Naoko T. Onodera

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.

Paratrypanosoma Is a Novel Early-Branching Trypanosomatid

The kinetoplastids are a widespread and important group of single-celled eukaryotes, many of which are devastating parasites of animals, including humans. We have discovered a new insect trypanosomatid in the gut of Culex pipiens mosquitoes. Glyceraldehyde-3-phosphate dehydrogenase- and SSU rRNA-based phylogenetic analyses show this parasite to constitute a distinct branch between the free-living Bodo saltans and the obligatory parasitic clades represented by the genus Trypanosoma and other trypanosomatids. From draft genome sequence data, we identified 114 protein genes shared among the new flagellate, 15 trypanosomatid species, B. saltans, and the heterolobosean Naegleria gruberi, as well as 129 protein genes shared with the basal kinetoplastid Perkinsela sp. Individual protein phylogenies together with analyses of concatenated alignments show that the new species, here named Paratrypanosoma confusum n. gen., n. sp., branches with very high support at the base of the family Trypanosomatidae. P. confusum thus represents a long-sought-after missing link between the ancestral free-living bodonids and the derived parasitic trypanosomatids. Further analysis of the P. confusum genome should provide insight into the emergence of parasitism in the medically important trypanosomatids.

Complete Nucleomorph Genome Sequence of the Nonphotosynthetic Alga Cryptomonas paramecium Reveals a Core Nucleomorph Gene Set

Nucleomorphs are the remnant nuclei of algal endosymbionts that were engulfed by nonphotosynthetic host eukaryotes. These peculiar organelles are found in cryptomonad and chlorarachniophyte algae, where they evolved from red and green algal endosymbionts, respectively. Despite their independent origins, cryptomonad and chlorarachniophyte nucleomorph genomes are similar in size and structure: they are both <1 million base pairs in size (the smallest nuclear genomes known), comprised three chromosomes, and possess subtelomeric ribosomal DNA operons. Here, we report the complete sequence of one of the smallest cryptomonad nucleomorph genomes known, that of the secondarily nonphotosynthetic cryptomonad Cryptomonas paramecium. The genome is 486 kbp in size and contains 518 predicted genes, 466 of which are protein coding. Although C. paramecium lacks photosynthetic ability, its nucleomorph genome still encodes 18 plastid-associated proteins. More than 90% of the “conserved” protein genes in C. paramecium (i.e., those with clear homologs in other eukaryotes) are also present in the nucleomorph genomes of the cryptomonads Guillardia theta and Hemiselmis andersenii. In contrast, 143 of 466 predicted C. paramecium proteins (30.7%) showed no obvious similarity to proteins encoded in any other genome, including G. theta and H. andersenii. Significantly, however, many of these “nucleomorph ORFans” are conserved in position and size between the three genomes, suggesting that they are in fact homologous to one another. Finally, our analyses reveal an unexpected degree of overlap in the genes present in the independently evolved chlorarachniophyte and cryptomonad nucleomorph genomes: ∼80% of a set of 120 conserved nucleomorph genes in the chlorarachniophyte Bigelowiella natans were also present in all three cryptomonad nucleomorph genomes. This result suggests that similar reductive processes have taken place in unrelated lineages of nucleomorph-containing algae.

The Complete Plastid Genome Sequence of the Secondarily Nonphotosynthetic Alga Cryptomonas paramecium: Reduction, Compaction, and Accelerated Evolutionary Rate

The cryptomonads are a group of unicellular algae that acquired photosynthesis through the engulfment of a red algal cell, a process called secondary endosymbiosis. Here, we present the complete plastid genome sequence of the secondarily nonphotosynthetic species Cryptomonas paramecium CCAP977/2a. The ∼78 kilobase pair (Kbp) C. paramecium genome contains 82 predicted protein genes, 29 transfer RNA genes, and a single pseudogene (atpF). The C. paramecium plastid genome is approximately 50 Kbp smaller than those of the photosynthetic cryptomonads Guillardia theta and Rhodomonas salina; 71 genes present in the G. theta and/or R. salina plastid genomes are missing in C. paramecium. The pet, psa, and psb photosynthetic gene families are almost entirely absent. Interestingly, the ribosomal RNA operon, present as inverted repeats in most plastid genomes (including G. theta and R. salina), exists as a single copy in C. paramecium. The G + C content (38%) is higher in C. paramecium than in other cryptomonad plastid genomes, and C. paramecium plastid genes are characterized by significantly different codon usage patterns and increased evolutionary rates. The content and structure of the C. paramecium plastid genome provides insight into the changes associated with recent loss of photosynthesis in a predominantly photosynthetic group of algae and reveals features shared with the plastid genomes of other secondarily nonphotosynthetic eukaryotes.

Genome Sequence of Shigella flexneri Serotype 5a Strain M90T Sm

Bacteria of the genus Shigella are a major cause of death worldwide (L. von Seidlein et al., PLoS Med. 3:e353, 2006). We sequenced the genome of Shigella flexneri strain M90T Sm (serotype 5a) and compared it to the published genome sequence of S. flexneri strain 8401 (serotype 5b).

Proposal of a Twin Aarginine Translocator System-Mediated Constraint against Loss of ATP Synthase Genes from Nonphotosynthetic Plastid Genomes

Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of these organelles. Yet the functional roles of some retained genes-such as those coding for ATP synthase-remain mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel loss of photosynthesis in multiple independent lineages of algae/plants.

Genomic Characterization of Neoparamoeba pemaquidensis (Amoebozoa) and Its Kinetoplastid Endosymbiont

ABSTRACT We have performed a genomic characterization of a kinetoplastid protist living within the amoebozoan Neoparamoeba pemaquidensis. The genome of this “Ichthyobodo-related organism” was found to be unexpectedly large, with at least 11 chromosomes between 1.0 and 3.5 Mbp and a total genome size of at least 25 Mbp.

Nucleomorph genome diversity and its phylogenetic implications in cryptomonad algae

The relationship between phylogeny and nucleomorph genome size was examined in 16 strains of cryptomonad algae using pulsed‐field gel electrophoresis, Southern hybridization and phylogenetic analyses. Our results suggest that all cryptomonads examined in this study contain three nucleomorph chromosomes and their total genome size ranges from 495 to 750 kb. In addition, we estimated the plastid genome size of the respective organisms. The plastid genomes of photosynthetic strains were approximately 120–160 kb in size, whereas the non‐photosynthetic Cryptomonas paramecium NIES715 possesses a genome of approximately 70 kb. Phylogenetic analysis of the nuclear small subunit ribosomal DNA (SSU rDNA) gene showed that nucleomorph genome size varies considerably within closely related strains. This result indicates that the reduction of nucleomorph genomes is a rapid phenomenon that occurred multiple times independently during cryptomonad evolution. The nucleomorph genome sizes of Cryptomonas rostratiformis NIES277 appeared to be approximately 495 kb. This is smaller than that of Guillardia theta CCMP327, which until now was thought to have the smallest known nucleomorph genome size among photosynthetic cryptomonads.

Complex array of endobionts in Petalomonas sphagnophila, a large heterotrophic euglenid protist from Sphagnum-dominated peatlands.

Petalomonas sphagnophila is a poorly studied plastid-lacking euglenid flagellate living in Sphagnum-dominated peatlands. Here we present a broad-ranging microscopic, molecular and microspectrophotometric analysis of uncultured P. sphagnophila collected from four field locations in Nova Scotia, Canada. Consistent with its morphological characteristics, 18S ribosomal DNA (rDNA) phylogenies indicate that P. sphagnophila is specifically related to Petalomonas cantuscygni, the only other Petalomonas species sequenced to date. One of the peculiar characteristics of P. sphagnophila is the presence of several green-pigmented particles ∼5 μm in diameter in its cytoplasm, which a previously published study suggested to be cyanobacterial endosymbionts. New data presented here, however, suggest that the green intracellular body may not be a cyanobacterium but rather an uncharacterized prokaryote yet to be identified by molecular sequencing. 16S rDNA library sequencing and fluorescence in situ hybridizations show that P. sphagnophila also harbors several other endobionts, including bacteria that represent five novel genus-level groups (one firmicute and four different proteobacteria). 16S rDNA phylogenies suggest that three of these endobionts are related to obligate intracellular bacteria such as Rickettsiales and Coxiella, while the others are related to the Daphnia pathogen Spirobacillus cienkowskii or belong to the Thermoactinomycetaceae. TEM, 16S rDNA library sequencing and a battery of PCR experiments show that the presence of the five P. sphagnophila endobionts varies markedly among the four geographic collections and even among individuals collected from the same location but at different time points. Our study adds significantly to the growing evidence for complex and dynamic protist–bacterial associations in nature.

Genome sequencing reveals metabolic and cellular interdependence in an amoeba-kinetoplastid symbiosis

Endosymbiotic relationships between eukaryotic and prokaryotic cells are common in nature. Endosymbioses between two eukaryotes are also known; cyanobacterium-derived plastids have spread horizontally when one eukaryote assimilated another. A unique instance of a non-photosynthetic, eukaryotic endosymbiont involves members of the genus Paramoeba, amoebozoans that infect marine animals such as farmed fish and sea urchins. Paramoeba species harbor endosymbionts belonging to the Kinetoplastea, a diverse group of flagellate protists including some that cause devastating diseases. To elucidate the nature of this eukaryote-eukaryote association, we sequenced the genomes and transcriptomes of Paramoeba pemaquidensis and its endosymbiont Perkinsela sp. The endosymbiont nuclear genome is ~9.5 Mbp in size, the smallest of a kinetoplastid thus far discovered. Genomic analyses show that Perkinsela sp. has lost the ability to make a flagellum but retains hallmark features of kinetoplastid biology, including polycistronic transcription, trans-splicing, and a glycosome-like organelle. Mosaic biochemical pathways suggest extensive ‘cross-talk’ between the two organisms, and electron microscopy shows that the endosymbiont ingests amoeba cytoplasm, a novel form of endosymbiont-host communication. Our data reveal the cell biological and biochemical basis of the obligate relationship between Perkinsela sp. and its amoeba host, and provide a foundation for understanding pathogenicity determinants in economically important Paramoeba.