Publications by authors named "Harry R Harhangi"

27 Publications

  • Page 1 of 1

Expanding the verrucomicrobial methanotrophic world: description of three novel species of Methylacidimicrobium gen. nov.

Appl Environ Microbiol 2014 Nov 29;80(21):6782-91. Epub 2014 Aug 29.

Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Nijmegen, the Netherlands.

Methanotrophic Verrucomicrobia have been found in geothermal environments characterized by high temperatures and low pH values. However, it has recently been hypothesized that methanotrophic Verrucomicrobia could be present under a broader range of environmental conditions. Here we describe the isolation and characterization of three new species of mesophilic acidophilic verrucomicrobial methanotrophs from a volcanic soil in Italy. The three new species showed 97% to 98% 16S rRNA gene identity to each other but were related only distantly (89% to 90% on the 16S rRNA level) to the thermophilic genus Methylacidiphilum. We propose the new genus Methylacidimicrobium, including the novel species Methylacidimicrobium fagopyrum, Methylacidimicrobium tartarophylax, and Methylacidimicrobium cyclopophantes. These mesophilic Methylacidimicrobium spp. were more acid tolerant than their thermophilic relatives; the most tolerant species, M. tartarophylax, still grew at pH 0.5. The variation in growth temperature optima (35 to 44°C) and maximum growth rates (µmax; 0.013 to 0.040 h(-1)) suggested that all species were adapted to a specific niche within the geothermal environment. All three species grew autotrophically using the Calvin cycle. The cells of all species contained glycogen particles and electron-dense particles in their cytoplasm as visualized by electron microscopy. In addition, the cells of one of the species (M. fagopyrum) contained intracytoplasmic membrane stacks. The discovery of these three new species and their growth characteristics expands the known diversity of verrucomicrobial methanotrophs and shows that they are present in many more ecosystems than previously assumed.
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http://dx.doi.org/10.1128/AEM.01838-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249049PMC
November 2014

An ecogenomic analysis of herbivore-induced plant volatiles in Brassica juncea.

Mol Ecol 2013 Dec 13;22(24):6179-96. Epub 2013 Nov 13.

Department of Zoology, Sri Venkateswara College, University of Delhi, Benito Juarez Marg, Dhaula kuan, New Delhi, 11002, India.

Upon herbivore feeding, plants emit complex bouquets of induced volatiles that may repel insect herbivores as well as attract parasitoids or predators. Due to differences in the temporal dynamics of individual components, the composition of the herbivore-induced plant volatile (HIPV) blend changes with time. Consequently, the response of insects associated with plants is not constant either. Using Brassica juncea as the model plant and generalist Spodoptera spp. larvae as the inducing herbivore, we investigated herbivore and parasitoid preference as well as the molecular mechanisms behind the temporal dynamics in HIPV emissions at 24, 48 and 72 h after damage. In choice tests, Spodoptera litura moth preferred undamaged plants, whereas its parasitoid Cotesia marginiventris favoured plants induced for 48 h. In contrast, the specialist Plutella xylostella and its parasitoid C. vestalis preferred plants induced for 72 h. These preferences matched the dynamic changes in HIPV blends over time. Gene expression analysis suggested that the induced response after Spodoptera feeding is mainly controlled by the jasmonic acid pathway in both damaged and systemic leaves. Several genes involved in sulphide and green leaf volatile synthesis were clearly up-regulated. This study thus shows that HIPV blends vary considerably over a short period of time, and these changes are actively regulated at the gene expression level. Moreover, temporal changes in HIPVs elicit differential preferences of herbivores and their natural enemies. We argue that the temporal dynamics of HIPVs may play a key role in shaping the response of insects associated with plants.
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http://dx.doi.org/10.1111/mec.12555DOI Listing
December 2013

Evidence that the catenane form of CS2 hydrolase is not an artefact.

Chem Commun (Camb) 2013 Sep;49(71):7770-2

Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.

CS2 hydrolase, a zinc-dependent enzyme that converts carbon disulfide to carbon dioxide and hydrogen sulfide, exists as a mixture of octameric ring and hexadecameric catenane forms in solution. A combination of size exclusion chromatography, multi-angle laser light scattering, and mass spectrometric analyses revealed that the unusual catenane structure is not an artefact, but a naturally occurring structure.
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http://dx.doi.org/10.1039/c3cc43219jDOI Listing
September 2013

Enrichment of an anammox bacterial community from a flooded paddy soil.

Environ Microbiol Rep 2013 Jun 28;5(3):483-9. Epub 2013 Feb 28.

Department of Environmental Engineering, Zhejiang University, Hangzhou, China.

This study describes the enrichment of anammox bacteria in a column simulating oxygen limited flooded paddy soils, which are important man-made ecosystems that receive substantial amounts of fixed nitrogen. The upper 50 cm of the paddy soil, containing a high amount of ammonium [1.6-10.4 mmol N kg (dry weight)(-1)], was selected as the inoculum for anammox enrichment. After 18 months of incubation with freshwater from the paddy soil ecosystem, the enrichment culture consumed approximately 4 mmol ammonium l(-1) day(-1) and 5 mmol nitrite l(-1) day(-1). The maximum specific anammox activity of the culture was 35.7 μmol N g (dry weight)(-1) h(-1). Fluorescence in situ hybridization indicated that anammox cells constituted 50% ± 10% of the enrichment culture. The phylogenetic analyses of 16S rRNA and the diagnostic hydrazine synthase (hzsA) genes showed that two dominant anammox species were enriched from paddy soil. The enriched Candidatus Anammoxoglobus-like organisms showed a 16S rRNA gene similarity of 97.5-99.2% to Candidatus Anammoxoglobus propionicus and the Candidatus Jettenia-like organisms showed 92.1-93.1% 16S rRNA gene identity to Candidatus Jettenia asiatica. Real-time quantitative PCR of hzsA gene suggested that up to 10(10) copies g (dry weight)(-1) of soil anammox bacteria were present in the enrichment culture.
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http://dx.doi.org/10.1111/1758-2229.12038DOI Listing
June 2013

Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane-oxidizing bacteria in a paddy soil.

FEMS Microbiol Lett 2012 Nov 18;336(2):79-88. Epub 2012 Sep 18.

State Key Laboratory of Environmental Aquatic Quality, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China.

The anaerobic ammonium-oxidizing (anammox) and nitrite-dependent anaerobic methane-oxidizing (n-damo) bacteria in a paddy soil core (0-100 cm) were investigated with newly designed primers targeting the hydrazine synthase β-subunit (hzsB) of anammox bacteria and the recently published primers targeting the pmoA and 16S rRNA genes of n-damo bacteria. The hzsB gene was identified as a proper biomarker to explore the anammox bacterial biodiversity and abundance in soil. The anammox bacteria were present throughout the soil core with the highest abundance of 2.7 × 10(6) hzsB copies g(-1) dry soil at 40-50 cm and were not detectable below 70 cm. Sequences related to at least three species of known anammox bacteria, 'Brocadia anammoxidans', 'Brocadia fulgida', and 'Jettenia asiatica' were detected. By combining the analysis of pmoA and 16S rRNA genes, the n-damo bacteria were observed to be present in 30-70 cm with abundance from 6.5 × 10(3) (60-70 cm) to 7.5 × 10(4) (30-40 cm) copies g(-1) dry soil. The pmoA sequences retrieved from different depths closely related to each other and formed a unique clade. Our results showed that anammox and n-damo bacteria co-occurred in the paddy soil. Both of them were abundant in deep layers (30-60 cm) and the community structures changed along depths in the soil core.
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http://dx.doi.org/10.1111/j.1574-6968.2012.02654.xDOI Listing
November 2012

Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis.

Arch Microbiol 2012 Nov 1;194(11):943-8. Epub 2012 Jul 1.

Department of Microbiology, IWWR, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.
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http://dx.doi.org/10.1007/s00203-012-0829-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3477478PMC
November 2012

Draft genome sequence of the volcano-inhabiting thermoacidophilic methanotroph Methylacidiphilum fumariolicum strain SolV.

J Bacteriol 2012 Jul;194(14):3729-30

Department of Microbiology, IWWR, Radboud University Nijmegen, Nijmegen, The Netherlands.

The draft genome of Methylacidiphilum fumariolicum SolV, a thermoacidophilic methanotroph of the phylum Verrucomicrobia, is presented. Annotation revealed pathways for one-carbon, nitrogen, and hydrogen catabolism and respiration together with central metabolic pathways. The genome encodes three orthologues of particulate methane monooxygenases. Sequencing of this genome will help in the understanding of methane cycling in volcanic environments.
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http://dx.doi.org/10.1128/JB.00501-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393509PMC
July 2012

Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria.

Appl Environ Microbiol 2012 Feb 2;78(3):752-8. Epub 2011 Dec 2.

Department of Microbiology, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Nijmegen, The Netherlands.

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.
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http://dx.doi.org/10.1128/AEM.07113-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264106PMC
February 2012

Anammox bacteria in different compartments of recirculating aquaculture systems.

Biochem Soc Trans 2011 Dec;39(6):1817-21

Department of Microbiology, IWWR, Radboud University Nijmegen, Heyendaalseweg 135, NL-6525 AJ, Nijmegen, The Netherlands.

Strict environmental restrictions force the aquaculture industry to guarantee optimal water quality for fish production in a sustainable manner. The implementation of anammox (anaerobic ammonium oxidation) in biofilters would result in the conversion of both ammonium and nitrite (both toxic to aquatic animals) into harmless dinitrogen gas. Both marine and freshwater aquaculture systems contain populations of anammox bacteria. These bacteria are also present in the faeces of freshwater and marine fish. Interestingly, a new planctomycete species appears to be present in these recirculation systems too. Further exploitation of anammox bacteria in different compartments of aquaculture systems can lead to a more environmentally friendly aquaculture practice.
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http://dx.doi.org/10.1042/BST20110743DOI Listing
December 2011

Molecular mechanism of anaerobic ammonium oxidation.

Nature 2011 Oct 2;479(7371):127-30. Epub 2011 Oct 2.

Institute for Water and Wetland Research, Department of Microbiology, Radboud University Nijmegen, Heyendaalseweg 135, 6525AJ Nijmegen, The Netherlands.

Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N(2)) to the atmosphere. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N(2)H(4)). Here we show that N(2)H(4) is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N(2)H(4). We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N(2)H(4) synthesis and its oxidation to N(2). These results present a new biochemical reaction forging an N-N bond and fill a lacuna in our understanding of the biochemical synthesis of the N(2) in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.
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http://dx.doi.org/10.1038/nature10453DOI Listing
October 2011

FACIL: Fast and Accurate Genetic Code Inference and Logo.

Bioinformatics 2011 Jul 8;27(14):1929-33. Epub 2011 Jun 8.

Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA, Nijmegen, Germany.

Motivation: The intensification of DNA sequencing will increasingly unveil uncharacterized species with potential alternative genetic codes. A total of 0.65% of the DNA sequences currently in Genbank encode their proteins with a variant genetic code, and these exceptions occur in many unrelated taxa.

Results: We introduce FACIL (Fast and Accurate genetic Code Inference and Logo), a fast and reliable tool to evaluate nucleic acid sequences for their genetic code that detects alternative codes even in species distantly related to known organisms. To illustrate this, we apply FACIL to a set of mitochondrial genomic contigs of Globobulimina pseudospinescens. This foraminifer does not have any sequenced close relative in the databases, yet we infer its alternative genetic code with high confidence values. Results are intuitively visualized in a Genetic Code Logo.

Availability And Implementation: FACIL is available as a web-based service at http://www.cmbi.ru.nl/FACIL/ and as a stand-alone program.
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http://dx.doi.org/10.1093/bioinformatics/btr316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129529PMC
July 2011

Intracellular localization of membrane-bound ATPases in the compartmentalized anammox bacterium 'Candidatus Kuenenia stuttgartiensis'.

Mol Microbiol 2010 Aug 9;77(3):701-15. Epub 2010 Jun 9.

Department of Microbiology, Institute for Water and Wetland Research, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands.

Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium 'Candidatus Kuenenia stuttgartiensis'. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07242.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2936114PMC
August 2010

Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia.

Environ Microbiol Rep 2009 Oct 3;1(5):293-306. Epub 2009 Mar 3.

Department of Microbiology, IWWR, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands. Department of Biology and Centre for Geobiology, University of Bergen, P.O. Box 7800, N-5020 Bergen, Norway. GNS Science, Extremophile Research Group, Private Bag 2000, 3352 Taupo, New Zealand. Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada, T2N 1N4. NIOZ Royal Netherlands Institute for Sea Research, Department of Marine Organic Biogeochemistry, P.O. Box 59, 1790 AB Den Burg, Texel, The Netherlands.

Aerobic methanotrophic bacteria are capable of utilizing methane as their sole energy source. They are commonly found at the oxic/anoxic interfaces of environments such as wetlands, aquatic sediments, and landfills, where they feed on methane produced in anoxic zones of these environments. Until recently, all known species of aerobic methanotrophs belonged to the phylum Proteobacteria, in the classes Gammaproteobacteria and Alphaproteobacteria. However, in 2007-2008 three research groups independently described the isolation of thermoacidophilic methanotrophs that represented a distinct lineage within the bacterial phylum Verrucomicrobia. Isolates were obtained from geothermal areas in Italy, New Zealand and Russia. They are by far the most acidophilic methanotrophs known, with a lower growth limit below pH 1. Here we summarize the properties of these novel methanotrophic Verrucomicrobia, compare them with the proteobacterial methanotrophs, propose a unified taxonomic framework for them and speculate on their potential environmental significance. New genomic and physiological data are combined with existing information to allow detailed comparison of the three strains. We propose the new genus Methylacidiphilum to encompass all three newly discovered bacteria.
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http://dx.doi.org/10.1111/j.1758-2229.2009.00022.xDOI Listing
October 2009

Anaerobic oxidation of dimethylsulfide and methanethiol in mangrove sediments is dominated by sulfate-reducing bacteria.

FEMS Microbiol Ecol 2009 Dec 19;70(3):483-92. Epub 2009 Aug 19.

Department of Microbiology, IWWR, Radboud University Nijmegen, Nijmegen, The Netherlands.

The oxidation of dimethylsulfide and methanethiol by sulfate-reducing bacteria (SRB) was investigated in Tanzanian mangrove sediments. The rate of dimethylsulfide and methanethiol accumulation in nonamended sediment slurry (control) incubations was very low while in the presence of the inhibitors tungstate and bromoethanesulfonic acid (BES), the accumulation rates ranged from 0.02-0.34 to 0.2-0.4 nmol g FW sediment(-1) h(-1), respectively. Degradation rates of methanethiol and dimethylsulfide added were 2-10-fold higher. These results point to a balance of production and degradation. Degradation was inhibited much stronger by tungstate than by BES, which implied that SRB were more important. In addition, a new species of SRB, designated strain SD1, was isolated. The isolate was a short rod able to utilize a narrow range of substrates including dimethylsulfide, methanethiol, pyruvate and butyrate. Strain SD1 oxidized dimethylsulfide and methanethiol to carbon dioxide and hydrogen sulfide with sulfate as the electron acceptor and exhibited a low specific growth rate of 0.010 +/- 0.002 h(-1), but a high affinity for its substrates. The isolated microorganism could be placed in the genus Desulfosarcina (the most closely related cultured species was Desulfosarcina variabilis, 97% identity). Strain SD1 represents a member of the dimethylsulfide/methanethiol-consuming SRB population in mangrove sediments.
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http://dx.doi.org/10.1111/j.1574-6941.2009.00765.xDOI Listing
December 2009

Cell division ring, a new cell division protein and vertical inheritance of a bacterial organelle in anammox planctomycetes.

Mol Microbiol 2009 Sep 24;73(6):1009-19. Epub 2009 Aug 24.

Department of Microbiology, Institute for Water and Wetland Research, Faculty of Science, Radboud University Nijmegen, Nijmegen, The Netherlands.

Anammox bacteria are members of the phylum Planctomycetes that oxidize ammonium anaerobically and produce a significant part of the atmosphere's dinitrogen gas. They contain a unique bacterial organelle, the anammoxosome, which is the locus of anammox catabolism. While studying anammox cell and anammoxosome division with transmission electron microscopy including electron tomography, we observed a cell division ring in the outermost compartment of dividing anammox cells. In most Bacteria, GTP hydrolysis drives the tubulin-analogue FtsZ to assemble into a ring-like structure at the cell division site where it functions as a scaffold for the molecular machinery that performs cell division. However, the genome of the anammox bacterium 'Candidatus Kuenenia stuttgartiensis' does not encode ftsZ. Genomic analysis of open reading frames with potential GTPase activity indicated a possible novel cell division ring gene: kustd1438, which was unrelated to ftsZ. Immunogold localization specifically localized kustd1438 to the cell division ring. Genomic analyses of other members of the phyla Planctomycetes and Chlamydiae revealed no putative functional homologues of kustd1438, suggesting that it is specific to anammox bacteria. Electron tomography also revealed that the bacterial organelle was elongated along with the rest of the cell and divided equally among daughter cells during the cell division process.
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http://dx.doi.org/10.1111/j.1365-2958.2009.06841.xDOI Listing
September 2009

Bacteria associated with iron seeps in a sulfur-rich, neutral pH, freshwater ecosystem.

ISME J 2008 Dec 28;2(12):1231-42. Epub 2008 Aug 28.

Department of Microbiology, IWWR, Radboud University Nijmegen, Toernooiveld 1, Nijmegen, The Netherlands.

The freshwater nature reserve De Bruuk is an iron- and sulfur-rich minerotrophic peatland containing many iron seeps and forms a suitable habitat for iron and sulfur cycle bacteria. Analysis of 16S rRNA gene-based clone libraries showed a striking correlation of the bacterial population of samples from this freshwater ecosystem with the processes of iron reduction (genus Geobacter), iron oxidation (genera Leptothrix and Gallionella) and sulfur oxidation (genus Sulfuricurvum). Results from fluorescence in situ hybridization analyses with a probe specific for the beta-1 subgroup of Proteobacteria, to which the genera Leptothrix and Gallionella belong, and newly developed probes specific for the genera Geobacter and Sulfuricurvum, supported the clone library data. Molecular data suggested members of the epsilonproteobacterial genus Sulfuricurvum as contributors to the oxidation of reduced sulfur compounds in the iron seeps of De Bruuk. In an evaluation of anaerobic dimethyl sulfide (DMS)-degrading activity of sediment, incubations with the electron acceptors sulfate, ferric iron and nitrate were performed. The fastest conversion of DMS was observed with nitrate. Further, a DMS-oxidizing, nitrate-reducing enrichment culture was established with sediment material from De Bruuk. This culture was dominated by dimorphic, prosthecate bacteria, and the 16S rRNA gene sequence obtained from this enrichment was closely affiliated with Hyphomicrobium facile, which indicates that the Hyphomicrobium species are capable of both aerobic and nitrate-driven DMS degradation.
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http://dx.doi.org/10.1038/ismej.2008.75DOI Listing
December 2008

A serpin in the cellulosome of the anaerobic fungus Piromyces sp. strain E2.

Mycol Res 2008 Aug 16;112(Pt 8):999-1006. Epub 2008 Feb 16.

Department of Microbiology, IWWR, Radboud University Nijmegen, Toernooiveld 1, NL-6525ED Nijmegen, The Netherlands.

A gene encoding a novel component of the cellulolytic complex (cellulosome) of the anaerobic fungus Piromyces sp. strain E2 was identified. The encoded 538 amino acid protein, named celpin, consists of a signal peptide, a positively charged domain of unknown function followed by two fungal dockerins, typical for components of the extracellular fungal cellulosome. The C-terminal end consists of a 380 amino acid serine proteinase inhibitor (or serpin) domain homologue, sharing 30% identity and 50% similarity to vertebrate and bacterial serpins. Detailed protein sequence analysis of the serpin domain revealed that it contained all features of a functional serpin. It possesses the conserved amino acids present in more than 70% of known serpins, and it contained the consensus of inhibiting serpins. Because of the confined space of the fungal cellulosome inside plant tissue and the auto-proteolysis of plant material in the rumen, the fungal serpin is presumably involved in protection of the cellulosome against plant proteinases. The celpin protein of Piromyces sp. strain E2 is the first non-structural, non-hydrolytic fungal cellulosome component. Furthermore, the celpin protein of Piromyces sp. strain E2 is the first representative of a serine proteinase inhibitor of the fungal kingdom.
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http://dx.doi.org/10.1016/j.mycres.2008.01.021DOI Listing
August 2008

Methanotrophy below pH 1 by a new Verrucomicrobia species.

Nature 2007 Dec 14;450(7171):874-8. Epub 2007 Nov 14.

Department of Microbiology, IWWR, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands.

Mud volcanoes, mudpots and fumaroles are remarkable geological features characterized by the emission of gas, water and/or semi-liquid mud matrices with significant methane fluxes to the atmosphere (10(-1) to 10(3) t y(-1)). Environmental conditions in these areas vary from ambient temperature and neutral pH to high temperatures and low pH. Although there are strong indications for biological methane consumption in mud volcanoes, no methanotrophic bacteria are known that would thrive in the hostile conditions of fumaroles (temperatures up to 70 degrees C and pH down to 1.8). The first step in aerobic methane oxidation is performed by a soluble or membrane-bound methane mono-oxygenase. Here we report that pmoA (encoding the beta-subunit of membrane-bound methane mono-oxygenase) clone libraries, made by using DNA extracted from the Solfatara volcano mudpot and surrounding bare soil near the fumaroles, showed clusters of novel and distant pmoA genes. After methanotrophic enrichment at 50 degrees C and pH 2.0 the most distant cluster, sharing less than 50% identity with any other described pmoA gene, was represented in the culture. Finally we isolated an acidiphilic methanotrophic bacterium Acidimethylosilex fumarolicum SolV belonging to the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, 'outside' the subphyla of the Alpha- and Gammaproteobacteria containing the established methanotrophs. This bacterium grows under oxygen limitation on methane as the sole source of energy, down to pH 0.8--far below the pH optimum of any previously described methanotroph. A. fumarolicum SolV has three different pmoA genes, with two that are very similar to sequences retrieved from the mudpot. Highly homologous environmental 16S rRNA gene sequences from Yellowstone Park show that this new type of methanotrophic bacteria may be a common inhabitant of extreme environments. This is the first time that a representative of the widely distributed Verrucomicrobia phylum, of which most members remain uncultivated, is coupled to a geochemically relevant reaction.
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http://dx.doi.org/10.1038/nature06222DOI Listing
December 2007

Close relationship of RNase P RNA in Gemmata and anammox planctomycete bacteria.

FEMS Microbiol Lett 2007 Mar 10;268(2):244-53. Epub 2007 Jan 10.

School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland, Australia.

The relationship of RNase P RNA from anammox bacteria 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis' with that from other Planctomycetes was investigated. Newly identified rnpB gene sequences were aligned against existing planctomycete RNase P RNA sequences and secondary structures deduced by a comparative approach. Deduced secondary structures were similar in both anammox bacteria and both possessed an insert within the P13 helix analogous to that present in all Gemmata isolates. Phylogenetic analysis also revealed a possible relationship between the RNase P RNA molecules of the two anammox organisms and the genus Gemmata.
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http://dx.doi.org/10.1111/j.1574-6968.2006.00597.xDOI Listing
March 2007

Expression and characterisation of a major c-type cytochrome encoded by gene kustc0563 from Kuenenia stuttgartiensis as a recombinant protein in Escherichia coli.

Protein Expr Purif 2007 Jan 9;51(1):28-33. Epub 2006 Sep 9.

Department of Biology (Area 10), University of York, Heslington, York YO10 5YW, UK.

The purification of small quantities of a major small c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis has recently been reported. In order to characterise this protein further we have expressed the gene encoding this cytochrome in Escherichia coli and have purified the protein to homogeneity. The protein is directed to the E. coli periplasm using its native signal sequence suggesting that it may be translocated via a Sec-type system in K. stuttgartiensis. The cytochrome has the visible spectroscopic properties typical of a low-spin c-type cytochrome, but these spectroscopic features broaden in high salt solutions. The oxidised cytochrome was able to bind the ligands NO and cyanide. A redox potential of +230 mV suggests that the protein is suitable to act as an electron carrier protein that may be involved in the respiratory chain between hydrazine oxidation and the reduction of nitrite. The predicted protein sequence for the cytochrome suggests it to be a predominantly alpha-helical protein, and this is supported by circular dichroism.
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http://dx.doi.org/10.1016/j.pep.2006.06.026DOI Listing
January 2007

Deciphering the evolution and metabolism of an anammox bacterium from a community genome.

Nature 2006 Apr;440(7085):790-4

Department of Microbiology, IWWR, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics--the reconstruction of genomic data directly from the environment--to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organism's special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.
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http://dx.doi.org/10.1038/nature04647DOI Listing
April 2006

High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

FEMS Yeast Res 2003 Oct;4(1):69-78

Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.
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http://dx.doi.org/10.1016/S1567-1356(03)00141-7DOI Listing
October 2003

Genomic DNA analysis of genes encoding (hemi-)cellulolytic enzymes of the anaerobic fungus Piromyces sp. E2.

Gene 2003 Sep;314:73-80

Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands.

Anaerobic fungi contain more than one copy of genes encoding (hemi-)cellulases in their genome. The arrangement of these genes on the chromosomes was not known. A genomic DNA (gDNA) library of Piromyces sp. E2 was screened with different probes specific for (hemi-)cellulolytic enzymes. This screening resulted in three gDNA clones with genes encoding glycoside hydrolase enzymes of families 1 (beta-glucosidase), 6 (exoglucanase) and 26 (mannanase). Each clone contained two or more genes of the same family. Comparison of the gene copies on a clone revealed that they were highly homologous, and in addition, 54-75% of the substitutions was synonymous. One of the mannanase genes contained an intron. PCR with selected primers resulted in a gDNA clone with a new representative (cel9B) of glycoside hydrolase family 9 (endoglucanase). Comparison with cel9A revealed that cel9B had 67% homology on the nucleotide level. Furthermore, three introns were present. All results of this paper taken together provided evidence for duplications of (hemi-)cellulolytic genes, which resulted in clusters of almost identical genes arranged head-to-tail on the genome. In contrast to other eukaryotes, this phenomenon appears frequently in anaerobic fungi.
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http://dx.doi.org/10.1016/s0378-1119(03)00705-4DOI Listing
September 2003

Cel6A, a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp. E2 and Piromyces equi.

Biochim Biophys Acta 2003 Jul;1628(1):30-9

Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands.

Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.
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http://dx.doi.org/10.1016/s0167-4781(03)00112-xDOI Listing
July 2003

Xylose metabolism in the anaerobic fungus Piromyces sp. strain E2 follows the bacterial pathway.

Arch Microbiol 2003 Aug 13;180(2):134-41. Epub 2003 Jun 13.

Department of Microbiology, University of Nijmegen, Toernooiveld 1, Faculty of Science, 6525 ED, Nijmegen, The Netherlands.

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25+/-13 nmol min(-1)mg protein(-1)). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.
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http://dx.doi.org/10.1007/s00203-003-0565-0DOI Listing
August 2003

beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase.

Biochem J 2003 Mar;370(Pt 3):963-70

Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands.

The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a beta-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular beta-glucosidase activity. To identify the component in the fungal cellulosome responsible for the beta-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737 bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for beta-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent K (m) and K (i) values of heterologous Cel3A and the fungal cellulosome for p -nitrophenyl-beta-D-glucopyranoside and D-glucono-1,5-delta-lactone respectively indicated that cel3A encodes the beta-glucosidase activity of the Piromyces sp. strain E2 cellulosome.
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http://dx.doi.org/10.1042/BJ20021767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223235PMC
March 2003

A highly expressed family 1 beta-glucosidase with transglycosylation capacity from the anaerobic fungus Piromyces sp. E2.

Biochim Biophys Acta 2002 Apr;1574(3):293-303

Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands.

Anaerobic fungi have very high cellulolytic activities and thus degrade cellulose very efficiently. In cellulose hydrolysis, beta-glucosidases play an important role in prevention of product inhibition because they convert oligosaccharides to glucose. A beta-glucosidase gene (cel1A) was isolated from a cDNA library of the anaerobic fungus Piromyces sp. E2. Sequence analysis revealed that the gene encodes a modular protein with a calculated mass of 75800 Da and a pI of 5.05. A secretion signal was followed by a negatively charged domain with unknown function. This domain was coupled with a short linker to a catalytic domain that showed high homology with glycosyl hydrolases belonging to family 1. Southern blot analysis revealed the multiplicity of the gene in the genome. Northern analysis showed that growth on fructose resulted in a high expression of cel1A. The cel1A gene was successfully expressed in Pichia pastoris. The purified heterologously expressed protein was shown to be encoded by the cel1A gene by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of a tryptic digest. Purified heterologous Cel1A was active towards several artificial and natural substrates with beta-1-4 linked glucose molecules with a remarkably high activity on cellodextrins. The enzyme was strongly inhibited by D-glucono-1,5-delta-lactone (K(i)=22 microM), but inhibition by glucose was much less (K(i)=9.5 mM). pH and temperature optimum were 6 and 39 degrees C, respectively. The enzyme was fairly stable, retaining more than 75% of its activity when incubated at 37 degrees C for 5 weeks. Transglycosylation activity could be demonstrated by MALDI-TOF MS analysis of products formed during degradation of cellopentaose.
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http://dx.doi.org/10.1016/s0167-4781(01)00380-3DOI Listing
April 2002