Schink, Bernhard

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Schink
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Bernhard
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Anaerobic dissimilatory phosphite oxidation, an extremely efficient concept of microbial electron economy

2023-08, Mao, Zhuqing, Müller, Nicolai, Borusak, Sabrina, Schleheck, David, Schink, Bernhard

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (−690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD+-dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO2 fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.

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Isophthalate:coenzyme A ligase initiates anaerobic degradation of xenobiotic isophthalate

2022-09-28, Junghare, Madan, Frey, Jasmin, Naji, Khalid M., Spiteller, Dieter, Vaaje-Kolstad, Gustav, Schink, Bernhard

Background: Environmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA.

Results: Isophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activates isophthalate to isophthalyl-CoA was heterologously expressed in E. coli (49.6 kDa) for biochemical characterization. IPCL is homologous to phenylacetate- CoA ligase that belongs to the family of ligases that form carbon-sulfur bonds. In the presence of coenzyme A, Mg2+ and ATP, IPCL converts isophthalate to isophthalyl-CoA, AMP and pyrophosphate (PPi). The enzyme was specifically induced after anaerobic growth of S. aromaticivorans in a medium containing isophthalate as the sole carbon source. Therefore, IPCL exhibited high substrate specificity and affinity towards isophthalate. Only substrates that are structurally related to isophthalate, such as glutarate and 3-hydroxybenzoate, could be partially converted to the respective coenzyme A esters. Notably, no activity could be measured with substrates such as phthalate, terephthalate and benzoate. Acetyl-CoA or succinyl-CoA did not serve as CoA donors. The enzyme has a theoretical pI of 6.8 and exhibited optimal activity between pH 7.0 to 7.5. The optimal temperature was between 25 °C and 37 °C. Denaturation temperature (Tm) of IPCL was found to be at about 63 °C. The apparent KM values for isophthalate, CoA, and ATP were 409 μM, 642 μM, and 3580 μM, respectively. Although S. aromaticivorans is a strictly anaerobic bacterium, the enzyme was found to be oxygen-insensitive and catalysed isophthalyl-CoA formation under both anoxic and oxic conditions.

Conclusion: We have successfully cloned the ipcl gene, expressed and characterized the corresponding IPCL enzyme, which plays a key role in isophthalate activation that initiates its activation and further degradation by S. aromaticivorans. Its biochemical characterization represents an important step in the elucidation of the complete degradation pathway of isophthalate.

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Phosphitispora fastidiosa gen. nov. sp. nov., a new dissimilatory phosphite-oxidizing anaerobic bacterium isolated from anaerobic sewage sludge

2021-12, Mao, Zhuqing, Gräßle, Fabian, Frey, Jasmin, Franchini, Paolo, Schleheck, David, Müller, Nicolai, Schink, Bernhard

A new strictly anaerobic bacterium, strain DYL19T, was enriched and isolated with phosphite as the sole electron donor and CO2 as a single carbon source and electron acceptor from anaerobic sewage sludge sampled at a sewage treatment plant in Constance, Germany. It is a Gram-positive, spore-forming, slightly curved, rod-shaped bacterium which oxidizes phosphite to phosphate while reducing CO2 to biomass and small amounts of acetate. Optimal growth is observed at 30 °C, pH 7.2, with a doubling time of 3 days. Beyond phosphite, no further inorganic or organic electron donor can be used, and no other electron acceptor than CO2 is reduced. Sulphate inhibits growth with phosphite and CO2. The G+C content is 45.95 mol%, and dimethylmenaquinone-7 is the only quinone detectable in the cells. On the basis of 16S rRNA gene sequence analysis and other chemotaxonomic properties, strain DYL19T is described as the type strain of a new genus and species, Phosphitispora fastidiosa gen. nov., sp. nov.

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Activation of short-chain ketones and isopropanol in sulfate-reducing bacteria

2021-02-16, Frey, Jasmin, Kaßner, Sophie, Spiteller, Dieter, Mergelsberg, Mario, Boll, Matthias, Schleheck, David, Schink, Bernhard

Background:
Degradation of acetone by aerobic and nitrate-reducing bacteria can proceed via carboxylation to acetoacetate and subsequent thiolytic cleavage to two acetyl residues. A different strategy was identified in the sulfate-reducing bacterium Desulfococcus biacutus that involves formylation of acetone to 2-hydroxyisobutyryl-CoA.

Results:
Utilization of short-chain ketones (acetone, butanone, 2-pentanone and 3-pentanone) and isopropanol by the sulfate reducer Desulfosarcina cetonica was investigated by differential proteome analyses and enzyme assays. Two-dimensional protein gel electrophoresis indicated that D. cetonica during growth with acetone expresses enzymes homologous to those described for Desulfococcus biacutus: a thiamine diphosphate (TDP)-requiring enzyme, two subunits of a B12-dependent mutase, and a NAD+-dependent dehydrogenase. Total proteomics of cell-free extracts confirmed these results and identified several additional ketone-inducible proteins. Acetone is activated, most likely mediated by the TDP-dependent enzyme, to a branched-chain CoA-ester, 2-hydroxyisobutyryl-CoA. This compound is linearized to 3-hydroxybutyryl-CoA by a coenzyme B12-dependent mutase followed by oxidation to acetoacetyl-CoA by a dehydrogenase. Proteomic analysis of isopropanol- and butanone-grown cells revealed the expression of a set of enzymes identical to that expressed during growth with acetone. Enzyme assays with cell-free extract of isopropanol- and butanone-grown cells support a B12-dependent isomerization. After growth with 2-pentanone or 3-pentanone, similar protein patterns were observed in cell-free extracts as those found after growth with acetone.

Conclusions:
According to these results, butanone and isopropanol, as well as the two pentanone isomers, are degraded by the same enzymes that are used also in acetone degradation. Our results indicate that the degradation of several short-chain ketones appears to be initiated by TDP-dependent formylation in sulfate-reducing bacteria.

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AMP-dependent phosphite dehydrogenase, a phosphorylating enzyme in dissimilatory phosphite oxidation

2023, Mao, Zhuqing, Fleming, Jennifer R., Mayans, Olga, Frey, Jasmin, Schleheck, David, Schink, Bernhard, Müller, Nicolai

Oxidation of phosphite (HPO32−) to phosphate (HPO42−) releases electrons at a very low redox potential (E0′= −690 mV) which renders phosphite an excellent electron donor for microbial energy metabolism. To date, two pure cultures of strictly anaerobic bacteria have been isolated that run their energy metabolism on the basis of phosphite oxidation, the Gram-negative Desulfotignum phosphitoxidans (DSM 13687) and the Gram-positive Phosphitispora fastidiosa (DSM 112739). Here, we describe the key enzyme for dissimilatory phosphite oxidation in these bacteria. The enzyme catalyzed phosphite oxidation in the presence of adenosine monophosphate (AMP) to form adenosine diphosphate (ADP), with concomitant reduction of oxidized nicotinamide adenine dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). The enzyme of P. fastidiosa was heterologously expressed in Escherichia coli. It has a molecular mass of 35.2 kDa and a high affinity for phosphite and NAD+. Its activity was enhanced more than 100-fold by addition of ADP-consuming adenylate kinase (myokinase) to a maximal activity between 30 and 80 mU x mg protein−1. A similar NAD-dependent enzyme oxidizing phosphite to phosphate with concomitant phosphorylation of AMP to ADP is found in D. phosphitoxidans, but this enzyme could not be heterologously expressed. Based on sequence analysis, these phosphite-oxidizing enzymes are related to nucleotide-diphosphate-sugar epimerases and indeed represent AMP-dependent phosphite dehydrogenases (ApdA). A reaction mechanism is proposed for this unusual type of substrate-level phosphorylation reaction.

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Mikroorganismen und Klimawandel : gibt’s was Neues?

2022-09, Schink, Bernhard

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Pelorhabdus rhamnosifermentans gen. nov., sp. nov.,a strictly anaerobic rhamnose degrader from freshwater lake sediment

2021-07, Gräßle, Fabian, Plugge, Caroline, Franchini, Paolo, Schink, Bernhard, Schleheck, David, Müller, Nicolai

A rhamnose-degrading bacterium, strain BoRhaAT, was isolated from profundal sediment of Lake Constance in agar dilution series with L-rhamnose as substrate and with a background lawn of Methanospirillum hungatei. The isolated strain was a motile rod that stained Gram positive. Growth was observed within a pH range of 4.0 - 7.5 and a temperature range of 15 to 30 °C. Fermentation products of rhamnose or glucose were acetate, propionate, ethanol, butyrate, and 1-propanol. The G+C content was 40.6% G+C. The dominant fatty acids are C16:1ω9c, i-C13:03OH, C16:0 and C17:1ω8c with 8-21% relative abundance. Polar lipids were glycolipids, phosphatidylethanolamine, phosphoaminolipid and other lipids, of which phosphatidylethanolamine was most abundant. The sequence of the 16S rRNA gene of the new isolate matches the sequence of its closest relative Anaerosporomusa subterranea to 92.4%. A comparison of the genome with this strain showed 60.2% genome-wide average amino acid identity (AAI), comparisons with other type strains showed a maximum of 62.7% AAI. Thus, the definition of a new genus is justified for which we propose the name Pelorhabdus. For strain BoRhaAT, we propose the name Pelorhabdus rhamnosifermentans gen. nov., sp. nov., with strain BoRhaAT (DSM 111565T = JCM 39158T) as the type strain.

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Methyloglobulus

2022-11, Schink, Bernhard, Deutzmann, Jörg S.

Me.thy.lo.glo'bu.lus. N.L. neut. n. methylum, the methyl group; L. masc. dim. n. globulus little ball, globule; N.L. masc. n. Methyloglobulus little round methyl-using bacterium. Proteobacteria / Gammaproteobacteria / Methylococcales / Methylococcaceae The genus Methyloglobulus belongs to the Gammaproteobacteria and consists so far of only one species, M. morosus. Cells are microaerobic and use only methane or methanol as substrate. Only a particulate methane monooxygenase was found. Closest phylogenetic relatives are the genera Methylosoma and Methylovulum. DNA G + C content (mol%) : 47.7 (HPLC determination).

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Quantification of archaea-driven freshwater nitrification from single cell to ecosystem levels

2022-06, Klotz, Franziska, Kitzinger, Katharina, Ngugi, David Kamanda, Büsing, Petra, Littmann, Sten, Kuypers, Marcel M. M., Schink, Bernhard, Pester, Michael

Deep oligotrophic lakes sustain large populations of the class Nitrososphaeria (Thaumarchaeota) in their hypolimnion. They are thought to be the key ammonia oxidizers in this habitat, but their impact on N-cycling in lakes has rarely been quantified. We followed this archaeal population in one of Europe’s largest lakes, Lake Constance, for two consecutive years using metagenomics and metatranscriptomics combined with stable isotope-based activity measurements. An abundant (8–39% of picoplankton) and transcriptionally active archaeal ecotype dominated the nitrifying community. It represented a freshwater-specific species present in major inland water bodies, for which we propose the name “Candidatus Nitrosopumilus limneticus”. Its biomass corresponded to 12% of carbon stored in phytoplankton over the year´s cycle. Ca. N. limneticus populations incorporated significantly more ammonium than most other microorganisms in the hypolimnion and were driving potential ammonia oxidation rates of 6.0 ± 0.9 nmol l‒1 d‒1, corresponding to potential cell-specific rates of 0.21 ± 0.11 fmol cell–1 d–1. At the ecosystem level, this translates to a maximum capacity of archaea-driven nitrification of 1.76 × 109 g N-ammonia per year or 11% of N-biomass produced annually by phytoplankton. We show that ammonia-oxidizing archaea play an equally important role in the nitrogen cycle of deep oligotrophic lakes as their counterparts in marine ecosystems.

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Two Marine Desulfotomaculum spp. of Different Origin are Capable of Utilizing Acetone and Higher Ketones

2021-05, Frey, Jasmin, Kaßner, Sophie, Schink, Bernhard

Degradation of acetone and higher ketones has been described in detail for aerobic and nitrate-reducing bacteria. Among sulfate-reducing bacteria, degradation of acetone and other ketones is still an uncommon ability and has not been understood completely yet. In the present work, we show that Desulfotomaculum arcticum and Desulfotomaculum geothermicum are able to degrade acetone and butanone. Total proteomics of cell-free extracts of both organisms indicated an involvement of a thiamine diphosphate-dependent enzyme, a B12-dependent mutase, and a specific dehydrogenase during acetone degradation. Similar enzymes were recently described to be involved in acetone degradation by Desulfococcus biacutus. As there are so far only two described sulfate reducers able to degrade acetone, D. arcticum and D. geothermicum represent two further species with this capacity. All these bacteria appear to degrade acetone via the same set of enzymes and therefore via the same pathway.