Early branching eukaryotes?

Recent phylogenetic analyses suggest that Giardia, Trichomonas and Microsporidia contain genes of mitochondrial origin and are thus unlikely to be primitively amitochondriate as previously thought. Furthermore, phylogenetic analyses of multiple data sets suggest that Microsporidia are related to Fungi rather than being deep branching as depicted in trees based upon SSUrRNA analyses. There is also room for doubt, on the basis of a lack of consistent support from analyses of other genes, whether Giardia or Trichomonas branch before other eukaryotes. So, at present, we cannot be sure which eukaryotes are descendants of the earliest-branching organisms in the eukaryote tree. Future resolution of the order of emergence of eukaryotes will depend upon a more critical phylogenetic analysis of new and existing data than hitherto. Hypotheses of branching order should preferably be based upon congruence between independent data sets, rather than on single gene trees.

Transporter gene acquisition and innovation in the evolution of Microsporidia intracellular parasites.

The acquisition of genes by horizontal transfer can impart entirely new biological functions and provide an important route to major evolutionary innovation. Here we have used ancient gene reconstruction and functional assays to investigate the impact of a single horizontally transferred nucleotide transporter into the common ancestor of the Microsporidia, a major radiation of intracellular parasites of animals and humans. We show that this transporter provided early microsporidians with the ability to steal host ATP and to become energy parasites. Gene duplication enabled the diversification of nucleotide transporter function to transport new substrates, including GTP and NAD+, and to evolve the proton-energized net import of nucleotides for nucleic acid biosynthesis, growth and replication. These innovations have allowed the loss of pathways for mitochondrial and cytosolic energy generation and nucleotide biosynthesis that are otherwise essential for free-living eukaryotes, resulting in the highly unusual and reduced cells and genomes of contemporary Microsporidia.

A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria.

Microsporidia are small (1-20 micron) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2,3], and are reported to lack mitochondria [2,4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2,4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5-9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.

Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis.

Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced. GPI of the parabasalid Trichomonas vaginalis was also sequenced. The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T. vaginalis, a Type II amitochondriate protist. These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica. G. intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs. The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes. The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes. The observation that the two diplomonads and T. vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids. The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria.

Biochemical and genetic evidence for a family of heterotrimeric G-proteins in Trichomonas vaginalis.

We have cloned a single copy gene from the human parasite Trichomonas vaginalis that encodes a putative protein of 402 amino acids with approximately 35% sequence identity to known alpha subunits of heterotrimeric G-proteins. It contains the characteristic GTP binding domains G-1 to G-5 with the key residues conserved. The new sequence has an unusual N-terminal extension of approximately 70 residues that cannot be aligned to reference G-proteins and which is characterised by proline-rich repeats. To investigate the expression and cellular localisation of the protein we produced specific antisera against a recombinant fusion protein. The antisera recognised a protein of an apparent molecular mass of 51 kDa in protein extracts from T. vaginalis and immunofluorescent microscopy established that the protein is localised to discrete endomembranes. Using a protocol designed to purify mammalian heterotrimeric G-proteins incorporating a GTPgammaS binding assay, we isolated two proteins from Trichomonas that are recognised by an heterologous GA/1 antisera raised to a peptide of the conserved G-1 domain of G-protein alpha subunits. These two proteins have an apparent molecular mass of 61 and 48 kDa, respectively, larger and smaller than the translation product of the cloned gene. Consistent with these results, the GA/1 antisera did not cross-react with the fusion protein produced from the gene we have cloned. These data suggest T. vaginalis possesses more than one heterotrimeric G-protein alpha subunit. Based on the sequence features of the cloned gene and the biochemical properties of the purified proteins, we suggest that these alpha subunits are likely to be part of classic heterotrimeric G-protein complexes.

Molecular data suggest an early acquisition of the mitochondrion endosymbiont.

The three deepest branching eucaryotic lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microspora, Metamonada and Parabasala. They are followed by either the Euglenozoa (e.g. Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eucaryotes. To investigate the hypothesis of an even earlier timing of the mitochondrion endosymbiosis we have amplified a partial cpn-60 coding region from the parabasalid Trichomonas vaginalis and the first such sequence from a percolozoan, Naegleria fowleri. Analysis of predicted protein sequences reveals a high degree of sequence similarity (> or = 40%) with a selection of published bacterial and mitochondrial cpn-60s for both taxa. Both sequences were recovered within a strongly supported monophyletic group, otherwise defined by mitochondrial sequences, which systematically clustered with alpha-proteobacteria. These results provide compelling evidence that the ancestor of T. vaginalis once contained the endosymbiont which gave rise to mitochondria, and suggest that this symbiosis probably occurred before the Trichomonas lineage diverged from the main eukaryote trunk. It also makes feasible the published hypothesis that the Trichomonas hydrogenosome might represent a biochemically modified mitochondrion. Analysis of the N. fowleri cpn-60 did not support the hypothesis that the mitochondrion-containing Percolozoa represent an earlier branch in the cpn-60 tree than Trichomonas or Trypanosoma.

Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates.

Some ciliates live anaerobically and lack mitochondria, but possess hydrogenosomes: organelles that contain hydrogenase and produce hydrogen. The origin of hydrogenosomes has been explained by two competing hypotheses: (i) they are biochemically modified mitochondria; or (ii) they are derived from endosymbiotic association(s) of ciliates and anaerobic eubacteria that possessed the hydrogenosome biochemistry. Phylogenetic analyses of representative aerobic, and anaerobic hydrogenosomal ciliates using host nuclear SSU rDNA sequences indicate a minimum of three, but more likely four, separate origins of hydrogenosomes. Whereas this does not refute either hypothesis, the implausibility of multiple convergent endosymbioses gives further support to the view that hydrogenosomes in ciliates derive from an existing organelle, which ultrastructural evidence suggests is the mitochondrion. Our results indicate a considerable potential for physiological-biochemical plasticity among a group of predominantly aerobic eucaryotes, and provide a phylogenetic framework to further refine and test hypotheses of the origins of the hydrogenosomal enzymes.

RNA sequence analysis shows that the symbionts in the ciliate Metopus contortus are polymorphs of a single methanogen species.

The polymerase chain reaction was used to amplify and partially sequence the 16S ribosomal RNA genes of symbiotic bacteria within the anaerobic ciliate Metopus contortus. In situ probing with fluorescent oligonucleotides showed that the amplified sequences originated from a single species of archaebacterium which is closely related to Methanocorpusculum parvum. The probed symbionts exhibited a variety of shapes and sizes. These data support the hypothesis, first proposed on the basis of electron microscopy, that the symbionts undergo a morphological transformation as part of the symbiotic process.

Molecular analysis of enrichment cultures of marine ammonia oxidisers.

Marine ammonia oxidising bacteria were enriched by incubation of sea water, amended with ammonium sulphate, and subsequent subculture in liquid inorganic medium. PCR primers were designed to be specific for rDNA sequences from ammonia oxidisers belonging to the beta-sub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PCR products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the presence of three new lineages within the clade defined by sequences of cultured beta-sub-group ammonia oxidisers. Two of the new lineages showed moderate similarity to sequences from pure cultures of ammonia oxidisers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira, which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the beta-proteobacteria ammonia oxidisers. The results also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.

Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia.

DNA coding for the 16S rRNA of an intracellular bacterium was directly amplified from lysed cells of a host amoebae using the polymerase chain reaction and primers specific for eubacteria. The amoebae had been used to recover an uncultured bacterium observed in the sputum of a patient with pneumonia. The amplified DNA was sequenced directly and compared with published 16S rRNA sequences. The analysis revealed that the intracellular bacterium is a member of the genus Legionella and that it is different from species, including L. pneumophila, for which 16S ribosomal RNA sequence data are available.

Some rumen ciliates have endosymbiotic methanogens.

Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

Use of 16S rRNA-targeted oligonucleotide probes to investigate function and phylogeny of sulphate-reducing bacteria and methanogenic archaea in a UK estuary.

Abstract Sulphate-reducing bacteria (SRB) and methanogenic archaea (MA) are important anaerobic terminal oxidisers of organic matter. However, we have little knowledge about the distribution and types of SRB and MA in the environment or the functional role they play in situ. Here we have utilised sediment slurry microcosms amended with ecologically significant substrates, including acetate and hydrogen, and specific functional inhibitors, to identify the important SRB and MA groups in two contrasting sites on a UK estuary. Substrate and inhibitor additions had significant effects on methane production and on acetate and sulphate consumption in the slurries. By using specific 16S-targeted oligonucleotide probes we were able to link specific SRB and MA groups to the use of the added substrates. Acetate consumption in the freshwater-dominated sediments was mediated by Methanosarcinales under low-sulphate conditions and Desulfobacter under the high-sulphate conditions that simulated a tidal incursion. In the marine-dominated sediments, acetate consumption was linked to Desulfobacter. Addition of trimethylamine, a non-competitive substrate for methanogenesis, led to a large increase in Methanosarcinales signal in marine slurries. Desulfobulbus was linked to non-sulphate-dependent H(2) consumption in the freshwater sediments. The addition of sulphate to freshwater sediments inhibited methane production and reduced signal from probes targeted to Methanosarcinales and Methanomicrobiales, while the addition of molybdate to marine sediments inhibited Desulfobulbus and Desulfobacterium. These data complement our understanding of the ecophysiology of the organisms detected and make a firm connection between the capabilities of species, as observed in the laboratory, to their roles in the environment.

Species Diversity of Uncultured and Cultured Populations of Soil and Marine Ammonia Oxidizing Bacteria.

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.

Cyclidium porcatum n. sp.: a Free-living anaerobic scuticociliate containing a stable complex of hydrogenosomes, eubacteria and archaeobacteria.

A new ciliate species (Cyclidium porcatum) is the first freshwater anaerobic scuticociliate to be cultured and described. It contains a unique tripartite structure consisting of hydrogenosomes (confirmed by cytochemical staining for hydrogenase), interspersed with methanogens (confirmed by auto fluorescence and in situ hybridisation with an archaeobacterial 16S rRNA-specific probe) and unidentified eubacteria (confirmed with a eubacterial 16S rRNA-specific probe). This complex structure is stable and persistent, indicating that it is an anaerobic symbiotic consortium incorporating three functional partners.

The linear PCR reaction: a simple and robust method for sequencing amplified rRNA genes.

The linear polymerase chain reaction was used to sequence amplified RNA genes from strains of Bacillus, Thermus and Legionella. The technique described is simple and reproducible and it works well with double standard product which has been PEG precipitated directly from PCR reactions.

Systematic and morphological diversity of endosymbiotic methanogens in anaerobic ciliates.

The identities and taxonomic diversity of the endosymbiotic methanogens from the anaerobic protozoa Metopus contortus, Metopus striatus, Metopus palaeformis, Trimyema sp. and Pelomyxa palustris were determined by comparative analysis of their 16S ribosomal RNA sequences. Fluorescent oligonucleotide probes were designed to bind to the symbiont rRNA sequences and to provide direct visual evidence of their origins from methanogenic archaea contained within the host cells. Confocal microscopy was used to analyze the morphology of the endosymbionts in whole cells of Metopus palaeformis, Metopus contortus, Trimyema sp, and Cyclidium porcatum. The endosymbionts are taxonomically diverse and are drawn from three different genera; Methanobacterium, Methanocorpusculum and Methanoplanus. In every case the symbionts are closely related to, but different from, free-living methanogens for which sequences are available. It is thus apparent that symbioses have been formed repeatedly and independently. Ciliates which are unrelated to each other (Trimyema sp. and Metopus contortus) may contain symbionts which are closely related, and congeneric ciliates (Metopus palaeformis and M. contortus) may contain symbionts which are distantly related to each other. This suggests that some of the symbiotic associations must be relatively recent. For example, at least one of the symbioses in Metopus must postdate the speciation of M. palaeformis and M. contortus. Despite this, Metopus contortus, Trimyema sp., Cyclidium porcatum and their respective endosymbionts show sophisticated morphological interactions which probably facilitate the exchange of materials between the partners.

The molecular phylogeny and systematics of the actinomycetes.

Sequences of 16S ribosomal RNA have provided actinomycetologists with a phylogenetic tree that allows the investigation of the evolution of actinomycetes and also provides a basis for classification. The origin of actinomycetes and, except for bifidobacteria, the order by which the main sublines evolved, cannot yet be determined with certainty. However, calibration of rRNA sequence divergence with palaeochemical data, and previously published substitution rates of endosymbiotic bacteria, suggest that the main radiation occurred less than 1 billion years ago. Within this radiation, several phylogenetically homogeneous, but sometimes phenotypically heterogeneous, clades appear to have diverged over a short evolutionary period. The resolution of the 16S rRNA molecule appears to be insufficient to clearly determine the branching patterns between clades in this area of the phylogenetic tree. The distribution of some morphological and chemotaxonomic traits such as types of peptidoglycan, menaquinone, phospholipids, cell wall sugars, and fatty acids facilitate the phenotypic delineation of genera within each clade. At higher taxonomic levels, e.g. at the family level, phenotypic similarities are unpredictable and tend to be less conserved. With the exception of mycolic acids, most traits are polyphyletic--hence they are unreliable indicators per se of phylogenetic relationships. Nevertheless, combinations of phenotypic properties are invaluable for predicting whether a new organism is likely to be a member of an established or a novel taxon. Current knowledge about the phylogenetic structure of the actinomycetes provides not only a sound basis for future taxonomic work but also a framework for the rational exploration of their ecology and biotechnological potential.

Iron hydrogenases and the evolution of anaerobic eukaryotes.

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain.

Biodiversity at the molecular level: the domains, kingdoms and phyla of life.

The results of comparative sequence analysis, mainly of small subunit (SSU) ribosomal (r)RNA sequences, have suggested that all of cellular life can be placed in one of three domains: the Archaea, Bacteria or Eucarya. There is some evidence that the Archaea may not be a monophyletic assemblage, but as yet this issue has not been resolved. Most of the lineages, and all of the deepest ones, in the tree based upon SSU rRNA sequences, are microbial. Traditional ideas of classification such as Whittaker's five kingdom scheme do not adequately describe life's diversity as revealed by sequence comparisons. There are many microbial groups that demonstrate much greater amounts of SSU rRNA sequence divergence than do members of the classical kingdoms, Animalia, Plantae and Fungi. The old microbial kingdoms Monera and Protista are clearly paraphyletic but as yet there is no consensus as to how they should be reorganized in taxonomic terms. New data from environmental analysis suggests that much of the microbial world is unknown. Every environment which has been analysed by molecular methods has revealed many previously unrecorded lineages. Some of these show great divergence from the sequences of cultured microorganisms suggesting that fundamentally new microbial groups remain to be isolated. The relationships of some of these new lineages may be expected to affect how the tree of life is organized into higher taxa, and to also influence which features will be recognized as synapomorphies. There is currently no objective measure whereby microbial diversity can be quantified and compared to the figures which are widely quoted for arthropods and other Metazoa.