34 results on '"Türkowsky, Dominique"'
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2. Thermal proteome profiling allows quantitative assessment of interactions between tetrachloroethene reductive dehalogenase and trichloroethene
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Türkowsky, Dominique, Lohmann, Patrick, Mühlenbrink, Marie, Schubert, Torsten, Adrian, Lorenz, Goris, Tobias, Jehmlich, Nico, and von Bergen, Martin
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- 2019
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3. Risk mitigation measures for pesticide runoff: How effective are they?
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Klein, Michael, primary, Klein, Judith, additional, Flade, Jens, additional, Großmann, Dietlinde, additional, Türkowsky, Dominique, additional, O'Connor, Isabel, additional, Spycher, Simon, additional, Reichenberger, Stefan, additional, Sittig, Stephan, additional, Multsch, Sebastian, additional, and Thomas, Kai, additional
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- 2023
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4. A Retentive Memory of Tetrachloroethene Respiration in Sulfurospirillum halorespirans - involved Proteins and a possible link to Acetylation of a Two-Component Regulatory System
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Türkowsky, Dominique, Esken, Jens, Goris, Tobias, Schubert, Torsten, Diekert, Gabriele, Jehmlich, Nico, and von Bergen, Martin
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- 2018
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5. A H2‐oxidizing, 1,2,3‐trichlorobenzene‐reducing multienzyme complex isolated from the obligately organohalide‐respiring bacterium Dehalococcoides mccartyi strain CBDB1
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Hartwig, Stefanie, Dragomirova, Nadya, Kublik, Anja, Türkowsky, Dominique, von Bergen, Martin, Lechner, Ute, Adrian, Lorenz, and Sawers, R. Gary
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- 2017
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6. Akkurate Bestimmung der Plasmidkopienzahl pro Zelle
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Jahn, Michael, Vorpahl, Carsten, Türkowsky, Dominique, and Müller, Susann
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- 2016
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7. Crop monoculture rather than agriculture reduces the spatial turnover of soil bacterial communities at a regional scale
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Figuerola, Eva L. M., Guerrero, Leandro D., Türkowsky, Dominique, Wall, Luis G., and Erijman, Leonardo
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- 2015
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8. Interspecies metabolite transfer and aggregate formation in a co-culture of Dehalococcoides and Sulfurospirillum dehalogenating tetrachloroethene to ethene
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Kruse, S., Türkowsky, Dominique, Birkigt, Jan, Matturro, B., Franke, Steffi, Jehmlich, Nico, von Bergen, Martin, Westermann, M., Rossetti, S., Nijenhuis, Ivonne, Adrian, Lorenz, Diekert, G., Goris, T., Kruse, S., Türkowsky, Dominique, Birkigt, Jan, Matturro, B., Franke, Steffi, Jehmlich, Nico, von Bergen, Martin, Westermann, M., Rossetti, S., Nijenhuis, Ivonne, Adrian, Lorenz, Diekert, G., and Goris, T.
- Abstract
Microbial communities involving dehalogenating bacteria assist in bioremediation of areas contaminated with halocarbons. To understand molecular interactions between dehalogenating bacteria, we co-cultured Sulfurospirillum multivorans, dechlorinating tetrachloroethene (PCE) to cis−1,2-dichloroethene (cDCE), and Dehalococcoides mccartyi strains BTF08 or 195, dehalogenating PCE to ethene. The co-cultures were cultivated with lactate as electron donor. In co-cultures, the bacterial cells formed aggregates and D. mccartyi established an unusual, barrel-like morphology. An extracellular matrix surrounding bacterial cells in the aggregates enhanced cell-to-cell contact. PCE was dehalogenated to ethene at least three times faster in the co-culture. The dehalogenation was carried out via PceA of S. multivorans, and PteA (a recently described PCE dehalogenase) and VcrA of D. mccartyi BTF08, as supported by protein abundance. The co-culture was not dependent on exogenous hydrogen and acetate, suggesting a syntrophic relationship in which the obligate hydrogen consumer D. mccartyi consumes hydrogen and acetate produced by S. multivorans. The cobamide cofactor of the reductive dehalogenase—mandatory for D. mccartyi—was also produced by S. multivorans. D. mccartyi strain 195 dechlorinated cDCE in the presence of norpseudo-B12 produced by S. multivorans, but D. mccartyi strain BTF08 depended on an exogenous lower cobamide ligand. This observation is important for bioremediation, since cofactor supply in the environment might be a limiting factor for PCE dehalogenation to ethene, described for D. mccartyi exclusively. The findings from this co-culture give new insights into aggregate formation and the physiology of D. mccartyi within a bacterial community.
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- 2021
9. Changes of the Proteome and Acetylome during Transition into the Stationary Phase in the Organohalide-Respiring Dehalococcoides mccartyi Strain CBDB1
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Greiner-Haas, Franziska, primary, Bergen, Martin von, additional, Sawers, Gary, additional, Lechner, Ute, additional, and Türkowsky, Dominique, additional
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- 2021
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10. The glyphosate formulation Roundup® LB plus influences the global metabolome of pig gut microbiota in vitro
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Krause, Jannike Lea, Haange, Sven Bastiaan, Schäpe, Stephanie, Engelmann, Beatrice, Rolle-Kampczyk, Ulrike, Fritz-Wallace, Katarina, Wang, Zhipeng, Jehmlich, Nico, Türkowsky, Dominique, Schubert, Kristin, Pöppe, J., Bote, K., Rösler, U., Herberth, Gunda, von Bergen, Martin, Krause, Jannike Lea, Haange, Sven Bastiaan, Schäpe, Stephanie, Engelmann, Beatrice, Rolle-Kampczyk, Ulrike, Fritz-Wallace, Katarina, Wang, Zhipeng, Jehmlich, Nico, Türkowsky, Dominique, Schubert, Kristin, Pöppe, J., Bote, K., Rösler, U., Herberth, Gunda, and von Bergen, Martin
- Abstract
Glyphosate is the world's most widely used herbicide, and its potential side effects on the intestinal microbiota of various animals, from honeybees to livestock and humans, are currently under discussion. Pigs are among the most abundant livestock animals worldwide and an impact of glyphosate on their intestinal microbiota function can have serious consequences on their health, not to mention the economic effects. Recent studies that addressed microbiota-disrupting effects focused on microbial taxonomy but lacked functional information.Therefore, we chose an experimental design with a short incubation time in which effects on the community structure are not expected, but functional effects can be detected. We cultivated intestinal microbiota derived from pig colon in chemostats and investigated the acute effect of 228 mg/d glyphosate acid equivalents from Roundup® LB plus, a frequently applied glyphosate formulation. The applied glyphosate concentration resembles a worst-case scenario for an 8–9 week-old pig and relates to the maximum residue levels of glyphosate on animal fodder. The effects were determined on the functional level by metaproteomics, targeted and untargeted meta-metabolomics, while variations in community structure were analyzed by 16S rRNA gene profiling and on the single cell level by microbiota flow cytometry.Roundup® LB plus did not affect the community taxonomy or the enzymatic repertoire of the cultivated microbiota in general or on the expression of the glyphosate target enzyme 5-enolpyruvylshikimate-3-phosphate synthase in detail. On the functional level, targeted metabolite analysis of short chain fatty acids (SCFAs), free amino acids and bile acids did not reveal significant changes, whereas untargeted meta-metabolomics did identify some effects on the functional level.This multi-omics approach provides evidence for subtle metabolic effects of Roundup® LB plus under the conditions applied.
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- 2020
11. The glyphosate formulation Roundup® LB plus influences the global metabolome of pig gut microbiota in vitro
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Krause, Jannike L., primary, Haange, Sven-Bastiaan, additional, Schäpe, Stephanie S., additional, Engelmann, Beatrice, additional, Rolle-Kampczyk, Ulrike, additional, Fritz-Wallace, Katarina, additional, Wang, Zhipeng, additional, Jehmlich, Nico, additional, Türkowsky, Dominique, additional, Schubert, Kristin, additional, Pöppe, Judith, additional, Bote, Katrin, additional, Rösler, Uwe, additional, Herberth, Gunda, additional, and von Bergen, Martin, additional
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- 2020
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12. Interspecies metabolite transfer in a co-culture of Dehalococcoides and Sulfurospirillum leads to rapid and complete tetrachloroethene dechlorination
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Kruse, Stefan, Türkowsky, Dominique, Birkigt, Jan, Matturro, Bruna, Franke, Steffi, Jehmlich, Nico, Bergen, Martin von, Westermann, Martin, Rossetti, Simona, Nijenhuis, Ivonne, Adrian, Lorenz, Diekert, Gabriele, and Goris, Tobias
- Abstract
Microbial communities involving dehalogenating bacteria assist in bioremediation of areas contaminated with chlorinated hydrocarbons. To understand molecular interactions between dehalogenating bacteria, we co-cultured two bacterial species dechlorinating chloroethenes: Sulfurospirillum multivorans , dechlorinating tetrachloroethene (PCE) to cis -1,2-dichloroethene ( c DCE) and Dehaloccoides mccartyi strains BTF08 or 195, transforming PCE via c DCE to ethene. The interaction of these bacteria cultivated with lactate as electron donor and PCE as electron acceptor was investigated using growth studies, metabolite analysis, microscopy, isotope fractionation and proteomics. Co-cultures exhibited more than 3-fold higher PCE to ethene dechlorination rates than D. mccartyi pure cultures. S. multivorans provided hydrogen, acetate and the reductive dehalogenase cobamide cofactor to D. mccartyi . While D. mccartyi 195 dechlorinated c DCE in the presence of norpseudo-B 12 produced by S. multivorans , cDCE dechlorination by D. mccartyi BTF08 depended on the supply of 5,6-dimethylbenzimidazole for producing functional cobamides. Co-cultures were characterized by the formation of aggregates and electron microscopy revealed an extracellular matrix enabling cell-to-cell contact. D. mccartyi showed an unusual barrel-like morphology, probably dependent on down-regulation of cell division gene expression, as observed in the co-culture proteome. Only the reductive dehalogenases PteA and VcrA were found in the proteomes of D. mccartyi BTF08 during dehalogenation of PCE to ethene.
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- 2019
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13. Regulation of expression and activity of reductive dehalogenases in organohalide-respiring bacteria
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Türkowsky, Dominique and Universität Leipzig
- Subjects
ddc:570 ,Proteomics, Reductive Dehalogenation, Thermal Proteome Profiling, Acetylome, Sulfurospirillum - Abstract
Organohalides have been abundantly utilized as pesticides and in industrial processes for the past 100 years, with over 30 000 sites in Europe still being contaminated today. Because of their recalcitrance, large quantities have accumulated in soils, sediments, and groundwater. Many organohalides can cause multiple adverse health effects, including neurological damage, congenital malformations, and a variety of human cancers. Fortunately, bacterial genera from a diverse range of phyla are capable of detoxifying these organohalides via anaerobic respiration, i.e., by using them as their terminal electron acceptor. These metabolic pathways involve a reductive dehalogenation reaction, during which a chlorine atom dissociates and thereby either immediately reduces the toxicity of the organohalide, or enables it to be further degraded by a broader range of organisms. Thus, organohalide-respiring bacteria can be used for the bioremediation of contaminated environments. To be able to support this application, fundamental research on these reactions and the metabolism of organohalide-respiring bacteria is a prerequisite. Many aspects of the physiology of organohalide-respiring bacteria are unresolved. Organohalide-respiring bacteria harbor up to 38 reductive dehalogenase homologous genes, which putatively encode the key enzymes of reductive dehalogenation. However, the regulation, protein-coding ability, the function of these enzymes as well as their interactions with other proteins has yet to be elucidated. Organohalide-respiring bacteria are difficult to study due to their slow growth, low biomass yields, oxygen sensitivity and genetic inaccessibility. The aim of this thesis was to circumvent these obstacles by introducing new methods for studying organohalide respiration and thereby enabling the formulation of informed predictions about the functions of reductive dehalogenases and the identity of their regulators. For this, obligate and facultative organohalide-respiring bacteria were assessed. To form a basis of the current research in the field, all available genomic, transcriptomic and proteomic literature on organohalide-respiring bacteria were reviewed and compared. Through combining quantitative expression data of hundreds of orthologs and subjecting them to statistical analyses, many new aspects of the metabolism of organohalide-respiring bacteria were uncovered. Especially notable were the unclear expression patterns of reductive dehalogenases and their accessory proteins. An important conclusion from this review was that shotgun proteomics is essential to reveal how many reductive dehalogenase proteins are produced in parallel, but this approach alone cannot clarify the function of these enzymes nor their underlying regulation processes. Therefore, the next chapter of this thesis aimed to extend and refine the standard proteomics approaches. First, proteomics conducted via mass spectrometry requires optimization of sample processing and analysis. Utilizing harsher conditions for protein extraction and digestion substantially improved proteome coverage compared to previous studies, especially of membrane proteins. The combination of this approach with a highly stringent statistical filtering procedure allowed a more detailed, reliable and thus more valid view of the proteome to be obtained from the model organism Sulfurospirillum halorespirans. The quantification of the putative protein histidine kinase provided the first evidence of its involvement in controlling organohalide respiration together with the putative response regulator, forming a complete two-component regulatory system. The quantification of the putative quinol dehydrogenase membrane subunit also supported its involvement in the organohalide respiratory chain of this genus. We observed that S. halorespirans undergoes the same type of peculiar memory-effect as Sulfurospirillum multivorans, that is, continuing to produce its complete dehalogenating machinery even after prolonged cultivation on a non-halogenated electron acceptor. To reveal the underlying mechanism, protein lysine acetylation was additionally measured, which is an important post-translational modification involved in many regulatory processes across all living organisms. Lysine acetylations are, e.g., known to alter the binding properties of DNA-interacting proteins like transcription factors or response regulators but have a range of other regulatory effects. In the first ‘acetylome’ study of an organohalide-respiring bacterium and an Epsilonproteobacterium, one-third of all S. halorespirans proteins were found to be acetylated at one point over the course of a long-term cultivation experiment. Interestingly, the putative response regulator of the two-component regulatory system described earlier was acetylated during the metabolic transition phase, after short-term adaptation to a non-halogenated electron acceptor. Another advancement of shotgun proteomics was its combination with thermal proteome profiling to elucidate substrate specificities of reductive dehalogenases and their regulators. The underlying principle behind thermal proteome profiling is to identify the interaction of a protein with a binding ligand through its impact on the thermal stability of the protein. The thermal stability of hundreds of proteins can be measured in parallel by a proteomics approach. Aliquots of protein extract are first incubated at different temperatures, and the non-denatured fraction of each protein is then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), thus allowing the composition of melting curves of each protein to be determined. With this unbiased approach, unknown protein-ligand interactions can also be identified. In a proof-of-concept study on S. multivorans, we adapted the method to anaerobic conditions and showed that this technique is suitable for the detection of interactions between enzymes and their specific substrates. For example, a melting curve shift was detected when the tetrachloroethene reductive dehalogenase, PceA, bound to its known substrate, trichloroethene. Furthermore, the melting curve shift of the putative response regulator in the two-component regulatory system indicated at least an indirect interaction between it and trichloroethene, providing the first biochemical evidence of its role in organohalide respiration besides mere expression data. In conclusion, this work not only includes the first systematic analysis of all omics-based studies conducted to date but substantially advanced the methods for assessing organohalide-respiring bacteria by providing a more detailed picture of their physiology. Besides methodological advances, it was demonstrated that the two-component regulatory system interacts with halogenated compounds and that its post-translational modification might impact long-term downregulation of the organohalide respiratory apparatus in Sulfurospirillum spp. The insights into the involvement of the two-component regulatory system in the organohalide respiration of Sulfurospirillum spp. would not have been uncovered by using less complex standard shotgun proteomics measurements. In the future, our findings will help to further elucidate regulators and functioning of reductive dehalogenases also in other organohalide-respiring bacteria.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128 Während der letzten einhundert Jahre wurden halogenierte organische Verbindungen großflächig in Industrie und Landwirtschaft eingesetzt, wodurch heute mehr als 30 000 Flächen in Europa kontaminiert sind. Aufgrund ihrer eingeschränkten Abbaubarkeit konnten sich riesige Mengen in Böden, Sedimenten und Grundwasser ausbreiten. Viele halogenierte organische Verbindungen können erhebliche nachteilige Auswirkungen auf die Gesundheit des Menschen haben, u.a. neurologische Schäden, Fehlbildungen und eine Vielzahl von Krebserkrankungen. Glücklicherweise sind bestimmte Bakterientypen unterschiedlicher Phyla in der Lage, diese Stoffe mittels anaerober Atmung, d.h. über deren Nutzung als terminalen Elektronenakzeptor, umzuwandeln. Diese reduktive Dehalogenierung, bei der ein Chlor-Rest abgespalten wird, vermindert die Toxizität der meisten Organohalide bzw. macht sie zugänglich für den Abbau durch ein breiteres Organismenspektrum. Demgemäß können Organohalid-atmende Bakterien für die Bioremediation kontaminierter Flächen genutzt werden. Voraussetzung für deren Einsatz ist jedoch das Verständnis der zugrundeliegenden biochemischen Reaktionen und des Metabolismus der Organohalid-Atmer. Viele Aspekte der Physiologie Organohalid-atmender Bakterien sind noch ungeklärt. Die Organismen besitzen bis zu 38 unterschiedliche Gene, die reduktive Dehalogenasen, die Schlüsselenzyme der Organohalid-Atmung, kodieren. Allerdings sind deren Regulation, Proteinkodierung, die Funktion der einzelnen Enzyme sowie deren Interaktionen mit anderen Proteinen noch unbekannt. Die Forschung an Organohalid-atmenden Bakterien wird durch deren langsames Wachstum, die geringen Zelldichten, die hohe Sensitivität gegenüber Sauerstoff und fehlende gentechnische Methoden erschwert. Ziel dieser Arbeit war es, die genannten Hindernisse mittels neuartiger Methoden an Organohalid-Atmern zu umgehen und damit Regulatoren und Funktionsweise der reduktiven Dehalogenasen zu bestimmen. Hierfür wurden sowohl obligate als auch fakultative Organohalid-atmende Bakterien herangezogen. Als Grundlage führte ich zunächst alle bisher durchgeführten Genomik-, Transkriptomik- und Proteomikstudien zu Organohalid-atmenden Bakterien zusammen. Hunderte zu Orthologen kombinierte und statistisch analysierte quantitative Expressionsdaten lieferten dabei ein umfassendes Bild vom Metabolismus der Organohalid-Atmer. Insbesondere die unklaren Expressionsmuster der reduktiven Dehalogenasen und ihrer akzessorischen Proteine wurden offenbar. Eine wichtige Erkenntnis des Review-Prozesses war, dass Standard-Proteomikansätze zwar unerlässlich sind, um beispielsweise die gleichzeitige Produktion mehrerer reduktiver Dehalogenasen offenzulegen, aber weder deren Funktionen noch Regulation aufklären können. Aus diesem Grund sollten im weiteren Verlauf dieser Arbeit die bisher genutzten Shotgun-Proteomikmethoden weiterentwickelt werden. Für eine umfassende Proteinanalyse mittels Massenspektrometrie müssen zunächst Probenaufarbeitung und Analyse optimiert werden. Durch die Verwendung harscherer Bedingungen bei Proteinextraktion und -verdau konnten wir die Proteomabdeckung, insbesondere unter Membranproteinen, im Vergleich zu früheren Studien erheblich verbessern. In Kombination mit einem sehr stringenten statistischen Filterprozess erlaubte dies einen detaillierten und validen Blick auf das Proteom des Modellorganismus Sulfurospirillum halorespirans. Die Quantifizierung der mutmaßlichen Protein-Histidinkinase ist der erste Beleg dafür, dass diese zusammen mit dem Regulationsprotein im Zweikomponentensystem an der Kontrolle der Organohalid-Atmung in Sulfurospirillum spp. beteiligt ist. Die quantifizierte Membranuntereinheit der Quinoldehydrogenase stützt die Annahme zu deren Beteiligung an der Atmungskette dieses Organismus. Wir konnten weiterhin zeigen, dass in S. halorespirans die gleiche außergewöhnliche Langzeitregulation wie in Sulfurospirillum multivorans wirksam ist, sodass auch nach langanhaltender Kultivierung auf nicht-halogenierten Substraten der komplette Organohalid-Atmungsapparat synthetisiert wird. Zur Aufklärung der zugrundeliegenden Regulation erweiterten wir unsere Analyse um Protein-Lysin-Acetylierungen, wichtige posttranslationale Modifikationen, die an verschiedensten regulatorischen Prozessen in allen Lebewesen beteiligt sind. Protein-Lysin-Acetylierungen beeinflussen z.B. die Wechselwirkungen zwischen Transkriptionsfaktoren oder Regulationsproteinen und der DNA, aber haben noch viele weitere regulatorische Effekte. In dieser ersten „Acetylom“-Studie an einem Organohalid-atmenden Bakterium bzw. einem Epsilonproteobacterium, konnten wir zeigen, dass ein Drittel aller S. halorespirans-Proteine im Verlauf der Langzeitkultivierung mindestens einmal acetyliert wurden. Interessanterweise war auch das mutmaßliche Regulatorprotein des oben erwähnten Zweikomponentensystems während der metabolischen Umstellungsphase, d.h. nach Kurzzeitanpassung an den nicht-halogenierten Elektronenakzeptor, acetyliert. Eine zusätzliche Weiterentwicklung der klassischen proteomischen Messungen war deren Kombination mit Thermal Proteome Profiling, um Substratspezifitäten und Regulatoren von reduktiven Dehalogenasen zu bestimmen. Zugrundeliegendes Prinzip des Thermal Proteome Profiling ist die Identifikation eines Proteinbindungspartners über dessen Einfluss auf die Thermostabilität der Faltung eines Proteins. Die Thermostabilität tausender Proteine kann mit Hilfe eines Proteomikansatzes bestimmt werden. Hierfür werden extrahierte Proteine zunächst aufgeteilt und unterschiedlichen Temperaturen ausgesetzt. Die nicht-denaturierte Fraktion jedes Proteins kann mittels Flüssigchromatographie mit Tandemmassenspektrometrie-Kopplung (LC-MS/MS) quantifiziert und zu Schmelzkurven zusammengesetzt werden. Mit dieser Methode können auch unbekannte Protein-Liganden-Interaktionen identifiziert werden. In unserer Machbarkeitsstudie an S. multivorans konnten wir zeigen, dass die von uns modifizierte Technik auch zur Aufklärung von Enzym-Substrat-Interaktionen und sogar unter anaeroben Bedigungen eingesetzt werden kann. So konnte nachgewiesen werden, dass die Schmelzkurve der reduktiven Tetrachlorethen-Dehalogenase PceA durch Bindung ihres bekannten Substrates Trichlorethen signifikant verschoben wurde. Außerdem deutet die Verschiebung der Schmelzkurve des mutmaßlichen Regulatorproteins des Zweikomponentensystems zumindest auf eine indirekte Interaktion mit Trichlorethen hin und ist damit, abgesehen von bloßen Expressionsdaten, der erste biochemische Beleg für dessen Rolle bei der Organohalid-Atmung. Zusammenfassend beinhaltet diese Arbeit nicht nur die erste systematische Analyse und Kombination aller bisher verfügbaren „Omics“-Studien, sondern auch deren Weiterenwiclung für die Untersuchung organohalid-atmender Bakterien, wodurch ein detailliertes Bild von deren Physiologie geschaffen werden konnte. Neben den technischen Neuerungen konnte gezeigt werden, dass das Zweikomponentensystem von Sulfurospirillum sp. mit halogenierten organischen Verbindungen interagiert und dass dessen posttranslationale Modifikation die Langzeitreulation des Organohalid-Atmungsapparates beeinflussen könnte. Die Einblicke in die Beteiligung des Zweikomponentensystems an der Organohalidatmung in Sulfurospirillum sp. wären durch Nutzung von weniger komplexen Standard-Proteomikmethoden unentdeckt geblieben. In Zukunft können uns diese neu entwickelten Methoden dabei unterstützen, Funktionalität und Regulation von reduktiven Dehalogenasen in anderen Organohalid-Atmern aufzuklären.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128
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- 2018
14. Interspecies metabolite transfer and aggregate formation in a co-culture of Dehalococcoides and Sulfurospirillum dehalogenating tetrachloroethene to ethene
- Author
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Kruse, Stefan, primary, Türkowsky, Dominique, additional, Birkigt, Jan, additional, Matturro, Bruna, additional, Franke, Steffi, additional, Jehmlich, Nico, additional, von Bergen, Martin, additional, Westermann, Martin, additional, Rossetti, Simona, additional, Nijenhuis, Ivonne, additional, Adrian, Lorenz, additional, Diekert, Gabriele, additional, and Goris, Tobias, additional
- Published
- 2019
- Full Text
- View/download PDF
15. Regulation of expression and activity of reductive dehalogenases in organohalide-respiring bacteria
- Author
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Universität Leipzig, Türkowsky, Dominique, Universität Leipzig, and Türkowsky, Dominique
- Abstract
Organohalides have been abundantly utilized as pesticides and in industrial processes for the past 100 years, with over 30 000 sites in Europe still being contaminated today. Because of their recalcitrance, large quantities have accumulated in soils, sediments, and groundwater. Many organohalides can cause multiple adverse health effects, including neurological damage, congenital malformations, and a variety of human cancers. Fortunately, bacterial genera from a diverse range of phyla are capable of detoxifying these organohalides via anaerobic respiration, i.e., by using them as their terminal electron acceptor. These metabolic pathways involve a reductive dehalogenation reaction, during which a chlorine atom dissociates and thereby either immediately reduces the toxicity of the organohalide, or enables it to be further degraded by a broader range of organisms. Thus, organohalide-respiring bacteria can be used for the bioremediation of contaminated environments. To be able to support this application, fundamental research on these reactions and the metabolism of organohalide-respiring bacteria is a prerequisite. Many aspects of the physiology of organohalide-respiring bacteria are unresolved. Organohalide-respiring bacteria harbor up to 38 reductive dehalogenase homologous genes, which putatively encode the key enzymes of reductive dehalogenation. However, the regulation, protein-coding ability, the function of these enzymes as well as their interactions with other proteins has yet to be elucidated. Organohalide-respiring bacteria are difficult to study due to their slow growth, low biomass yields, oxygen sensitivity and genetic inaccessibility. The aim of this thesis was to circumvent these obstacles by introducing new methods for studying organohalide respiration and thereby enabling the formulation of informed predictions about the functions of reductive dehalogenases and the identity of their regulators. For this, obligate and facultative organohalide-respiring, Während der letzten einhundert Jahre wurden halogenierte organische Verbindungen großflächig in Industrie und Landwirtschaft eingesetzt, wodurch heute mehr als 30 000 Flächen in Europa kontaminiert sind. Aufgrund ihrer eingeschränkten Abbaubarkeit konnten sich riesige Mengen in Böden, Sedimenten und Grundwasser ausbreiten. Viele halogenierte organische Verbindungen können erhebliche nachteilige Auswirkungen auf die Gesundheit des Menschen haben, u.a. neurologische Schäden, Fehlbildungen und eine Vielzahl von Krebserkrankungen. Glücklicherweise sind bestimmte Bakterientypen unterschiedlicher Phyla in der Lage, diese Stoffe mittels anaerober Atmung, d.h. über deren Nutzung als terminalen Elektronenakzeptor, umzuwandeln. Diese reduktive Dehalogenierung, bei der ein Chlor-Rest abgespalten wird, vermindert die Toxizität der meisten Organohalide bzw. macht sie zugänglich für den Abbau durch ein breiteres Organismenspektrum. Demgemäß können Organohalid-atmende Bakterien für die Bioremediation kontaminierter Flächen genutzt werden. Voraussetzung für deren Einsatz ist jedoch das Verständnis der zugrundeliegenden biochemischen Reaktionen und des Metabolismus der Organohalid-Atmer. Viele Aspekte der Physiologie Organohalid-atmender Bakterien sind noch ungeklärt. Die Organismen besitzen bis zu 38 unterschiedliche Gene, die reduktive Dehalogenasen, die Schlüsselenzyme der Organohalid-Atmung, kodieren. Allerdings sind deren Regulation, Proteinkodierung, die Funktion der einzelnen Enzyme sowie deren Interaktionen mit anderen Proteinen noch unbekannt. Die Forschung an Organohalid-atmenden Bakterien wird durch deren langsames Wachstum, die geringen Zelldichten, die hohe Sensitivität gegenüber Sauerstoff und fehlende gentechnische Methoden erschwert. Ziel dieser Arbeit war es, die genannten Hindernisse mittels neuartiger Methoden an Organohalid-Atmern zu umgehen und damit Regulatoren und Funktionsweise der reduktiven Dehalogenasen zu bestimmen. Hierfür wurden sowohl obligate als auch
- Published
- 2018
16. Desulfitobacterium contributes to the microbial transformation of 2,4,5‐T by methanogenic enrichment cultures from a Vietnamese active landfill
- Author
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Lechner, U., Türkowsky, Dominique, Dinh, T.T.H., Al-Fathi, Hassan, Schwoch, S., Franke, S., Gerlach, M.-S., Koch, M., von Bergen, Martin, Jehmlich, Nico, Dang, T.C.H., Lechner, U., Türkowsky, Dominique, Dinh, T.T.H., Al-Fathi, Hassan, Schwoch, S., Franke, S., Gerlach, M.-S., Koch, M., von Bergen, Martin, Jehmlich, Nico, and Dang, T.C.H.
- Abstract
The herbicide 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T) was a major component of Agent Orange, which was used as a defoliant in the Vietnam War. Little is known about its degradation under anoxic conditions. Established enrichment cultures using soil from an Agent Orange bioremediation plant in southern Vietnam with pyruvate as potential electron donor and carbon source were shown to degrade 2,4,5‐T via ether cleavage to 2,4,5‐trichlorophenol (2,4,5‐TCP), which was further dechlorinated to 3,4‐dichlorophenol. Pyruvate was initially fermented to hydrogen, acetate and propionate. Hydrogen was then used as the direct electron donor for ether cleavage of 2,4,5‐T and subsequent dechlorination of 2,4,5‐TCP. 16S rRNA gene amplicon sequencing indicated the presence of bacteria and archaea mainly belonging to the Firmicutes, Bacteroidetes, Spirochaetes, Chloroflexi and Euryarchaeota. Desulfitobacterium hafniense was identified as the dechlorinating bacterium. Metaproteomics of the enrichment culture indicated higher protein abundances of 60 protein groups in the presence of 2,4,5‐T. A reductive dehalogenase related to RdhA3 of D. hafniense showed the highest fold change, supporting its function in reductive dehalogenation of 2,4,5‐TCP. Despite an ether‐cleaving enzyme not being detected, the inhibition of ether cleavage but not of dechlorination, by 2‐bromoethane sulphonate, suggested that the two reactions are catalysed by different organisms.
- Published
- 2018
17. An integrative overview of genomic, transcriptomic and proteomic analyses in organohalide respiration research
- Author
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Türkowsky, Dominique, Jehmlich, Nico, Diekert, G., Adrian, Lorenz, von Bergen, Martin, Goris, T., Türkowsky, Dominique, Jehmlich, Nico, Diekert, G., Adrian, Lorenz, von Bergen, Martin, and Goris, T.
- Abstract
Organohalide respiration (OHR) is a crucial process in the global halogen cycle and of interest for bioremediation. However, investigations on OHR are hampered by the restricted genetic accessibility and the poor growth yields of many organohalide-respiring bacteria (OHRB). Therefore, genomics, transcriptomics and proteomics are often used to investigate OHRB. In general, these gene expression studies are more useful when the data of the different ‘omics’ approaches are integrated and compared among a wide range of cultivation conditions and ideally involve several closely related OHRB. Despite the availability of a couple of proteomic and transcriptomic datasets dealing with OHRB, such approaches are currently not covered in reviews. Therefore, we here present an integrative and comparative overview of omics studies performed with the OHRB Sulfurospirillum multivorans, Dehalococcoides mccartyi, Desulfitobacterium spp. and Dehalobacter restrictus. Genes, transcripts, proteins and the regulatory and biochemical processes involved in OHR are discussed, and a comprehensive view on the unusual metabolism of D. mccartyi, which is one of the few bacteria possibly using a quinone-independent respiratory chain, is provided. Several ‘omics’-derived theories on OHRB, e.g. the organohalide-respiratory chain, hydrogen metabolism, corrinoid biosynthesis or one-carbon metabolism are critically discussed on the basis of this integrative approach.
- Published
- 2018
18. Thermal proteome profiling allows quantitative assessment of interactions between tetrachloroethene reductive dehalogenase and trichloroethene
- Author
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Türkowsky, Dominique, Lohmann, Patrick, Mühlenbrink, Marie, Schubert, T., Adrian, Lorenz, Goris, T., Jehmlich, Nico, von Bergen, Martin, Türkowsky, Dominique, Lohmann, Patrick, Mühlenbrink, Marie, Schubert, T., Adrian, Lorenz, Goris, T., Jehmlich, Nico, and von Bergen, Martin
- Abstract
Thermal proteome profiling (TPP) is increasingly applied in eukaryotes to investigate protein-ligand binding through protein melting curve shifts induced by the presence of a ligand. In anaerobic bacteria, identification of protein-substrate interactions is a major challenge. We applied TPP to Sulfurospirillum multivorans, which is able to use trichloroethene as electron acceptor for growth, to investigate the interaction of its tetrachloroethene reductive dehalogenase PceA with trichloroethene. Several modifications in the protocol (e.g., incubation under anaerobic conditions; increasing the temperature range up to 97 °C) extended the protein detection range and allowed the investigation of oxygen-sensitive proteins. Enzymatic reductive dehalogenation was prevented by omitting the electron donor during incubations. This enabled detecting the interaction of PceA with trichloroethene and confirmed that trichloroethene is a substrate of this enzyme. Interestingly, a putative response regulator showed a similar trend, which is the first biochemical hint for its proposed role in trichloroethene respiration. We proved that our TPP approach facilitates the identification of protein-substrate interactions of strictly anaerobic reductive dehalogenases and probably their regulators. This strategy can be used to identify yet unknown substrate specificities and possible signal-sensing proteins, and therefore has the potential to elucidate one of the unresolved fields in research on organohalide-respiring bacteria.SignificanceThe assessment of enzyme-substrate or protein-ligand interactions in organohalide-respiring bacteria is a fundamental challenge. Thermal proteome profiling (TPP) allows elucidating proteome-wide thermal stability changes relying on the sensitivity of modern mass spectrometry. This gives access to the identification of interactions not detectable with other methods. In this TPP study, we demonstrate the interactions of a chlorinated substrate with a reducti
- Published
- 2018
19. Interspecies metabolite transfer and aggregate formation in a co-culture of Dehalococcoidesand Sulfurospirillumdehalogenating tetrachloroethene to ethene
- Author
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Kruse, Stefan, Türkowsky, Dominique, Birkigt, Jan, Matturro, Bruna, Franke, Steffi, Jehmlich, Nico, von Bergen, Martin, Westermann, Martin, Rossetti, Simona, Nijenhuis, Ivonne, Adrian, Lorenz, Diekert, Gabriele, and Goris, Tobias
- Abstract
Microbial communities involving dehalogenating bacteria assist in bioremediation of areas contaminated with halocarbons. To understand molecular interactions between dehalogenating bacteria, we co-cultured Sulfurospirillum multivorans, dechlorinating tetrachloroethene (PCE) to cis−1,2-dichloroethene (cDCE), and Dehalococcoides mccartyistrains BTF08 or 195, dehalogenating PCE to ethene. The co-cultures were cultivated with lactate as electron donor. In co-cultures, the bacterial cells formed aggregates and D. mccartyiestablished an unusual, barrel-like morphology. An extracellular matrix surrounding bacterial cells in the aggregates enhanced cell-to-cell contact. PCE was dehalogenated to ethene at least three times faster in the co-culture. The dehalogenation was carried out via PceA of S. multivorans, and PteA (a recently described PCE dehalogenase) and VcrA of D. mccartyiBTF08, as supported by protein abundance. The co-culture was not dependent on exogenous hydrogen and acetate, suggesting a syntrophic relationship in which the obligate hydrogen consumer D. mccartyiconsumes hydrogen and acetate produced by S. multivorans. The cobamide cofactor of the reductive dehalogenase—mandatory for D. mccartyi—was also produced by S. multivorans. D. mccartyistrain 195 dechlorinated cDCE in the presence of norpseudo-B12produced by S. multivorans, but D. mccartyistrain BTF08 depended on an exogenous lower cobamide ligand. This observation is important for bioremediation, since cofactor supply in the environment might be a limiting factor for PCE dehalogenation to ethene, described for D. mccartyiexclusively. The findings from this co-culture give new insights into aggregate formation and the physiology of D. mccartyiwithin a bacterial community.
- Published
- 2021
- Full Text
- View/download PDF
20. Desulfitobacteriumcontributes to the microbial transformation of 2,4,5-T by methanogenic enrichment cultures from a Vietnamese active landfill
- Author
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Lechner, Ute, primary, Türkowsky, Dominique, additional, Dinh, Thi Thu Hang, additional, Al-Fathi, Hassan, additional, Schwoch, Stefan, additional, Franke, Stefan, additional, Gerlach, Michelle-Sophie, additional, Koch, Mandy, additional, von Bergen, Martin, additional, Jehmlich, Nico, additional, and Dang, Thi Cam Ha, additional
- Published
- 2018
- Full Text
- View/download PDF
21. 0656 - Thermal Proteome Profiling–A High-Throuput Technique for Screening the Proteome for Enzyme-Substrate Interactions
- Author
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Türkowsky, Dominique, primary
- Published
- 2018
- Full Text
- View/download PDF
22. An integrative overview of genomic, transcriptomic and proteomic analyses in organohalide respiration research
- Author
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Türkowsky, Dominique, primary, Jehmlich, Nico, additional, Diekert, Gabriele, additional, Adrian, Lorenz, additional, von Bergen, Martin, additional, and Goris, Tobias, additional
- Published
- 2018
- Full Text
- View/download PDF
23. A H2-oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1
- Author
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Hartwig, S., Dragomirova, N., Kublik, Anja, Türkowsky, Dominique, von Bergen, Martin, Lechner, U., Adrian, Lorenz, Sawers, R.G., Hartwig, S., Dragomirova, N., Kublik, Anja, Türkowsky, Dominique, von Bergen, Martin, Lechner, U., Adrian, Lorenz, and Sawers, R.G.
- Abstract
Dehalococcoides mccartyi is a small, slow-growing bacterium of the phylum Chloroflexi that conserves energy using aliphatic and aromatic organohalides as electron acceptors, and hydrogen as sole electron donor. A recent study identified a protein complex in the membrane of strain CBDB1 comprising a Hup hydrogenase, a complex iron-sulphur molybdoprotein and a reductive dehalogenase (RdhA) that catalyses reduction of 1,2,3,4-tetrachlorobenzene. Using a combination of size-exclusion chromatography, in-gel hydrogenase activity-staining, immunological analysis and mass spectrometry, we identified here a large molecular mass protein complex solubilized from the cytoplasmic membrane of D. mccartyi strain CBDB1 that catalysed H2-dependent reduction of 1,2,3-trichlorobenzene (1,2,3-TCB) to 1,3-DCB. In-gel zymographic staining revealed H2:benzyl viologen oxidoreductase activity associated with the complex and immunological analysis identified co-elution of CdbdA195, the predicted catalytic subunit of the iron-sulphur molybdoenzyme, the chlorobenzene-specific RdhA, CbrA, and traces of HupL, the catalytic subunit of the Hup hydrogenase. Quantitative reverse transcriptase PCR analyses indicated that the expression of the hupL and cbdbA195 genes was induced by 1,2,3-TCB but not by hydrogen. Together, these data identify and describe a protein-based electron-transfer complex catalysing H2 oxidation coupled to chlorobenzene reduction.
- Published
- 2017
24. A H2-oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacteriumDehalococcoides mccartyistrain CBDB1
- Author
-
Hartwig, Stefanie, primary, Dragomirova, Nadya, additional, Kublik, Anja, additional, Türkowsky, Dominique, additional, von Bergen, Martin, additional, Lechner, Ute, additional, Adrian, Lorenz, additional, and Sawers, R. Gary, additional
- Published
- 2017
- Full Text
- View/download PDF
25. Desulfitobacterium contributes to the microbial transformation of 2,4,5‐T by methanogenic enrichment cultures from a Vietnamese active landfill.
- Author
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Lechner, Ute, Al‐Fathi, Hassan, Schwoch, Stefan, Franke, Stefan, Gerlach, Michelle‐Sophie, Türkowsky, Dominique, Bergen, Martin, Jehmlich, Nico, Dinh, Thi Thu Hang, Dang, Thi Cam Ha, and Koch, Mandy
- Subjects
TRICHLOROPHENOXYACETIC acid ,TOXICOLOGY of Agent Orange ,BIOREMEDIATION ,HERBICIDES & the environment ,DEHALOGENATION - Abstract
Summary: The herbicide 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T) was a major component of Agent Orange, which was used as a defoliant in the Vietnam War. Little is known about its degradation under anoxic conditions. Established enrichment cultures using soil from an Agent Orange bioremediation plant in southern Vietnam with pyruvate as potential electron donor and carbon source were shown to degrade 2,4,5‐T via ether cleavage to 2,4,5‐trichlorophenol (2,4,5‐TCP), which was further dechlorinated to 3,4‐dichlorophenol. Pyruvate was initially fermented to hydrogen, acetate and propionate. Hydrogen was then used as the direct electron donor for ether cleavage of 2,4,5‐T and subsequent dechlorination of 2,4,5‐TCP. 16S rRNA gene amplicon sequencing indicated the presence of bacteria and archaea mainly belonging to the Firmicutes, Bacteroidetes, Spirochaetes, Chloroflexi and Euryarchaeota. Desulfitobacterium hafniense was identified as the dechlorinating bacterium. Metaproteomics of the enrichment culture indicated higher protein abundances of 60 protein groups in the presence of 2,4,5‐T. A reductive dehalogenase related to RdhA3 of D. hafniense showed the highest fold change, supporting its function in reductive dehalogenation of 2,4,5‐TCP. Despite an ether‐cleaving enzyme not being detected, the inhibition of ether cleavage but not of dechlorination, by 2‐bromoethane sulphonate, suggested that the two reactions are catalysed by different organisms. Anaerobic enrichment cultures from an Agent Orange‐treating active landfill degraded 2,4,5‐T via cleavage of the ether bond and subsequent reductive dechlorination. 16S rRNA gene amplicon sequencing and metaproteomics revealed the presence of diverse fermenting bacteria, which provided the community with hydrogen.The latter served as electron donor for both 2,4,5‐T degradation steps. Whereas the ether‐cleaving organism could not be identified, Desulfitobacterium hafniense harbouring the RdhA3 reductive dehalogenase was indicated as the reductively dehalogenating bacterium. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
26. Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family
- Author
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Hartwig, S., Thomas, C., Krumova, N., Quitzke, V., Türkowsky, Dominique, Jehmlich, Nico, Adrian, Lorenz, Sawers, R.G., Hartwig, S., Thomas, C., Krumova, N., Quitzke, V., Türkowsky, Dominique, Jehmlich, Nico, Adrian, Lorenz, and Sawers, R.G.
- Abstract
Six Hyp maturation proteins (HypABCDEF) are conserved in micro-organisms that synthesize [NiFe]-hydrogenases (Hyd). Of these, the HypC chaperones interact directly with the apo-form of the catalytically active large subunit of Hyd enzymes and are believed to transfer the Fe(CN)2CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In E. coli, HypC is specifically required for maturation of Hyd-3 while its paralogue, HybG, is specifically required for Hyd-2 maturation; either HypC or HybG can mature Hyd-1. In this study, we demonstrate that the products of the hypABFCDE operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an E. coli hyp mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of E. coli was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O2-sensitive H2-uptake HupLS [NiFe]-hydrogenase from D. mccartyi CBDB1 was also synthesized in an active form in E. coli. Together, these findings indicated that HypC from D. mccartyi CBDB1 exhibits promiscuity in its large subunit interaction in E. coli. Based on these findings, we generated amino acid variants of E. coli HybG capable of partial recovery of Hyd-3-dependent H2 production in a hypC hybG double null mutant. Together, these findings identify amino acid regions in HypC accessory proteins that specify interaction with the large subunits of hydrogenase and demonstrate functional compatibility of Hyp accessory protein machineries.
- Published
- 2015
27. Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family
- Author
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Hartwig, Stefanie, primary, Sawers, R. Gary, additional, Krumova, Nadya, additional, Türkowsky, Dominique, additional, Quitzke, Vivien, additional, Thomas, Claudia, additional, Jehmlich, Nico, additional, and Adrian, Lorenz, additional
- Published
- 2015
- Full Text
- View/download PDF
28. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR
- Author
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Jahn, Michael, Vorpahl, Carsten, Türkowsky, Dominique, Lindmeyer, M., Bühler, B., Harms, Hauke, Müller, Susann, Jahn, Michael, Vorpahl, Carsten, Türkowsky, Dominique, Lindmeyer, M., Bühler, B., Harms, Hauke, and Müller, Susann
- Abstract
Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.
- Published
- 2014
29. Population heterogeneity in Pseudomonas putida analyzed on the single cell level using proteomics and digital PCR
- Author
-
Jahn, Michael, Vorpahl, Carsten, Türkowsky, Dominique, Lindmeyer, M., Bühler, B., Harms, Hauke, Müller, Susann, Jahn, Michael, Vorpahl, Carsten, Türkowsky, Dominique, Lindmeyer, M., Bühler, B., Harms, Hauke, and Müller, Susann
- Abstract
no abstract
- Published
- 2014
30. A H2-oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1.
- Author
-
Hartwig, Stefanie, Dragomirova, Nadya, Kublik, Anja, Türkowsky, Dominique, von Bergen, Martin, Lechner, Ute, Adrian, Lorenz, and Sawers, R. Gary
- Subjects
DEHALOCOCCOIDES ,BACTERIA ,HYDROGEN ,MOLYBDOPROTEINS ,OXIDOREDUCTASES - Abstract
Dehalococcoides mccartyi is a small, slow-growing bacterium of the phylum Chloroflexi that conserves energy using aliphatic and aromatic organohalides as electron acceptors, and hydrogen as sole electron donor. A recent study identified a protein complex in the membrane of strain CBDB1 comprising a Hup hydrogenase, a complex iron-sulphur molybdoprotein and a reductive dehalogenase (RdhA) that catalyses reduction of 1,2,3,4-tetrachlorobenzene. Using a combination of size-exclusion chromatography, in-gel hydrogenase activity-staining, immunological analysis and mass spectrometry, we identified here a large molecular mass protein complex solubilized from the cytoplasmic membrane of D. mccartyi strain CBDB1 that catalysed H
2 -dependent reduction of 1,2,3-trichlorobenzene (1,2,3-TCB) to 1,3-DCB. In-gel zymographic staining revealed H2 :benzyl viologen oxidoreductase activity associated with the complex and immunological analysis identified co-elution of CdbdA195, the predicted catalytic subunit of the iron-sulphur molybdoenzyme, the chlorobenzene-specific RdhA, CbrA, and traces of HupL, the catalytic subunit of the Hup hydrogenase. Quantitative reverse transcriptase PCR analyses indicated that the expression of the hupL and cbdbA195 genes was induced by 1,2,3-TCB but not by hydrogen. Together, these data identify and describe a protein-based electron-transfer complex catalysing H2 oxidation coupled to chlorobenzene reduction. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
31. Population heterogeneity in Pseudomonas putida analyzed on the single cell level using proteomics and digital PCR
- Author
-
Jahn, Michael, primary, Vorpahl, Carsten, additional, Türkowsky, Dominique, additional, Lindmeyer, Martin, additional, Bühler, Bruno, additional, Harms, Hauke, additional, and Müller, Susann, additional
- Published
- 2014
- Full Text
- View/download PDF
32. Crop monoculture rather than agriculture reduces the spatial turnover of soil bacterial communities at a regional scale
- Author
-
Figuerola, Eva L. M., primary, Guerrero, Leandro D., additional, Türkowsky, Dominique, additional, Wall, Luis G., additional, and Erijman, Leonardo, additional
- Published
- 2014
- Full Text
- View/download PDF
33. A H 2 -oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1.
- Author
-
Hartwig S, Dragomirova N, Kublik A, Türkowsky D, von Bergen M, Lechner U, Adrian L, and Sawers RG
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Benzyl Compounds metabolism, Chlorobenzenes pharmacology, Chloroflexi genetics, Gene Expression Regulation, Bacterial, Operon, Chlorobenzenes metabolism, Chloroflexi metabolism, Multienzyme Complexes isolation & purification, Multienzyme Complexes metabolism, Oxidation-Reduction
- Abstract
Dehalococcoides mccartyi is a small, slow-growing bacterium of the phylum Chloroflexi that conserves energy using aliphatic and aromatic organohalides as electron acceptors, and hydrogen as sole electron donor. A recent study identified a protein complex in the membrane of strain CBDB1 comprising a Hup hydrogenase, a complex iron-sulphur molybdoprotein and a reductive dehalogenase (RdhA) that catalyses reduction of 1,2,3,4-tetrachlorobenzene. Using a combination of size-exclusion chromatography, in-gel hydrogenase activity-staining, immunological analysis and mass spectrometry, we identified here a large molecular mass protein complex solubilized from the cytoplasmic membrane of D. mccartyi strain CBDB1 that catalysed H
2 -dependent reduction of 1,2,3-trichlorobenzene (1,2,3-TCB) to 1,3-DCB. In-gel zymographic staining revealed H2 :benzyl viologen oxidoreductase activity associated with the complex and immunological analysis identified co-elution of CdbdA195, the predicted catalytic subunit of the iron-sulphur molybdoenzyme, the chlorobenzene-specific RdhA, CbrA, and traces of HupL, the catalytic subunit of the Hup hydrogenase. Quantitative reverse transcriptase PCR analyses indicated that the expression of the hupL and cbdbA195 genes was induced by 1,2,3-TCB but not by hydrogen. Together, these data identify and describe a protein-based electron-transfer complex catalysing H2 oxidation coupled to chlorobenzene reduction., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)- Published
- 2017
- Full Text
- View/download PDF
34. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.
- Author
-
Jahn M, Vorpahl C, Türkowsky D, Lindmeyer M, Bühler B, Harms H, and Müller S
- Subjects
- DNA genetics, Flow Cytometry, Gene Dosage, Plasmids, Polymerase Chain Reaction methods
- Abstract
Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.
- Published
- 2014
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