorarachniophytes.

Secondary plastids are acquired by the engulfment and retention of eukaryotic algae, which results in an additional surrounding membrane or pair of membranes relative to the more familiar primary plastids of land plants. In most cases, the endocytosed alga loses its eukaryotic genome as it becomes integrated, but in two algal groups, the cryptophytes and chlorarachniophytes, the secondary plastids retain a vestigial nucleus in the periplastidal compartment (PPC), the remnant eukaryotic cytoplasm between the inner and the outer membrane pairs. Many essential housekeeping genes are missing from these reduced genomes, suggesting that they are now encoded in the host nucleus and their products are targeted to the PPC. One such nucleus-encoded, PPC-targeted protein, the translation elongation factor like (EFL) was recently identified in chlorarachniophytes. It bears an N-terminal-targeting sequence comprising a signal peptide and a transit peptide-like sequence (TPL) similar to the plastid-targeted proteins of chlorarachniophytes as well as a hydrophilic C-terminal extension rich in lysine and aspartic acid. Here, we characterize the function of the N- and C-terminal extensions of PPC-targeted EFL in transformed chlorarachniophyte cells. Using green fluorescent protein as a reporter molecule, we demonstrate that several negatively charged amino acids within the TPL are essential for accurate targeting to the PPC. Our findings further reveal that the C-terminal extension functions as a PPC retention signal in combination with an N-terminal plastid-targeting peptide, which suggests that plastid and PPC proteins may be sorted in the PPC.