The genome of an endosymbiotic methanogen is very similar to those of its free-living relatives.

The methanogenic endosymbionts of anaerobic protists represent the only known intracellular archaea, yet, almost nothing is known about genome structure and content in these lineages. Here, an almost complete genome of an intracellular Methanobacterium species was assembled from a metagenome derived from its host ciliate, a Heterometopus species. Phylogenomic analysis showed that the endosymbiont was closely related to free-living Methanobacterium isolates, and when compared with the genomes of free-living Methanobacterium, the endosymbiont did not show significant reduction in genome size or GC content. Additionally, the Methanobacterium endosymbiont genome shared the majority of its genes with its closest relative, though it did also contain unique genes possibly involved in interactions with the host via membrane-associated proteins, the removal of toxic by-products from host metabolism and the production of small signalling molecules. Though anaerobic ciliates have been shown to transmit their endosymbionts to daughter cells during division, the results presented here could suggest that the endosymbiotic Methanobacterium did not experience significant genetic isolation or drift and/or that this lineage was only recently acquired. Altogether, comparative genomic analysis identified genes potentially involved in the establishment and maintenance of the symbiosis, as well provided insight into the genomic consequences for an intracellular archaeum.

Insights into the metabolic functioning of a multipartner ciliate symbiosis from oxygen-depleted sediments.

Symbioses between anaerobic or microaerophilic protists and prokaryotes are common in anoxic and oxygen-depleted habitats ranging from marine sediments to gastrointestinal tracts. Nevertheless, little is known about the mechanisms of metabolic interaction between partners. In these putatively syntrophic associations, consumption of fermentative end products (e.g., hydrogen) by the prokaryotic symbionts is thought to facilitate protistan anaerobic metabolism. Here, we employed metagenomic and metatranscriptomic sequencing of a microaerophilic or anaerobic karyorelictid ciliate and its prokaryotic symbionts from oxygen-depleted Santa Barbara Basin (CA, USA) sediments to assess metabolic coupling within this consortium. This sequencing confirmed the predominance of deltaproteobacterial symbionts from the Families Desulfobacteraceae and Desulfobulbaceae and suggested active symbiont reduction of host-provided sulphate, transfer of small organic molecules from host to symbionts and hydrogen cycling among the symbionts. In addition, patterns of gene expression indicated active cell division by the symbionts, their growth via autotrophic processes and nitrogen exchange with the ciliate host. Altogether, this research underscores the importance of symbiont metabolism to host fermentative metabolism and, thus, likely its success in anoxic and low-oxygen habitats, but also suggests ciliate-associated prokaryotes play a role in important biogeochemical processes.

Marine protist associations and environmental impacts across trophic levels in the twilight zone and below.

Marine protists are integral to marine food webs and exhibit complex relationships with other microbial taxa. Phagotrophic protists contribute significantly to carbon turnover in the sunlit ocean and evidence suggests grazing in the dark ocean can be significant as well. New in situ sampling technologies hold great promise for more accurately accessing these impacts. The molecular signatures of parasitic protists comprise significant fractions of many high-throughput sequencing datasets, suggesting a major role in controlling populations of their host(s). The prokaryotic symbionts of free-living protists can be numerous, and, particularly in low-oxygen to anoxic marine habitats, their collective metabolisms may contribute significantly to biogeochemical cycling. This short review addresses principally planktonic communities in the mesopelagic and bathypelagic dark ocean.

Molecular indicators of microbial diversity in oolitic sands of Highborne Cay, Bahamas.

Microbialites (stromatolites and thrombolites) are mineralized mat structures formed via the complex interactions of diverse microbial-mat communities. At Highborne Cay, in the Bahamas, the carbonate component of these features is mostly comprised of ooids. These are small, spherical to ellipsoidal grains characterized by concentric layers of calcium carbonate and organic matter and these sand-sized particles are incorporated with the aid of extra-cellular polymeric substances (EPS), into the matrix of laminated stromatolites and clotted thrombolite mats. Here, we present a comparison of the bacterial diversity within oolitic sand samples and bacterial diversity previously reported in thrombolitic and stromatolitic mats of Highborne Cay based on analysis of clone libraries of small subunit ribosomal RNA gene fragments and lipid biomarkers. The 16S-rRNA data indicate that the overall bacterial diversity within ooids is comparable to that found within thrombolites and stromatolites of Highborne Cay, and this significant overlap in taxonomic groups suggests that ooid sands may be a source for much of the bacterial diversity found in the local microbialites. Cyanobacteria were the most diverse taxonomic group detected, followed by Alphaproteobacteria, Gammaproteobacteria, Planctomyces, Deltaproteobacteria, and several other groups also found in mat structures. The distributions of intact polar lipids, the fatty acids derived from them, and bacteriohopanepolyols provide broad general support for the bacterial diversity identified through analysis of nucleic acid clone libraries.

Presence of the oral bacterium Capnocytophaga canimorsus in the tooth plaque of canines.

Capnocytophaga canimorsus is a potentially pathogenic microorganism when transmitted to humans from the oral cavity of canines. Although there is some knowledge about the frequency of occurrence in canines, it is uncertain whether there is a correlation between its occurrence and lifestyle, health, or breed of dog. Samples of tooth plaque from a total of 131 canines were collected, cultured on selective media, and tested using physiological and molecular analyses to help discern the presence of C. canimorsus. Phylogenetic analyses determined that 49.2% of canines sampled carried a species of Capnocytophaga and 21.7% of the canines sampled in this study carried C. canimorsus. Statistical analyses found that male dogs and those that are neutered and spayed are more likely to host Capnocytophaga species. The data also suggested that breed was a statistically significant predictor of C. canimorsus, with the smaller breeds more likely to carry the potential pathogen. In addition, three "human" species of Capnocytophaga; C. ochracea, C. haemolytica, and one isolate of either C. gingivalis or C. granulosa were cultured from five canines. Sixteen canines sampled carried an unidentified Capnocytophaga species, with the sequences from all isolates forming a well-defined phylogenetic clade with 100% bootstrap support that may well represent a new species of Capnocytophaga.

Identity of epibiotic bacteria on symbiontid euglenozoans in O2-depleted marine sediments: evidence for symbiont and host co-evolution.

A distinct subgroup of euglenozoans, referred to as the 'Symbiontida,' has been described from oxygen-depleted and sulfidic marine environments. By definition, all members of this group carry epibionts that are intimately associated with underlying mitochondrion-derived organelles beneath the surface of the hosts. We have used molecular phylogenetic and ultrastructural evidence to identify the rod-shaped epibionts of the two members of this group, Calkinsia aureus and B.bacati, hand-picked from the sediments of two separate oxygen-depleted, sulfidic environments. We identify their epibionts as closely related sulfur or sulfide-oxidizing members of the epsilon proteobacteria. The epsilon proteobacteria generally have a significant role in deep-sea habitats as primary colonizers, primary producers and/or in symbiotic associations. The epibionts likely fulfill a role in detoxifying the immediate surrounding environment for these two different hosts. The nearly identical rod-shaped epibionts on these two symbiontid hosts provides evidence for a co-evolutionary history between these two sets of partners. This hypothesis is supported by congruent tree topologies inferred from 18S and 16S rDNA from the hosts and bacterial epibionts, respectively. The eukaryotic hosts likely serve as a motile substrate that delivers the epibionts to the ideal locations with respect to the oxic/anoxic interface, whereby their growth rates can be maximized, perhaps also allowing the host to cultivate a food source. Because symbiontid isolates and additional small subunit rDNA gene sequences from this clade have now been recovered from many locations worldwide, the Symbiontida are likely more widespread and diverse than presently known.

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.

Phylogenetic relationships of class II fumarase genes from trichomonad species.

Class II fumarase sequences were obtained by polymerase chain reaction from five trichomonad species. All residues known to be highly conserved in this enzyme were present. Nuclear run-on assays showed that one of the two genes identified in Tritrichomonas foetus was expressed, whereas no fumarase transcripts were detected in the related species Trichomonas vaginalis. These findings corroborate previous biochemical data. Fumarase genes were also expressed in Monocercomonas sp. and Tetratrichomonas gallinarum but not in Pentatrichomonas hominis, Trichomonas gallinae, Trichomonas tenax, and Trichomitus batrachorum under the culture conditions used. Molecular trees inferred by likelihood methods reveal that trichomonad sequences have no affinity to described class II fumarase genes from other eukaryotes. The absence of functional mitochondria in protists such as trichomonads suggests that they diverged from other eukaryotes prior to the alpha-proteobacterial symbiosis that led to mitochondria. Furthermore, they are basal to other eukaryotes in rRNA analyses. However, support for the early-branching status of trichomonads and other amitochondriate protists based on phylogenetic analyses of multiple data sets has been equivocal. Although the presence of hydrogenosomes suggests that trichomonads once had mitochondria, their class II iron-independent fumarase sequences differ markedly from those of other mitochondriate eukaryotes. All of the class II fumarase genes described from other eukaryotes are of apparent alpha-proteobacterial origin and hence a marker of mitochondrial evolution. In contrast, the class II fumarase from trichomonads emerges among other eubacterial homologs. This is intriguing evidence for an independent acquisition of these genes in trichomonads apart from the mitochondrial endosymbiosis event that gave rise to the form present in other eukaryotes. The ancestral trichomonad class II fumarase may represent a prokaryotic form that was replaced in other eukaryotes after the divergence of trichomonads with the movement of endosymbiont genes into the nucleus. Alternatively, it may have been acquired via a separate endosymbiotic event or lateral gene transfer.

Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies.

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.

Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences.

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily.

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas faetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.

Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.