Formate dehydrogenases and hydrogenases in syntrophic propionate-oxidizing communities : gene analysis and transcritional profiling

Many places on earth are without oxygen (anaerobic) such as rice paddy fields, swamps and sediments of freshwater lakes and oceans. When oxygen, nitrate or other electron acceptors are not present, organic material is degraded to carbon dioxide and methane by mixed microbial species that each have their own specific function in degradation. Anaerobic microbial communities are used in anaerobic digesters all over the world to treat organic waste and wastewater. Propionate is one of the most important intermediates in anaerobic digestion. It can only be degraded by propionate oxidizing bacteria when methanogenic archaea keep the concentration of the interspecies electron carriers, hydrogen and formate, low. However, little is known about the molecular mechanism of hydrogen and formate transfer. Hydrogenases are involved in hydrogen transfer and require Fe, Ni and/or Se for catalysis. Formate dehydrogenases that are involved in formate transfer require the trace metals W or Mo and in some cases Se for catalysis. However, the effect of W, Mo and Se limitation on the propionate degrading community of a UASB reactor and the transcription of formate dehydrogenase and hydrogenase encoding genes in this community was never examined. This would give more insight in formate transfer in the propionate degrading community of the UASB reactor and provide a method to study depletion of these metals in the reactor sludge. We used the genome sequences of the propionate degrading Syntrophobacter fumaroxidans and its syntrophic methanogenic partner, Methanospirillum hungatei to study molecular mechanisms of hydrogen and formate transfer in syntrophic cocultures and UASB reactor sludge, by gene analysis and molecular techniques. Gene analysis and microarray data determined formate dehydrogenase and hydrogenase encoding gene clusters in S. fumaroxidans and M. hungatei (Chapter 4). When S. fumaroxidans oxidizes propionate, reducing equivalents are generated by three intermediate reactions in the form of FADH2, NADH and reduced ferredoxin. We found by gene analysis (Chapter 2) and RT qPCR (Chapter 3) that the genes coding for four formate dehydrogenases, six hydrogenases and one formate hydrogen lyase of S. fumaroxidans and five formate dehydrogenases and three hydrogenases of M. hungatei were all transcribed during syntrophic and axenic growth. However, the transcription levels were dependent on the growth condition. Comparison of transcription levels also revealed that electrons from ferredoxin and NADH are simultaneously confurcated for hydrogen production by a cytoplasmic [FeFe]-hydrogenase. Moreover, results indicated that during syntrophic growth electrons from ferredoxin and NADH are confurcated to formate via a cytoplasmic formate dehydrogenase (FDH1). During syntrophic growth, the electrons generated at the level of FADH2, travel via a cytoplasmic oriented succinate dehydrogenase, menaquinones, cytochrome b and c to the periplasmic formate dehydrogenase (FDH2) (Chapter 5). When S. fumaroxidans is grown in pure culture with alternative electron acceptors such as sulfate and fumarate, electrons flow partly to FDH2, and partly to the periplasmic hydrogenase (Hyn). The energy gained from propionate conversion to methane, acetate, and carbon dioxide has to be shared by S. fumaroxidans and M. hungatei. When M. hungatei takes more energy, less energy remains for S. fumaroxidans. In this situation S. fumaroxidans up-regulates transcription of genes coding for an additional cytoplasmic confurcating hydrogenase (Hox) and the periplasmic hydrogenase (Hyn) that is coupled to succinate oxidation. In addition, S. fumaroxidans induces transcription of genes coding for the Rnf-complex and ferredoxin dependent hydrogenases and formate dehydrogenases. This provides the possibility to use the membrane potential for the energy dependent coupling of ferredoxin reduction to NADH oxidation. The designed RT qPCR primers were used in UASB reactor sludge from the alcohol distillery NEDALCO in Bergen op Zoom (Netherlands) to investigate the effect of trace elements depletion. A lab-scale UASB reactor was fed with propionate and synthetic medium without added W, Mo and Se. During the reactor run, Syntrophobacter spp. were the dominant propionate-oxidizers and M. hungatei the dominant hydrogen and formate using methanogen. However, when propionate degradation decreased, two other propionate-oxidizers; Pelotomaculum propionicicum and Smithella propionica became abundant (Chapter 6). RT qPCR showed that in this reactor run the transcription of genes coding for formate dehydrogenases and hydrogenases in S. fumaroxidans decreased while transcription of genes coding for formate dehydrogenases and hydrogenases in M. hungatei were more stable (Chapter 7). This research shows that RT qPCR is a fast technique that can give information on the active processes in a UASB reactor, and that trace element limitation and possible malfunctioning of UASB reactors can be predicted. With this PhD research we gained insight in the molecular mechanisms of hydrogen and formate transfer between S. fumaroxidans an M. hungatei in defined cocultures and in a propionate-fed UASB reactor. This contributes to the understanding of similar molecular mechanisms in other syntrophic microorganisms and may improve the performance of anaerobic digesters in the future. <br/

Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei

Transcription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO2 to formylmethanofuran. The best candidates for F420-dependent N5,N10-methyl-H4 MPT and N5,N10,-methylene-H4MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenase

Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOB(T)).

Syntrophobacter fumaroxidans strain MPOB(T) is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project

Role of lipoxygenase products in the effects of angiotensin II in the isolated aorta and perfused heart of the rat

The objective of this study was to determine whether arachidonate metabolites are involved in the vasoconstrictive effects of angiotensin II in rats. In the isolated perfused heart, dexamethasone (4 mg/kg) significantly suppressed the maximal decreases in coronary flow induced by angiotensin II and vasopressin (reference drug). In the heart, the nonselective lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 1 μM) markedly suppressed the angiotensin II-induced decreases in coronary flow. NDGA (10 μM) inhibited both angiotensin II- and methoxamine- (reference drug) induced contractions in aortic rings with (in the presence of L-NAME) and without endothelium. In the heart, the leukotriene synthesis inhibitor MK-886 (0.3 μM) significantly reduced the maximal effects to angiotensin II, but the leukotriene antagonist FPL 55712 (0.1 and 0.3 μM) had no effect. We conclude that in the isolated perfused rat heart angiotensin II-induced decreases in coronary flow are in part mediated by Hpoxygenase products, which might be derived from the 5-Hpoxygenase pathway, but are probably not leukotrienes. Furthermore, endothelium independent Hpoxygenase products mediate part of the contractile responses to angiotensin II in the isolated rat aorta

The Influence of Radiation Damage on the Deflection of High-Energy Beams in Bent Silicon Crystals

Experimental results obtained for deflection of 450 GeV/c protons channeling along the {111} planes in a bent, strongly irradiated silicon crystal are presented. A comparison between the deflection efficiencies in irradiated areas and non-irradiated areas in the crystal shows that irradiation by 2.4 · 1020 protons/cm2 leads to a reduction of around 30 % in deflection efficiency. As a consequence, beam-splitting and extraction from an accelerator by means of a bent crystal are feasible solutions at high energies even for intense beams and during long periods

Food-induced fatal anaphylaxis: from epidemiological data to general prevention strategies

BACKGROUND: Anaphylaxis hospitalisations are increasing in many countries, in particular for medication and food triggers in young children. Food-related anaphylaxis remains an uncommon cause of death, but a significant proportion of these are preventable. AIM: To review published epidemiological data relating to food-induced anaphylaxis and potential risk factors of fatal and/or near-fatal anaphylaxis cases, in order to provide strategies to reduce the risk of severe adverse outcomes in food anaphylaxis. METHODS: We identified 32 published studies available in MEDLINE (1966-2017), EMBASE (1980-2017), CINAHL (1982-2017), using known terms and synonyms suggested by librarians and allergy specialists. RESULTS: Young adults with a history of asthma, previously known food allergy particularly to peanut/tree nuts are at higher risk of fatal anaphylaxis reactions. In some countries, cow's milk and seafood/fish are also becoming common triggers of fatal reactions. Delayed adrenaline injection is associated with fatal outcomes, but timely adrenaline alone may be insufficient. There is still a lack of evidence regarding the real impact of these risk factors and co-factors (medications and/or alcohol consumption, physical activities, and mast cell disorders). CONCLUSIONS: General strategies should include optimization of the classification and coding for anaphylaxis (new ICD 11 anaphylaxis codes), dissemination of international recommendations on the treatment of anaphylaxis, improvement of the prevention in food and catering areas and, dissemination of specific policies for allergic children in schools. Implementation of these strategies will involve national and international support for ongoing local efforts in relationship with networks of centres of excellence to provide personalized management (which might include immunotherapy) for the most at-risk patients. This article is protected by copyright. All rights reserved

Record deflection efficiencies measured for high energy protons in a bent germanium crystal

New experimental results on the deflection of high energy protons in a bent germanium crystal are presented. At 450 GeV/c, the 50 mm long crystal gave record deflection efficiencies up to 60% for small angles (1 mrad), while at angles as large as 12 mrad, the efficiency is about 25 times larger than for a silicon crystal of the same size. The experimental results are in good agreement with a model for channeling and deflection developed by Ellison and give - together with a similar comparison for a 200 GeV/c beam - confidence in extrapolations to higher energies (e.g. to LHC), other crystal materials or different deflection angles