J. H. P. Hackstein

Radboud University Nijmegen, Nymegen, Gelderland, Netherlands

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Publications (113)393.94 Total impact

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    ABSTRACT: 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<0.05). The influence of xylan depended on its dose. The enzymatic studies confirmed the fibrolytic and amylolytic abilities of ciliates. Neither the solubility nor the increase of the supplemented dose of purified protein influenced the density of the ciliate population. The recommended food consisted of meadow hay, wheat gluten, crystalline cellulose and barley flour when supplied in the proportions of 0.6, 0.16, 0.12 and 0.12mg/mL per day. We observed morphological variation of the ciliates, involving partial or complete reduction of the caudal lobes. Copyright © 2014 Elsevier GmbH. All rights reserved.
    European Journal of Protistology 11/2014; 51(1):109-117. DOI:10.1016/j.ejop.2014.11.002 · 2.34 Impact Factor
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    ABSTRACT: 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.
    European Journal of Protistology 04/2014; 50(2):166-173. DOI:10.1016/j.ejop.2014.01.004 · 2.34 Impact Factor
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    ABSTRACT: 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.
    Molecular Biology and Evolution 03/2011; 28(8):2379-91. DOI:10.1093/molbev/msr059 · 14.31 Impact Factor
  • Rob M. De Graaf · Johannes H. P. Hackstein
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    ABSTRACT: The adaptation of unicellular eukaryotes to life under permanently anaerobic conditions involves substantial changes in mitochondrial morphology and metabolism. The analysis of 16 independently arisen adaptations reveals that losses of genes of the organellar genome initiate a pathway that eventually leads to the complete loss of the genome. Simultaneously, the mitochondrial electron transport chain undergoes changes that lastly lead to a complete loss of the subunits and the loss of the ability to generate ATP by using a proton gradient. In addition, many genes found in textbook mitochondria become lost, leading to a rainbow of mitochondrion-derived organelles with characteristic proteomes. These derived organelles are named hydrogenosomes if they generate hydrogen and ATP, and mitosomes if they produce neither of the two. In essence, these organelles are mitochondria that adapted to life under anaerobic conditions by reductive evolutionary tinkering.
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    Johannes H. P. Hackstein
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    ABSTRACT: Many anaerobic ciliates possess hydrogenosomes, and consequently, they have the potential to host endosymbiotic methanogens. The endosymbiotic methanogens are vertically transmitted and even the cyst stages carry methanogens. Accordingly, the analysis of the SSU rRNA genes of ciliates and their methanogenic endosymbionts revealed that the endosymbionts are specific for their hosts and not identical with free-living methanogens. Notably, the endosymbionts of a monophyletic group of ciliates that thrive in either freshwater environments or intestinal tracts are substantially different. Ciliates from freshwater sediments host methanogens belonging to the Methanomicrobiales, while ciliates thriving in the intestinal tracts of cockroaches, millipedes and frogs host methanogens that belong to the Methanobacteriales. Comparative analysis of free-living and gut-dwelling ciliates and their corresponding endosymbionts reveals only a limited co-evolution suggesting infrequent endosymbiont replacements. Such an endosymbiont replacement is clearly the reason for the very distant endosymbionts of free-living and gut-dwelling ciliates: the endosymbionts are related to the methanogens in the particular environments, in which the hosts live.
    09/2010: pages 13-23;
  • Johannes H. P. Hackstein · Aloysius G. M. Tielens
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    ABSTRACT: “Hydrogenosomes” are mitochondrion-derived, double membrane-bounded organelles that produce hydrogen and ATP. These properties discriminate them from the likewise mitochondrion-derived “mitosomes” that produce neither hydrogen nor ATP. The only character that is most likely shared by mitochondria, hydrogenosomes, and mitosomes is their involvement in the Fe–S metabolism. Hydrogenosomes and mitosomes are found in a broad spectrum of rather unrelated species of unicellular, anaerobic eukaryotes, suggesting that hydrogenosomes and mitosomes evolved repeatedly and independently in the various taxonomic groups. With the exception of two hydrogenosomes, all these organelles lack a genome and an electron transport chain, which makes it sometimes difficult to trace their origins back to their mitochondrial origins. However, genomic evidence, EST studies, and the analysis of the organellar metabolism clearly reveal both a mitochondrial descent and individual differences in the properties of the various organelles. In this paper, we describe the diversity of hydrogenosomes based predominantly on information that became available recently. We also pay attention to the fact that certain hydrogenosomes are found in close association with endosymbiotic methanogens.
    09/2010: pages 175-206;
  • Johannes H. P. Hackstein · Theo A. van Alen
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    ABSTRACT: 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. Also invertebrates can host methanogens in their gastro-intestinal tract. In contrast to the vertebrates, only a few taxa of arthropods emit methane: millipedes, termites, cockroaches and scarab beetles. All other arthropods in our study did not emit methane and did not host even traces of methanogens. As in vertebrates, the diet of the animals is not crucial for the presence of methanogens. Again, a genetic factor seems to control the presence or absence of methanogens. Methanogenesis is also a prerequisite for the presence of intestinal anaerobic protozoa with endosymbiotic methanogens, but not for the presence of impressive structural differentiations of the hindgut epithelium, which – in methanogenic taxa – host enormous amounts of methanogens.
    09/2010: pages 115-142;
  • J. H. P. Hackstein · T. A. van Alen
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    ABSTRACT: Hydrogenosomes are organelles that produce molecular hydrogen and ATP. The broad phylogenetic distribution of their hosts suggests that the hydrogenosomes of these organisms evolved several times independently from the mitochondria of aerobic progenitors. Morphology and 18S rRNA phylogeny suggest that the microaerophilic amoeboflagellate Psalteriomonas lanterna, which possesses hydrogenosomes and elusive "modified mitochondria", belongs to the Heterolobosea, a taxon that consists predominantly of aerobic, mitochondriate organisms. This taxon is rather unrelated to taxa with hitherto studied hydrogenosomes. Electron microscopy of P. lanterna flagellates reveals a large globule in the centre of the cell that is build up from stacks of some 20 individual hydrogenosomes. The individual hydrogenosomes are surrounded by a double membrane that encloses a homogeneous, dark staining matrix lacking cristae. The "modified mitochondria" are found in the cytoplasm of the cell and are surrounded by 1-2 cisterns of rough endoplasmatic reticulum, just as the mitochondria of certain related aerobic Heterolobosea. The ultrastructure of the "modified mitochondria" and hydrogenosomes is very similar, and they have the same size distribution as the hydrogenosomes that form the central stack.The phylogenetic analysis of selected EST sequences (Hsp60, Propionyl-CoA carboxylase) supports the phylogenetic position of P. lanterna close to aerobic Heterolobosea (Naegleria gruberi). Moreover, this analysis also confirms the identity of several mitochondrial or hydrogenosomal key-genes encoding proteins such as a Hsp60, a pyruvate:ferredoxin oxidoreductase, a putative ADP/ATP carrier, a mitochondrial complex I subunit (51 KDa), and a [FeFe] hydrogenase. Comparison of the ultrastructure of the "modified mitochondria" and hydrogenosomes strongly suggests that both organelles are just two morphs of the same organelle. The EST studies suggest that the hydrogenosomes of P. lanterna are physiologically similar to the hydrogenosomes of Trichomonas vaginalis and Trimastix pyriformis. Phylogenetic analysis of the ESTs confirms the relationship of P. lanterna with its aerobic relative, the heterolobosean amoeboflagellate Naegleria gruberi, corroborating the evolution of hydrogenosomes from a common, mitochondriate ancestor.
    BMC Evolutionary Biology 12/2009; 9(1):287. DOI:10.1186/1471-2148-9-287 · 3.41 Impact Factor
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    ABSTRACT: 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. 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. 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.
    BMC Genomics 11/2009; 10(1):514. DOI:10.1186/1471-2164-10-514 · 4.04 Impact Factor
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    ABSTRACT: 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. 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. The combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.
    BMC Genomics 01/2009; 9:587. DOI:10.1186/1471-2164-9-587 · 4.04 Impact Factor
  • Johannes H. P. Hackstein · Scott E. Baker · Jaap J. van Hellemond · Aloysius G. M. Tielens
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    ABSTRACT: Fungi form avery diverse group of eukaryotes. The majority of investigated fungi contain mitochondria and are capable of oxidative phosphorylation. On the other hand, anaerobically functioning chytridiomycete fungi, found as symbionts in the gastrointestinal tract of many herbivorous mammals, contain hydrogenosomes. These organelles are found in multiple classes of protozoa and catabolize glycolytic end products and produce hydrogen and ATP by substrate-level phosphorylation. However, in contrast to the hydrogenosomes of trichomonads and anaerobic ciliates, the hydrogenosomes of the anaerobic chytrids Neocallimastix and Piromyces lack pyruvate dehydrogenase (PDH) and pyruvate-ferrodoxin oxidoreductase (PFO) and instead contain pyruvate-formate lyase (PFL). The function in carbohydrate metabolism of these hydrogenosomes of anaerobic chytridiomycete fungi and their evolutionary relation to fungal mitochondria is discussed.
    08/2008: pages 147-162;
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    ABSTRACT: 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 agenome and this genome is clearly of mitochondrial origin. In fact, the hydrogenosome of N.ovalis unites typical mitochondrial features such as agenome 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.
    01/2008: pages 97-112;
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    Wander W Sprenger · Johannes H P Hackstein · Jan T Keltjens
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    ABSTRACT: 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<1 microM) and hydrogen (K(m)=200 Pa; threshold <0.7 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.
    FEMS Microbiology Ecology 06/2007; 60(2):266-75. DOI:10.1111/j.1574-6941.2007.00287.x · 3.88 Impact Factor
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    ABSTRACT: 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. 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. 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.
    BMC Evolutionary Biology 02/2007; 7:230. DOI:10.1186/1471-2148-7-230 · 3.41 Impact Factor
  • J. H. P. Hackstein · J. Tjaden · W. J. H. Koopman · M. A. Huijnen
    World Views Environment Culture Religion 01/2007;
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    Johannes H P Hackstein · Joachim Tjaden · Martijn Huynen
    Current Genetics 11/2006; 50(4):225-45. DOI:10.1007/s00294-006-0088-8 · 2.68 Impact Factor

Publication Stats

2k Citations
393.94 Total Impact Points


  • 1987–2014
    • Radboud University Nijmegen
      • • Institute for Water and Wetland Research
      • • Faculty of Science
      • • Department of Microbiology
      • • Department of Molecular and Laser Physics
      • • Department of Genetics
      Nymegen, Gelderland, Netherlands
  • 2000
    • Utrecht University
      Utrecht, Utrecht, Netherlands
    • Universität Stuttgart
      Stuttgart, Baden-Württemberg, Germany