Eukaryotic Fe-hydrogenases -- old eukaryotic heritage or adaptive acquisitions?

All eukaryotes seem to possess proteins that most probably evolved from an ancestral Fe-hydrogenase. These proteins, known as NARF or Nar, do not produce hydrogen. Notably, a small group of rather unrelated unicellular anaerobes and a few algae possess Fe-hydrogenases, which produce hydrogen. In most, but not all organisms, hydrogen production occurs in membrane-bounded organelles, i.e. hydrogenosomes or plastids. Whereas plastids are monophyletic, hydrogenosomes evolved repeatedly and independently from mitochondria or mitochondria-like organelles. A systematic analysis of the various hydrogenosomes and their hydrogenases will contribute to an understanding of the evolution of the eukaryotic cell, and provide clues to the evolutionary origin(s) of the Fe-hydrogenase.

A re-appraisal of the diversity of the methanogens associated with the rumen ciliates.

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.

Assessment of ciliates in the sheep rumen by DGGE.

AIMS: This work was carried out to develop a rapid molecular profiling technique to screen ciliate populations in the rumen of sheep. METHODS AND RESULTS: DGGE was used to study the ciliate diversity in the rumen of sheep. There was considerable variation between sheep which were co-housed, and fed the same diet. However, no difference in the major banding patterns was detected, when samples were collected from a single sheep sampled at different points. Following dietary changes, use of a pair-wise comparison of lanes, demonstrated that although there was still diversity between the ciliate population of sheep, the effects as a result of dietary changes were greater. CONCLUSIONS: The technique generated molecular profiles which are sufficiently different to allow comparison between samples, and to permit molecular ecological studies on the rumen ciliate population. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study means that ciliate diversity in the rumen may now be studied by those unfamiliar with morphological identification of these organisms.

Cross-epithelial hydrogen transfer from the midgut compartment drives methanogenesis in the hindgut of cockroaches.

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 (<0.1 kPa) in the posterior hindgut. Moreover, living cockroaches emitted large quantities of CH(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.

Male sterile mutant casanova gives clues to mechanisms of sperm-egg interactions in Drosophila melanogaster.

The plasma membrane of the spermatozoa of Drosophila melanogaster contains two integral proteins with glycosidase activity, beta-N-acetylglucosaminidase and alpha-D-mannosidase. Biochemical analysis and ultrastructural cytochemistry of spermatozoa of the autosomal male sterile mutant casanova reveal that at least one of these enzymes, beta-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 alpha-D-mannosidase activity as wild-type sperm, whereas beta-N-acetylglucosaminidase activity reaches only 51% of the wild-type level. The biochemical characteristics of alpha-D-mannosidase and of the residual beta-N-acetylglucosaminidase are the same as in wild-type males. Ultrastructural localization of the enzymes indicated that casanova spermatozoa lacks beta-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 beta-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.

Hydrogenosomes: convergent adaptations of mitochondria to anaerobic environments.

Hydrogenosomes are membrane-bound organelles that compartmentalise the final steps of energy metabolism 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 (Nyctotherus 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.

Towards an understanding of the genetics of human male infertility: lessons from flies.

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.

Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates.

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.

Methanomicrococcus blatticola gen. nov., sp. nov., a methanol- and methylamine-reducing methanogen from the hindgut of the cockroach Periplaneta americana.

A small irregular coccoid methanogenic bacterium (PAT) was isolated from the hindgut of the cockroach Periplaneta americana. Fluorescence microscopy and transmission electron microscopy of the hindgut of P. americana suggest that the organism occurs abundantly in the microbiota attached to the hindgut wall. The strain produces methane by the reduction of methanol and methylated amines with molecular hydrogen. Acetate, coenzyme M, yeast extract, tryptic soy broth and vitamins are required for growth. The cells lack a rigid cell wall and lyse immediately in buffers of low ionic strength. Maximum rate of growth (specific growth rate, 0.22 h(-1)) occurs in a rich medium at 39 degrees C, at a pH range of 7.2-7.7 and at a salt concentration below 100 mM NaCl. Sequence analysis of the small-subunit rDNA indicates that strain PAT is related to the family Methanosarcinaceae but does not belong to any previously described genus. Therefore, it is proposed that strain PAT be classified in a new genus, related to the Methanosarcinaceae, as Methanomicrococcus blatticola (type strain PAT = DSM 13328T).

Hydrogenosomes: eukaryotic adaptations to anaerobic environments.

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 beta-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae.

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.

Voltage-dependent reversal of anodic galvanotaxis in Nyctotherus ovalis.

Aerobic and anaerobic ciliates swim towards the cathode when they are exposed to a constant DC field. Nyctotherus ovalis from the intestinal tract of cockroaches exhibits a different galvanotactic response: at low strength of the DC field the ciliates orient towards the anode whereas DC fields above 2-4 V/cm cause cathodic swimming. This reversal of the galvanotactic response is not due to backward swimming. Rather the ciliates turn around and orient to the cathode with their anterior pole. Exposure to various cations, chelators, and Ca(2+)-channel inhibitors suggests that Ca(2+)-channels similar to the "long lasting" Ca(2+)-channels of vertebrates are involved in the voltage-dependent anodic galvanotaxis. Evidence is presented that host-dependent epigenetic factors can influence the voltage-threshold for the switch from anodic to cathodic swimming.

A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism.

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives.

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.

Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2.

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.

Eukaryotic molecular biodiversity: systematic approaches for the assessment of symbiotic associations.

'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.

Endosymbiotic interactions in anaerobic protozoa.

Several aspects of the endosymbiosis of methanogenic archaea with anaerobic protozoa are reviewed. Special attention is played 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 machinery 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.