Rob M. de Graaf

An anaerobic mitochondrion that produces hydrogen.

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product—biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.

A Metagenomics-Based Metabolic Model of Nitrate-Dependent Anaerobic Oxidation of Methane by Methanoperedens-Like Archaea

Methane oxidation is an important process to mitigate the emission of the greenhouse gas methane and further exacerbating of climate forcing. Both aerobic and anaerobic microorganisms have been reported to catalyze methane oxidation with only a few possible electron acceptors. Recently, new microorganisms were identified that could couple the oxidation of methane to nitrate or nitrite reduction. Here we investigated such an enrichment culture at the (meta) genomic level to establish a metabolic model of nitrate-driven anaerobic oxidation of methane (nitrate-AOM). Nitrate-AOM is catalyzed by an archaeon closely related to (reverse) methanogens that belongs to the ANME-2d clade, tentatively named Methanoperedens nitroreducens. Methane may be activated by methyl-CoM reductase and subsequently undergo full oxidation to carbon dioxide via reverse methanogenesis. All enzymes of this pathway were present and expressed in the investigated culture. The genome of the archaeal enrichment culture encoded a variety of enzymes involved in an electron transport chain similar to those found in Methanosarcina species with additional features not previously found in methane-converting archaea. Nitrate reduction to nitrite seems to be located in the pseudoperiplasm and may be catalyzed by an unusual Nar-like protein complex. A small part of the resulting nitrite is reduced to ammonium which may be catalyzed by a Nrf-type nitrite reductase. One of the key questions is how electrons from cytoplasmically located reverse methanogenesis reach the nitrate reductase in the pseudoperiplasm. Electron transport in M. nitroreducens probably involves cofactor F420 in the cytoplasm, quinones in the cytoplasmic membrane and cytochrome c in the pseudoperiplasm. The membrane-bound electron transport chain includes F420H2 dehydrogenase and an unusual Rieske/cytochrome b complex. Based on genome and transcriptome studies a tentative model of how central energy metabolism of nitrate-AOM could work is presented and discussed.

The Organellar Genome and Metabolic Potential of the Hydrogen-Producing Mitochondrion of Nyctotherus ovalis

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.

Macronuclear genome structure of the ciliate Nyctotherus ovalis : Single-gene chromosomes and tiny introns

BackgroundNyctotherus 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.ResultsWe 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.ConclusionThe combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.

The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin

BackgroundThe 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.ResultsThe [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.ConclusionThe 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.

Plasmids from the gut microbiome of cabbage root fly larvae encode SaxA that catalyses the conversion of the plant toxin 2-phenylethyl isothiocyanate

Cabbage root fly larvae (Delia radicum) cause severe crop losses (≥ 50%) of rapeseed/ canola and cabbages used in the food and biofuel industries. These losses occur despite the fact that cabbages produce insecticidal toxins such as isothiocyanates. Here we describe the cabbage root fly larval gut microbiome as a source of isothiocyanate degrading enzymes. We sequenced the microbial gut community of the larvae and analysed phylogenetic markers and functional genes. We combined this with the isolation of several microbial strains representing the phylogenetic distribution of the metagenome. Eleven of those isolates were highly resistant towards 2-phenylethyl isothiocyanate, a subset also metabolized 2-phenylethyl isothiocyanate. Several plasmids appeared to be shared between those isolates that metabolized the toxin. One of the plasmids harboured a saxA gene that upon transformation gave resistance and enabled the degradation of 2-phenylethyl isothiocyanate in Escherichia coli. Taken together, the results showed that the cabbage root fly larval gut microbiome is capable of isothiocyanate degradation, a characteristic that has not been observed before, and may help us understand and design new pest control strategies.

Hydrogenosomes of Anaerobic Ciliates

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.

Membrane-bound electron transport systems of an anammox bacterium: A complexome analysis.

Electron transport, or oxidative phosphorylation, is one of the hallmarks of life. To this end, prokaryotes evolved a vast variety of protein complexes, only a small part of which have been discovered and studied. These protein complexes allow them to occupy virtually every ecological niche on Earth. Here, we applied the method of proteomics-based complexome profiling to get a better understanding of the electron transport systems of the anaerobic ammonium-oxidizing (anammox) bacteria, the N2-producing key players of the global nitrogen cycle. By this method nearly all respiratory complexes that were previously predicted from genome analysis to be involved in energy and cell carbon fixation were validated. More importantly, new and unexpected ones were discovered. We believe that complexome profiling in concert with (meta)genomics offers great opportunities to expand our knowledge on bacterial respiratory processes at a rapid and massive pace, in particular in new and thus far poorly investigated non-model and environmentally-relevant species.

Isolation and identification of 4-α-rhamnosyloxy benzyl glucosinolate in Noccaea caerulescens showing intraspecific variation

Glucosinolates are secondary plant compounds typically found in members of the Brassicaceae and a few other plant families. Usually each plant species contains a specific subset of the ∼ 130 different glucosinolates identified to date. However, intraspecific variation in glucosinolate profiles is commonly found. Sinalbin (4-hydroxybenzyl glucosinolate) so far has been identified as the main glucosinolate of the heavy metal accumulating plant species Noccaea caerulescens (Brassicaceae). However, a screening of 13 N. caerulescens populations revealed that in 10 populations a structurally related glucosinolate was found as the major component. Based on nuclear magnetic resonance (NMR) and mass spectrometry analyses of the intact glucosinolate as well as of the products formed after enzymatic conversion by sulfatase or myrosinase, this compound was identified as 4-α-rhamnosyloxy benzyl glucosinolate (glucomoringin). So far, glucomoringin had only been reported as the main glucosinolate of Moringa spp. (Moringaceae) which are tropical tree species. There was no apparent relation between the level of soil pollution at the location of origin, and the presence of glucomoringin. The isothiocyanate that is formed after conversion of glucomoringin is a potent antimicrobial and antitumor agent. It has yet to be established whether glucomoringin or its breakdown product have an added benefit to the plant in its natural habitat.

The symbiotic intestinal ciliates and the evolution of their hosts

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.