Search

Ciliates are highly complex unicellular eukaryotes. Most of them live in aerobic environments and possess mitochondria. However, in several orders of ciliates, anaerobic species evolved that contain “hydrogenosomes”, organelles that produce hydrogen and ATP. These hydrogenosomes of ciliates have not been studied in the same detail as those of trichomonads and chytrid fungi. Therefore, generalizations on the characteristics of hydrogenosomes of ciliates are somewhat premature, especially since phylogenetic studies suggest that hydrogenosomes have arisen independently several times in ciliates. In this chapter, the hydrogenosomes of the anaerobic, heterotrichous ciliate Nyctotherus ovalis from the hindgut of cockroaches will mainly be described as these are the ones that are, at the moment, the most thoroughly studied. Thus far, this is the only hydrogenosome known to possess a genome and this genome is clearly of mitochondrial origin. In fact, the hydrogenosome of N. ovalis unites typical mitochondrial features such as a genome and an electron-transport chain with the most characteristic hydrogenosomal property, the production of hydrogen. The hydrogenosomal metabolism of N. ovalis will be compared with that of two other ciliates that have been studied in less detail, i.e. the holotrichous rumen ciliate Dasytricha, and the free-living plagiopylid ciliate Trimyema. All studies combined indicate that it is likely that the various types of hydrogenosomes in ciliates evolved by modifications of aerobic mitochondria when the different ciliates adapted to anaerobic or micro aerobic environments. Furthermore, it is clear that the hydrogenosomes of anaerobic ciliates are different from those of chytrid fungi and from the well-studied ones in trichomonads.

Nearly all vertebrates host methanogens in their gastrointestinal tracts. However, a great fraction of vertebrates emits only traces of methane from their faeces (approx. 1 nmol/g faeces/h) and has no significant amounts of methane in their breath. In contrast, many animals host some 100 times more methanogens in their gastrointestinal tract and emit methane with their breath. These substantial differences are not caused by different feeding habits; rather a genetic factor controls the presence of large amounts of methanogens. The attribute “methane production” is evolutionary stable, and the loss of this character obeys Dollo’s law: once lost in the course of evolution, this character cannot be acquired another time.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis1. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds 1. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology2,3. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs)4. However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown5,6. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including conserved scaffolding proteins unique to the Neocallimastigomycota. High-quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single-molecule technology. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure - an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.

It is generally accepted that hydrogenosomes (hydrogen-producing organelles) evolved from a mitochondrial ancestor. However, until recently, only indirect evidence for this hypothesis was available. Here, we present the almost complete genome of the hydrogen-producing mitochondrion of the anaerobic ciliate Nyctotherus ovalis and show that, except for the notable absence of genes encoding electron transport chain components of Complexes III, IV, and V, it has a gene content similar to the mitochondrial genomes of aerobic ciliates. Analysis of the genome of the hydrogen-producing mitochondrion, in combination with that of more than 9,000 genomic DNA and cDNA sequences, allows a preliminary reconstruction of the organellar metabolism. The sequence data indicate that N. ovalis possesses hydrogen-producing mitochondria that have a truncated, two step (Complex I and II) electron transport chain that uses fumarate as electron acceptor. In addition, components of an extensive protein network for the metabolism of amino acids, defense against oxidative stress, mitochondrial protein synthesis, mitochondrial protein import and processing, and transport of metabolites across the mitochondrial membrane were identified. Genes for MPV17 and ACN9, two hypothetical proteins linked to mitochondrial disease in humans, were also found. The inferred metabolism is remarkably similar to the organellar metabolism of the phylogenetically distant anaerobic Stramenopile Blastocystis. Notably, the Blastocystis organelle and that of the related flagellate Proteromonas lacertae also lack genes encoding components of Complexes III, IV, and V. Thus, our data show that the hydrogenosomes of N. ovalis are highly specialized hydrogen-producing mitochondria.

Methanomicrococcus blatticola is an obligately anaerobic methanogen that derives the energy for growth exclusively from the reduction of methylated compounds to methane with molecular hydrogen as energy source. Competition for methanol (concentration below 10 microM) and H(2) (concentration below 500 Pa), as well as oxidative stress due to the presence of oxygen are likely to occur in the peripheral region of the cockroach hindgut, the species' normal habitat. We investigated the ecophysiological properties of M. blatticola to explain how it can successfully compete for its methanogenic substrates. The organism showed affinities for methanol (K(m)=5 microM; threshold 0.2 h(-1)) and specific growth yields (up to 7 g of dry cells per mole of methane formed) that are particularly high within the realm of mesophilic methanogens. Upon oxygen exposure, part of the metabolic activity may be diverted into oxygen removal, thus establishing appropriate anaerobic conditions for survival and growth.

The eukaryotic biodiversity in historical air-dried samples of Dutch agricultural soil has been assessed by random sequencing of an 18S rRNA gene library and by denaturing gradient gel electrophoresis. Representatives of nearly all taxa of eukaryotic soil microbes could be identified, demonstrating that it is possible to study eukaryotic microbiota in samples from soil archives that have been stored for more than 30 years at room temperature. In a pilot study, 41 sequences were retrieved that could be assigned to fungi and a variety of aerobic and anaerobic protists such as cercozoans, ciliates, xanthophytes (stramenopiles), heteroloboseans, and amoebozoans. A PCR-denaturing gradient gel electrophoresis analysis of samples collected between 1950 and 1975 revealed significant changes in the composition of the eukaryotic microbiota.

Up to half of the biomass in the rumen can be represented by ciliates, which play an important role in the digestion processes of their hosts. In the literature, very little information can be found on determination of the diversity of complex rumen ciliate communities. The identification of these fast moving protists is mainly based on live observations and comparisons of their highly variable cell morphologies. It makes accurate identification and quantification of rumen ciliates very difficult if not impossible. The development of fluorescence in situ hybridization (FISH), established as a technique to identify prokaryotes and eukaryotes using ribosomal RNA-targeted fluorescently labeled oligonucleotide probes, is a promising approach to identify and investigate rumen ciliates. The present study, part of CIMES (ciliates as monitors for environmental safety), a project sponsored by the European Commission, shows the problems of applying FISH on rumen ciliates and how to solve them. Tests resulted in a new protocol, which recommends para-formaldehyde and formaldehyde at 1–2% final concentration to preserve the ciliates before applying FISH. Furthermore, seven new oligonucleotide probes could be developed and successfully be tested to identify different rumen ciliate taxa of the order Entodiniomorphida (class Litostomatea) by applying FISH. It is also shown, how FISH together with confocal laser scanning microscopy can improve analyses of ciliate cell morphologies. Thanks to Peter Pristas, Peter Javorsky, Ralf Einspanier, Susanne Ulbrich (providing rumen samples), Seung Yeo Moon-van der Staay, Georg van der Staay (providing unpublished 18S-rDNA sequences), the European Commission (financial support, project QLK3-CT-2002-02151).

The diversity of methanogenic archaea associated with different species of ciliated protozoa in the rumen was analysed. Partial fragments of archaeal SSU rRNA genes were amplified from DNA isolated from single cells from the rumen protozoal species Metadinium medium, Entodinium furca, Ophryoscolex caudatus and Diplodinium dentatum. Sequence analysis of these fragments indicated that although all of the new isolates clustered with sequences previously described for methanogens, there was a difference in the relative distribution of sequences detected here as compared to that of previous work. In addition, many of the novel sequences, although clearly of archaeal origin have relatively low identity to the sequences in database which are most closely related to them.

The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.

SUMMARY 1. The zebra mussel (Dreissena polymorpha) is an aquatic nuisance species that invaded Ireland around 1994. We studied the invasion of the zebra mussel combining field surveys and genetic studies, to determine the origin of invasion and the vector of introduction. 2. Field surveys showed that live zebra mussels, attached to the hulls of pleasure boats, were transported from Britain to Ireland. These boats were lifted from British waters onto trailers, transported to Ireland by ferry and lifted into Irish waters within a day. Lengthfrequency distributions of dead and living mussels on one vessel imported 3 months earlier revealed a traumatic occurrence caused by the overland, air-exposed transportation. Results show that a large number of individuals survived after re-immersion in Irish waters and continued to grow. 3. Zebra mussels from populations in Ireland, Great Britain, the Netherlands, France and North America, were analysed using amplified fragment length polymorphisms (AFLP)fingerprinting to determine the origin of the Irish invasion. Phylogenetic analysis revealed that Irish and British mussels clustered closely together, suggesting an introduction from Britain. 4. Ireland remained un-invaded by the zebra mussel for more than 150 year. The

Bromeliads are mainly epiphytic rosette plants occurring only in Central and South America. They collect rain water and particulate materials in tanks (cisterns) formed by the coalescing leaf axils. These tanks form an extensive, highly compartmentalized water and humus body above the ground and are inhabited by many ordinary and endemic organisms. We discovered at least 10 new ciliate species in the tanks of about 15 bromelian species from the Dominican Republic, Ecuador, and Brazil. Since there are 2000–3000 bromelian species with very different lifestyles, they are likely to contain hundreds of novel ciliate species. Two of the new species described here are real “flagships”, that is, they have such a large size that they would have been found in Europe, if they were there. Consequently, these species must have a restricted geographical distribution, disproving the old hypothesis that microscopic organisms are cosmopolitan. Also the smaller ciliates described here represent new genera and families, arguing for a long-lasting, independent evolution driven by ecological constraints and spatial isolation. Almost half of the new species can switch from bacteriophagous, microstome morphs to a predatory, macrostome lifestyle, likely due to the strong competition in these peculiar habitats. The high morphological and ecological diversity of tank bromeliad ciliates is only partially recovered by small-subunit (18S) ribosomal RNA (rRNA) gene sequences.

Intestinal microorganisms play an important role in plant fiber degradation by larvae of the rose chafer Pachnoda marginata. In the hindgut of the larvae 2.5 to 7.4 × 108 bacteria per ml of gut content with xylanase or endoglucanase activity were found. Bacteria in the midgut were not (hemi)cellulolytic, but the alkaline environment in this part of the intestinal tract functions as a precellulolytic phase, solubilizing part of the lignocellulosic material. Accordingly, the degradation of lignocellulose-rich material in Pachnoda marginata larvae appeared to be a combination of a physico-chemical and microbiological process. A number of different facultative anaerobic and strictly anaerobic bacteria with (hemi)cellulolytic activity were isolated from the hindgut. A dominant (hemi)cellulolytic species was a Gram positive, irregular shaped, facultative aerobic bacterium. Further physiological identification placed the isolate in the genus Promicromonospora. Comparative 16S rDNA analysis and phenotypic features revealed that the isolate represented a new species for which the name Promicromonospora pachnodae is proposed. P. pachnodae produced xylanases and endoglucanases on several plant derived polymers, both under aerobic and anaerobic conditions.

The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent Km values of about 40 µm for ADP, and 105 µm or 140 µm, respectively, for ATP have been determined, similar to the data reported for isolated rat mitochondria. The apparent Km for ATP decreased up to 10-fold in the presence of the protonophore m-chlorocarbonylcyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec. L2 exhibited the same characteristics, but the affinities for ADP (165 µm) and ATP (2.33 mm) were significantly lower. Notably, AAC1-3 from Arabidopsis thaliana and AAC1 from Solanum tuberosum (potato) showed significantly higher external affinities for both nucleotides (10–22 µm); they were only slightly influenced by CCCP. Studies on intact plant mitochondria confirmed these observations. Back exchange experiments with preloaded E. coli cells expressing AACs indicate a preferential export of ATP for all AACs tested. This is the first report of a functional integration of proteins belonging to the mitochondrial carrier family (MCF) into a bacterial cytoplasmic membrane. The technique described here provides a relatively simple and highly reproducible method for functional studies of individual mitochondrial-type carrier proteins from organisms that do not allow the application of sophisticated genetic techniques.

A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP--similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial-type AACs. Hydrogenosome-bearing anaerobic ciliates possess clearly distinct mitochondrial-type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial-type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.

The rumen ciliate Eremoplastron dilobum was isolated from sheep rumen fluid and cultivated in vitro as a species population. Four different salt solutions were used to prepare the culture media. However, only the "Artificial rumen fluid" composed of (g/L): K2HPO4-3.48, NaHCO3-2.1, NaCl-0.76, CaCl2×6H2O-0.33, CH3COONa-6.12, MgCl2×6H2O-0.3, Na2HPO4-1.71, NaHPO4×H2O-1.01 and distilled water enabled cultivation of this species for over 56 weeks. The protozoa were able to grow in a medium consisting of culture salt solution and powdered meadow hay (0.6mg/ml per d). The addition of wheat gluten did not increase the population density of E. dilobum whereas the supplemented crystalline cellulose and/or barley flour improved the growth of ciliates (P

Contains fulltext : 136128.pdf (Publisher’s version ) (Closed access) The evolution of sophisticated differentiations of the gastro-intestinal tract enabled herbivorous mammals to digest dietary cellulose and hemicellulose with the aid of a complex anaerobic microbiota. Distinctive symbiotic ciliates, which are unique to this habitat, are the largest representatives of this microbial community. Analyses of a total of 484 different 18S rRNA genes show that extremely complex, but related ciliate communities can occur in the rumen of cattle, sheep, goats and red deer (301 sequences). The communities in the hindgut of equids (Equus caballus, Equus quagga), and elephants (Elephas maximus, Loxodonta africanus; 162 sequences), which are clearly distinct from the ruminant ciliate biota, exhibit a much higher diversity than anticipated on the basis of their morphology. All these ciliates from the gastro-intestinal tract constitute a monophyletic group, which consists of two major taxa, i.e. Vestibuliferida and Entodiniomorphida. The ciliates from the evolutionarily older hindgut fermenters exhibit a clustering that is specific for higher taxa of their hosts, as extant species of horse and zebra on the one hand, and Africa and Indian elephant on the other hand, share related ciliates. The evolutionary younger ruminants altogether share the various entodiniomorphs and the vestibuliferids from ruminants.

In the intestinal tracts of animals, methanogenesis from CO 2 and other C 1 compounds strictly depends on the supply of electron donors by fermenting bacteria, but sources and sinks of reducing equivalents may be spatially separated. Microsensor measurements in the intestinal tract of the omnivorous cockroach Blaberus sp. showed that molecular hydrogen strongly accumulated in the midgut (H 2 partial pressures of 3 to 26 kPa), whereas it was not detectable (4 [105 ± 49 nmol (g of cockroach) −1 h −1 ] but only traces of H 2 . In vitro incubation of isolated gut compartments, however, revealed that the midguts produced considerable amounts of H 2 , whereas hindguts emitted only CH 4 [106 ± 58 and 71 ± 50 nmol (g of cockroach) −1 h −1 , respectively]. When ligated midgut and hindgut segments were incubated in the same vials, methane emission increased by 28% over that of isolated hindguts, whereas only traces of H 2 accumulated in the headspace. Radial hydrogen profiles obtained under air enriched with H 2 (20 kPa) identified the hindgut as an efficient sink for externally supplied H 2 . A cross-epithelial transfer of hydrogen from the midgut to the hindgut compartment was clearly evidenced by the steep H 2 concentration gradients which developed when ligated fragments of midgut and hindgut were placed on top of each other—a configuration that simulates the situation in vivo. These findings emphasize that it is essential to analyze the compartmentalization of the gut and the spatial organization of its microbiota in order to understand the functional interactions among different microbial populations during digestion.

The plasma membrane of the spermatozoa of Drosophila melanogaster contains two integral proteins with glycosidase activity, β-N-acetylglucosaminidase and α-D-mannosidase. Biochemical analysis and ultrastructural cytochemistry of spermatozoa of the autosomal male sterile mutant casanova reveal that at least one of these enzymes, β-N-acetylglucosaminidase, is crucial for sperm–egg interactions. casanova sperm are motile, morphologically normal, are transferred to the female at mating, but are unable to fertilize the eggs. The mutation was localised by deficiency mapping to the chromosomal region 95E8-F7. Fluorimetric assays showed that the mutant's sperm have the same level of α-D-mannosidase activity as wild-type sperm, whereas β-N-acetylglucosaminidase activity reaches only 51% of the wild-type level. The biochemical characteristics of α-D-mannosidase and of the residual β-N-acetylglucosaminidase are the same as in wild-type males. Ultrastructural localization of the enzymes indicated that casanova spermatozoa lacks β-N-acetylglucosaminidase on the plasma membrane covering the acrosome, whereas the location of this glycosidase at the terminal part of the sperm tail is indistinguishable from the wild-type situation. The results strongly suggest that in Drosophila the β-N-acetylglucosaminidase of the plasma membrane covering the acrosome functions as a receptor for the glycoconjugates on the egg surface. We named the putative egg receptor EROS. This is the first evidence for an egg/sperm recognition system in insects. The mechanism is similar to those known from higher animals. Mol. Reprod. Dev. 60: 248–259, 2001. © 2001 Wiley-Liss, Inc.

Hydrogenosomes are membrane-bound organelles that compartmentalise the Final steps of energy metabo I is in in a number of anaerobic eukaryotes. They produce hydrogen and ATP. Here we will review the data, which are relevant for the questions: how did the hydrogenosomes originate, and what was their ancestor? Notably, there is strong evidence that hydrogenosomes evolved several times as adaptations to anaerobic environments. Most likely, hydrogenosomes and mitochondria share a common ancestor, but an unequivocal proof for this hypothesis is difficult because hydrogenosomes lack an organelle genome - with one remarkable exception (Nyctoherus ovalis). In particular, the diversity of extant hydrogenosomes hampers a straightforward analysis of their origins. Nevertheless, it is conceivable to postulate that the common ancestor of mitochondria and hydrogenosomes was a facultative anaerobic organelle that participated in the early radiation of unicellular eukaryotes. Consequently, it is reasonable to assume that both, hydrogenosomes and mitochondria are evolutionary adaptations to anaerobic or aerobic environments, respectively.

It has been argued that about 4-5% of male adults suffer from infertility due to a genetic causation. From studies in the fruitfly Drosophila, there is evidence that up to 1500 recessive genes contribute to male fertility in that species. Here we suggest that the control of human male fertility is of at least comparable genetic complexity. However, because of small family size, conventional positional cloning methods for identifying human genes will have little impact on the dissection of male infertility. A critical selection of well-defined infertility phenotypes in model organisms, combined with identification of the genes involved and their orthologues in man, might reveal the genes that contribute to human male infertility.

Like mitochondria, hydrogenosomes compartmentalize crucial steps of eukaryotic energy metabolism; however, this compartmentalization differs substantially between mitochondriate aerobes and hydrogenosome-containing anaerobes. Because hydrogenosomes have arisen independently in different lineages of eukaryotic microorganisms, comparative analysis of the various types of hydrogenosomes can provide insights into the functional and evolutionary aspects of compartmentalized energy metabolism in unicellular eukaryotes.

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences.

Fibre digestion in vivo was studied in eight species of arthropods. The highest degree of fibre digestion (61–65%) was measured in Schistocerca gregaria , Eurycanta calcarata , and larvae of Pachnoda marginata . This approximates the fibre digestion of mammalian hindgut fermenters. The presence of propionic acid, butyric acid, and i -valeric acid, in the different compartments of the intestinal tract of arthropods appeared to be a good indicator of microbial fermentation. From the results it becomes clear that different strategies can be followed for the degradation of fibres. Cellulolytic enzymes can be produced by symbiotic microorganisms. In P. marginata larvae, fibre digestion was the result of this microbial activity. On the other hand, S. gregaria and E. calcarata , which possess low numbers of bacteria in their intestinal tract, degrade fibres with the aid of endogenously produced enzymes. The origin of cellulolytic enzymes did not influence the efficiency of fibre digestion.

Several aspects of the endosymbiosis of methanogenic archaea with anaerobic protozoa are reviewed. Special attention is payed to the role of hydrogenosomes and plastid-like organelles that seem to provide the substrates for the methanogenic endosymbionts. Evidence is presented that hydrogenosomes evolved several times in the various protoctistan taxa. Hydrogenosomes are seemingly different, and their common denominator is the production of hydrogen. The absence of nucleic acids and a protein-synthesizing machineny hampers the analysis of their divergent evolutionary history, and molecular genetic data argue not only for different but even a chimeric origin of the hydrogenosomes.

‘Biodiversity’ addresses the wealth of species that constitute the biosphere. Notwithstanding that they have been regarded as mental constructs in the past, species are really existing entities that form and disappear in the course of evolution. Molecular techniques allow to trace the dynamics of speciation and to determine the relatedness of species and the genetic diversity within populations. These techniques also permit to recognize the incredible diversity of protists: their importance for the global conversion of biomass and energy had been greatly underestimated until recently. Because it is not possible to ‘count’ all species living on earth, a variety of approaches have been used to estimate global biodiversity. Such estimations are extrapolations of historical trends or of punctual assessments of the biodiversity of selected ecosystems. Therefore, new concepts are required to calculate global biodiversity. Systematic approaches that evaluate small, complex biotopes exhaustively, or that calculate the number of symbionts or parasites on the basis of their potential hosts have already led to a substantial revision of earlier estimations. Here, an evaluation of potential animal hosts for methanogenic archaea and intestinal protists is described that reveals the importance of host taxonomy for the assessments. If molecular techniques can confirm the presumed specificity of symbiotic and parasitic associations a substantial revision of the current assumptions about the biodiversity of such organisms will be necessary.

The Y chromosomes of most Drosophila species are necessary for male fertility but they are not involved in sex determination. They have many puzzling properties that resemble the effects caused by B chromosomes. Classical genetic and molecular studies reveal substantial affinities between Y and B chromosomes and suggest that the Y chromosomes of Drosophila are not degenerated homologues of the X chromosomes, but rather that their Y chromosomes evolved as specialized supernumeraries similar to classical B chromosomes.

It has been assumed that the feeding habits of animals predispose the composition of the microbial biota living in their intestinal tracts. Here we show that in arthropods and vertebrates the presence of methanogenic bacteria requires a quality of the host that is under phylogenetic rather than dietary constraint: competence for intestinal methanogenic bacteria is a primitive-shared character among reptiles, birds, and mammals, and a shared-derived trait of millipedes, termites, cockroaches and scarab beetles. The presence of methanogenic bacteria seems to be a prerequisite for the evolution of anatomic specializations of the intestinal tract such as hindguts, caeca or rumina, and it is likely that it also has consequences for the reproductive strategies of the animals. Methanogenic animals contribute to atmospheric methane by their breath and faeces. Because the status as either methane-producer or non-producer is shared by most species belonging to a higher taxonomic unit, it is possible to calculate methane emissions that are characteristic for whole taxa. In combination with ecological field data on the biomass it is possible to arive at estimates concerning the global contributions by animals. The demonstration of a genetic basis for the symbiosis between methanogens and animals will allow new approaches for the reduction of methane emission by domestic animals.

The Y chromosomes of Drosophila are necessary for male fertility. They carry giant genes that have some unconventional properties besides controlling the motility of the spermatozoa. Classical genetic and molecular studies suggest that evolution has favoured the close association between these genes and repetitive DNA sequences with 'selfish' traits.

We have screened more than 110 representatives of the different taxa of terrestrial arthropods for methane production in order to obtain additional information about the origins of biogenic methane. Methanogenic bacteria occur in the hindguts of nearly all tropical representatives of millipedes (Diplopoda), cockroaches (Blattaria), termites (Isoptera), and scarab beetles (Scarabaeidae), while such methanogens are absent from 66 other arthropod species investigated. Three types of symbiosis were found: in the first type, the arthropod's hindgut is colonized by free methanogenic bacteria; in the second type, methanogens are closely associated with chitinous structures formed by the host's hindgut; the third type is mediated by intestinal anaerobic protists with intracellular methanogens. Such symbiotic associations are likely to be a characteristic property of the particular taxon. Since these taxa represent many families with thousands of species, the world populations of methane-producing arthropods constitute an enormous biomass. We show that arthropod symbionts can contribute substantially to atmospheric methane.

Free-Living Protozoa with Endosymbiotic Methanogens.- Anaerobic Ciliates and Their Methanogenic Endosymbionts.- Symbiotic Methanogens and Rumen Ciliates.- The Methanogenic and Eubacterial Endosymbionts of Trimyema.- Termite Gut Flagellates and Their Methanogenic and Eubacterial Symbionts.- Methanogens in the Digestive Tract of Termites.- Methanogenic Archaea in Humans and Other Vertebrates.- Methanogens in the Gastro-Intestinal Tract of Animals.- Syntrophy in Methanogenic Degradation.- Hydrogenosomes.- Evolution of Prokaryote-Animal Symbiosis from a Genomics Perspective.

Nearly all vertebrates host methanogens in their gastro-intestinal tracts. However, a great fraction of vertebrates emits only traces of methane from their faeces (∼1 nmol/g faeces/h) and has no significant amounts of methane in their breath. In contrast, many animals host some 100 times more methanogens in their gastro-intestinal tract and emit methane in their breath. These substantial differences are not caused by different feeding habits; rather a genetic factor controls the presence of large amounts of methanogens. The attribute “methane production” is evolutionarily stable, and the loss of this character obeys Dollo’s law: once lost in the course of evolution, this character cannot be acquired another time.

Background There are thousands of very diverse ciliate species from which only a handful mitochondrial genomes have been studied so far. These genomes are rather similar because the ciliates analysed (Tetrahymena spp. and Paramecium aurelia) are closely related. Here we study the mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus. These ciliates are only distantly related to Tetrahymena spp. and Paramecium aurelia, but more closely related to Nyctotherus ovalis, which possesses a hydrogenosomal (mitochondrial) genome. Results The linear mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus were sequenced and compared with the mitochondrial genomes of several Tetrahymena species, Paramecium aurelia and the partially sequenced mitochondrial genome of the anaerobic ciliate Nyctotherus ovalis. This study reports new features such as long 5'gene extensions of several mitochondrial genes, extremely long cox1 and cox2 open reading frames and a large repeat in the middle of the linear mitochondrial genome. The repeat separates the open reading frames into two blocks, each having a single direction of transcription, from the repeat towards the ends of the chromosome. Although the Euplotes mitochondrial gene content is almost identical to that of Paramecium and Tetrahymena, the order of the genes is completely different. In contrast, the 33273 bp (excluding the repeat region) piece of the mitochondrial genome that has been sequenced in both Euplotes species exhibits no difference in gene order. Unexpectedly, many of the mitochondrial genes of E. minuta encoding ribosomal proteins possess N-terminal extensions that are similar to mitochondrial targeting signals. Conclusion The mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus are rather different from the previously studied genomes. Many genes are extended in size compared to mitochondrial genes from other sources.

Two competing hypotheses have been proposed for the function of the Y chromosomal fertility factors in Drosophila, which form giant lampbrush loops during the primary spermatocyte stage. The first hypothesis suggests a conventional coding function, the second proposes an unconventional gene function mediated through protein binding by nascent transcripts. Therefore, we studied the genetics and cytogenetics of the two Y chromosomal fertility genes A and C of Drosophila hydei (which form the lampbrush loops threads and pseudonucleolus) in order to test the validity of these different hypotheses. Both lampbrush loops bind specific proteins, which are recognized by different antisera. Absence of either of the lampbrush loops does not interfere with the synthesis of the antigens but completely prevents the binding of the particular antigen to other lampbrush loops. Absence of the loops also does not interfere with the postmeiotic presence and localization of the particular antigen. Deletion (or inactivation) of either of the lampbrush loops threads or pseudonucleolus causes sterility of the male flies as do other male-sterile alleles of both fertility genes, which do not affect the morphology of the lampbrush loops. The phenotypic effects of these mutations on sperm morphogenesis are identical for all various male-sterile alleles of each of the fertility genes A and C, regardless of whether a particular allele leaves the loop intact, modifies that loop, or deletes (or inactivates) the loop completely. Finally, the isolation of fertile Y chromosomal mutations which modify the morphology of the lampbrush loops demonstrates that it is possible to uncouple loop morphology and genetic function. These findings do not support the hypothesis that the binding of proteins to a lampbrush loop has a substantial impact on spermiogenesis.

Background Nyctotherus ovalis is a single-celled eukaryote that has hydrogen-producing mitochondria and lives in the hindgut of cockroaches. Like all members of the ciliate taxon, it has two types of nuclei, a micronucleus and a macronucleus. N. ovalis generates its macronuclear chromosomes by forming polytene chromosomes that subsequently develop into macronuclear chromosomes by DNA elimination and rearrangement. Results We examined the structure of these gene-sized macronuclear chromosomes in N. ovalis. We determined the telomeres, subtelomeric regions, UTRs, coding regions and introns by sequencing a large set of macronuclear DNA sequences (4,242) and cDNAs (5,484) and comparing them with each other. The telomeres consist of repeats CCC(AAAACCCC)n, similar to those in spirotrichous ciliates such as Euplotes, Sterkiella (Oxytricha) and Stylonychia. Per sequenced chromosome we found evidence for either a single protein-coding gene, a single tRNA, or the complete ribosomal RNAs cluster. Hence the chromosomes appear to encode single transcripts. In the short subtelomeric regions we identified a few overrepresented motifs that could be involved in gene regulation, but there is no consensus polyadenylation site. The introns are short (21–29 nucleotides), and a significant fraction (1/3) of the tiny introns is conserved in the distantly related ciliate Paramecium tetraurelia. As has been observed in P. tetraurelia, the N. ovalis introns tend to contain in-frame stop codons or have a length that is not dividable by three. This pattern causes premature termination of mRNA translation in the event of intron retention, and potentially degradation of unspliced mRNAs by the nonsense-mediated mRNA decay pathway. Conclusion The combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.

Fungi form a very diverse group of eukaryotes. The majority of investigated fungi contain mitochondria and are capable of oxidative phosphorylation. On the other hand, anaerobically functioning fungi, found as symbionts in the gastrointestinal tract of many herbivorous mammals, contain hydrogenosomes. These organelles of mitochondrial origin are also found in multiple classes of anaerobically functioning protists. Hydrogenosomes produce hydrogen as an end product of a fermentative energy metabolism and produce ATP by substrate-level phosphorylation. However, the hydrogenosomes of the anaerobic fungi Neocallimastix and Piromyces differ from the hydrogenosomes of trichomonads and those of anaerobic ciliates in the way they convert pyruvate to acetyl-CoA. The hydrogenosomes of these anaerobic fungi use pyruvate:formate lyase (PFL), whereas trichomonads use pyruvate-ferredoxin oxidoreductase (PFO), and anaerobic ciliates use pyruvate dehydrogenase (PDH) for the degradation of pyruvate. The characteristics and role of these hydrogenosomes in the energy metabolism of anaerobic fungi are discussed.

We constructed balancer-chromosomes for the large autosomes ofDrosophila hydei and screened more than 16000 chromosomes for male sterile mutations in order to dissect spermatogenesis genetically. 365 mutants on the X chromosome and the autosomes 2, 3, and 4 were recovered and analysed cytologically in squash preparations under phase-contrast optics. The majority of the mutations allows a rather advanced differentiation of the spermatozoa. At the light-microscopical level, it is possible to classify these mutations with respect to individualization, coiling or motility of the mutant spermatozoa. In contrast, a small number of mutants exhibits conspicuous, pleiotropic phenotypes. Gonial divisions, the shaping of the spermatocyte nucleus and male meiotic divisions are controlled by X chromosomal or autosomal genes which can mutate to male sterile alleles. A number of nonallelic 3rd chromosome male sterile mutations interfere with the unfolding of the Y chromosomal lampbrush loops. Other autosomal male sterile mutations modify the morphology of these lampbrush loops. Another group of mutations inhibits the formation of the nebenkern while the development of the spermatid nucleus and the flagellum can proceed. Such male sterile mutations can decouple the development of nucleus, protein body, nebenkern, and flagellum of the spermatid. Thus, we can describe spermatogenesis inDrosophila as the coordinate execution of the individual developmental programs of the different components of the spermatozoon.

Methanomicrococcus blatticola is an obligately anaerobic methanogen that derives the energy for growth exclusively from the reduction of methylated compounds to methane with molecular hydrogen as energy source. Competition for methanol (concentration below 10 microM) and H(2) (concentration below 500 Pa), as well as oxidative stress due to the presence of oxygen are likely to occur in the peripheral region of the cockroach hindgut, the species' normal habitat. We investigated the ecophysiological properties of M. blatticola to explain how it can successfully compete for its methanogenic substrates. The organism showed affinities for methanol (K(m)=5 microM; threshold1 microM) and hydrogen (K(m)=200 Pa; threshold0.7 Pa) that are superior to other methylotrophic methanogens (Methanosphaera stadtmanae, Methanosarcina barkeri) investigated here. Thermodynamic considerations indicated that 'methanol respiration', i.e. the use of methanol as the terminal electron acceptor, represents an attractive mode of energy generation, especially at low hydrogen concentrations. Methanomicrococcus blatticola exploits the opportunities by specific growth rates (0.2 h(-1)) and specific growth yields (up to 7 g of dry cells per mole of methane formed) that are particularly high within the realm of mesophilic methanogens. Upon oxygen exposure, part of the metabolic activity may be diverted into oxygen removal, thus establishing appropriate anaerobic conditions for survival and growth.

Background The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I. Results The [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome. Conclusion The hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering.

The eukaryotic biodiversity in historical air-dried samples of Dutch agricultural soil has been assessed by random sequencing of an 18S rRNA gene library and by denaturing gradient gel electrophoresis. Representatives of nearly all taxa of eukaryotic soil microbes could be identified, demonstrating that it is possible to study eukaryotic microbiota in samples from soil archives that have been stored for more than 30 years at room temperature. In a pilot study, 41 sequences were retrieved that could be assigned to fungi and a variety of aerobic and anaerobic protists such as cercozoans, ciliates, xanthophytes (stramenopiles), heteroloboseans, and amoebozoans. A PCR-denaturing gradient gel electrophoresis analysis of samples collected between 1950 and 1975 revealed significant changes in the composition of the eukaryotic microbiota.

Background The horizontal transfer of expressed genes from Bacteria into Ciliates which live in close contact with each other in the rumen (the foregut of ruminants) was studied using ciliate Expressed Sequence Tags (ESTs). More than 4000 ESTs were sequenced from representatives of the two major groups of rumen Cilates: the order Entodiniomorphida (Entodinium simplex, Entodinium caudatum, Eudiplodinium maggii, Metadinium medium, Diploplastron affine, Polyplastron multivesiculatum and Epidinium ecaudatum) and the order Vestibuliferida, previously called Holotricha (Isotricha prostoma, Isotricha intestinalis and Dasytricha ruminantium). Results A comparison of the sequences with the completely sequenced genomes of Eukaryotes and Prokaryotes, followed by large-scale construction and analysis of phylogenies, identified 148 ciliate genes that specifically cluster with genes from the Bacteria and Archaea. The phylogenetic clustering with bacterial genes, coupled with the absence of close relatives of these genes in the Ciliate Tetrahymena thermophila, indicates that they have been acquired via Horizontal Gene Transfer (HGT) after the colonization of the gut by the rumen Ciliates. Conclusion Among the HGT candidates, we found an over-representation (>75%) of genes involved in metabolism, specifically in the catabolism of complex carbohydrates, a rich food source in the rumen. We propose that the acquisition of these genes has greatly facilitated the Ciliates' colonization of the rumen providing evidence for the role of HGT in the adaptation to new niches.