Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

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Prof. Dr.

Prof.

Gut bacteria metabolize natural and synthetic steroid hormones via the reductive OsrABC Pathway

Anaerobic metabolism

Steroid hormones, critical regulators of various physiological processes, are metabolized by both the host and gut bacteria, with profound implications for mammalian health. Due to their diverse effects, many natural and synthetic steroids are widely used to treat autoimmune diseases, inflammatory disorders, hormone deficiencies, and cancer. In this study, we isolated a novel gut bacterium, Clostridium steroidoreducens strain HCS.1, and identified a unique steroid metabolism pathway, OsrABC, capable of reducing a broad range of steroid hormones, including glucocorticoids, mineralocorticoids, androgens, and progestins.1 The OsrABC pathway consists of three key enzymes: OsrA, a 3-oxo-Δ1-steroid reductase that targets synthetic corticosteroids; OsrB, a versatile 3-oxo-Δ4-steroid reductase that produces 5β-dihydrosteroid intermediates; and OsrC, which further reduces these intermediates to 3β,5β-tetrahydrosteroid products. Our results suggest that this pathway may contribute to chronic inflammation by potentially interfering with glucocorticoid therapies and affecting drug bioavailability. Notably, osrA and osrB homologs were found to be highly enriched in active Crohn's disease patients, particularly in microbes such as Clostridium tertium, Clostridium_AQ innocuum, and Ruminococcus_B gnavus, the latter two of which have previously been associated with the disease. These microbes may exploit steroid metabolism to thrive in an inflamed environment, further perpetuating inflammation. In conclusion, our study highlights how microbial metabolism may influence the efficacy of steroid-based therapies in patients with inflammatory diseases, underscoring the need for personalized treatment approaches.Jacoby, C. et al. Gut Bacteria Metabolize Natural and Synthetic Steroid Hormones via the Reductive OsrABC Pathway. bioRxiv 2024.10.08.617280 (2024) doi:10.1101/2024.10.08.617280.

Short lecture

Unraveling chloromethane conversion in the acetogen Acetobacterium dehalogenans

Chloromethane (CM) is an abundant environmental pollutant that contributes significantly to ozone depletion [1]. Acetobacterium dehalogenans is one of the few isolated anaerobic bacteria shown to utilize CM as its carbon and energy source [2]. The enzyme system responsible for its CM demethylation/dehalogenation has not been identified so far. We aim to identify and characterize this system and to understand the metabolic changes of A. dehalogenans during growth on CM versus the methoxylated aromatic syringate. We analyzed the growth and substrate conversion of A. dehalogenans grown on CM, syringate and both substrates and used comparative transcriptomics to analyze the gene expression pattern of A. dehalogenans grown under these conditions. We identified a corrinoid-dependent methyltransferase system with three adjacent genes mtcABC which were highly upregulated during growth on CM in comparison to syringate. We heterologously produced and purified the corresponding proteins in E. coli, subsequently performed activity assays to determine their substrate specificity, and conducted X-ray crystallography. We discovered that this Mtc methyltransferase system demethylates methyl halides such as chloromethane and iodomethane. X-ray crystallography of the MtcB methyltransferase revealed a hydrophobic internal channelling system for methyl halides. Phylogenetic analysis showed that homologous proteins are encoded by several other anaerobic bacteria, especially Bacillota. Such organisms may also have the potential to convert methyl halides such as CM. The Mtc system is most closely related to the archaeal Mto system, which converts methoxylated aromatic compounds. In summary, we identified and characterized a novel enzyme system used for methyl halide demethylation in anaerobic microorganisms.[1] Harper, D. B. (2000). Nat Prod Rep, 17, 337-348.[2] Traunecker, J., et al. (1991). Arch Microbiol, 156, 416–421.

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus

Nitrogenases are the only enzymes able to "fix" atmospheric N2 into bioavailable NH3 and hence are essential for sustaining biological life. Nitrogenase catalysis is dependent on an ample supply of both ATP and electrons, the latter provided by low-potential redox proteins, often ferredoxins (Fds). Regarding this electron transport to nitrogenase proteins, many open questions remain; specifically electron transport to the iron (Fe-) nitrogenase had hardly been investigated. Our goal was to identify which Fds are important for nitrogen fixation by the Fe-nitrogenase and try to elucidate their functions.Highlighted in our recent study, we identified two essential Fds for nitrogen fixation by the Fe-nitrogenase in Rhodobacter capsulatus (R. capsulatus)[1]. Genetic deletions of the Fd genes (fdx) in R. capsulatus were constructed, in a molybdenum nitrogenase deletion strain, and characterised via diazotrophic growth rates and in vivo nitrogenase activity. Deletion of two fdx genes, fdxC and fdxN, slowed diazotrophic growth and lowered Fe-nitrogenase activities. The ∆fdxN and ∆fdxC deletion strains were further studied via whole cell proteomics and plasmid complementation experiments. Proteomics revealed both strains had an upregulation of proteins essential for electron delivery to nitrogenases, indicating an interruption in electron transport to the Fe-nitrogenase. Plasmid complementation patterns differed greatly for ∆fdxN and ∆fdxC, revealing the two proteins were likely not redundant in function, but instead performed distinct roles.To further explore the functions of FdN and FdC, our current research has focused on studying FdN and FdC in vitro. Specifically, FdN and FdC have been characterised spectroscopically, structurally and biochemically. Both Fds have been purified with redox-active FeS centres, shown by metal content analysis and UV-Vis spectroscopy, under reductive and oxidative conditions. Additionally, we have obtained an atomic resolution structure of one of the Fds, which has provided new details about the FeS cluster coordination, protein architecture and hydrogen bonding network. Overall, our findings have provided two key protein targets for the further study and bioengineering of nitrogen fixation systems, specifically those focussed on increasing the electron flux to nitrogenases for increased product formation.1 Addison, H., …, Rebelein J. (2024) Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus. mBio. 15, e03314-03323

Prof. Dr

O2-independent C-H activation by novel molybdenum-containing water dependent alkyl hydroxylases

Hydrocarbons are essential in numerous natural processes and represent the main components of crude oil.1 The conversion of hydrocarbons into downstream products represents the basis for the petrochemical, pharmaceutical and plastic industries. Made up primarily of hydrogen and carbon atoms, hydrocarbons exist in various forms, including alkanes, alkenes and aromatic compounds. Their degradation is essential for the bioremediation of polluted environments, as they can be toxic and persistent in nature1. On the other side, the initial steps of bacterial alkane degradation may be important for the functionalization of alkanes into chiral alcohols as building blocks for value-added products. The activation of non-polar C‑H bonds in hydrocarbons is a major challenge in both chemical and enzymatic syntheses due to high activation energies. In aerobic environments, bacteria use O2 for the hydroxylation of C-H bonds catalysed by oxygenases or peroxygenases. In contrast, anaerobic bacteria employ alternative strategies for the initial hydrocarbon activation. Here, molybdenum-dependent hydroxylases of the type II DMSO reductase family have been postulated as key enzymes that use water to hydroxylate unactivated C-H bonds.In this study, we isolated and characterized a p‑cymene dehydrogenase (CmdABC) from denitrifying Aromatoleum aromaticum pCyN12 as well as a putative alkane hydroxylase (AhyABC) from the sulfate-reducing bacterium Desulfococcus oleovorans Hxd33,4 after heterologous production in the β‑proteobacterium Thauera aromatica K172. Metal analyses and UV-vis spectroscopy of the heterotrimeric enzyme complexes indicate the presence of one molybdenum cofactor, five FeS clusters and one heme b, respectively. We used cryo-electron microscopy to obtain structural insights and combined these findings with biochemical studies and quantum mechanics/molecular mechanics (QM/MM) modelling to explore the limits of C-H bond activation by molybdenum-dependent hydroxylases. Taken together, the studies performed on both enzymes provide further insights into the mechanisms of water-dependent hydrocarbon activation in anoxic environments.1Rabus, R. et al., J Mol Microbiol Biotechnol, 26, 5-28 (2016)2Strijkstra, A. et al., AEM, 80, 7592–7603 (2014)3Aeckersberg, F. et al., Microbiol, 156, 5-14 (1991)4Callaghan A., Front Microbiol, 4, 1-9 (2013)

Enhancing Methanogenesis performance in the Power-to-Methane process: Addressing H2 and CO2 intermittency

Archaea

QuestionPower-to-Methane (PtM) technology stands at the forefront as a promising solution for the storage of surplus renewable energy aligned with CO2 reduction. In the biological PtM process, methanogens utilize H2 generated from water electrolysis powered by renewable energy and CO2 from industrial waste gas to produce CH4, namely methanogenesis. Our work aims to enhance the robustness of PtM processes by optimizing this methanogenesis step, addressing challenges related to intermittent gas feeding.MethodsThe model strain Methanococcus maripaludis MM901 was cultured in batch cultures to study its starvation-revival dynamics under H2- vs. CO2- starvation by monitoring OD600nm and specific CH4 production rate. To obtain mechanistic understanding of the observed difference, we also (1) measured NAD+ and NADH in the cells during starvation using bioluminescent assays, as well as (2) performed oxygen exposure experiments, where cultures were exposed to air for 1h during starvation followed by monitoring their revival dynamics after O2 removal.ResultsM. maripaludis MM901 exhibited a shorter lag time and higher metabolic activity after H2 starvation compared to CO2 starvation, and an increase in starvation time amplified the difference. We hypothesize that cells experience a reductive stress under CO2 starvation. We measured NAD+ and NADH in cells during starvation and observed significantly higher NADH/NAD+ ratio in CO2 starved cells indicating accumulation of reducing equivalence. O2 exposure also exerts greater inhibition on the revival of CO2-starved cultures over H2-starved cultures shown by a significantly longer lag phase.ConclusionsIn CO2-starved cells where there is H2 leftover, it is possible that O2 reacts with accumulated reducing equivalence, resulting in the production of highly oxidative reactive oxygen species, and subsequently damaged enzymes and DNA in cells. To ensure the process stability and gas product quality, where H2 leftover is preferred over CO2 leftover, further efforts are needed to adapt methanogens to starvation under CO2 limitation.

Studies on the substrate depending iron reduction and reductive bioleaching of Metallosphaera sedulaT

Since conventional leaching of nickel and copper from limonitic laterites via pyro- and hydrometallurgical processes requires large amounts of energy and results in high CO2 emissions, environmentally friendly alternatives like bioleaching are becoming increasingly important. The reductive bioleaching of limonitic laterites showed promising results using bacteria of the genera Acidithiobacillus and Sulfobacillus [1]. Thermoacidophilic archaea of the genus Metallosphaera are known to oxidize sulfur and iron as well as reduce iron and they are suggested to have a high potential for reductive bioleaching of laterites [2].The goal of this study was to investigate the influence of different substrates on the iron reduction of the thermoacidophilic archaeon Metallosphaera sedulaT and to evaluate its bioleaching potential in an initial leaching experiment.M. sedula was incubated under anaerobic conditions at 70°C in Norris Medium with 50 mM Fe(III) and one of the following substrates: (i) 0.1 % complex organics (e.g. yeast extract, casamino acids), (ii) 10 mM amino acids (e.g. L-alanine, L-cysteine), (iii) 10 mM D-mannose, (iv) 1% elemental sulfur. In total 11 different substrates were tested. Bioleaching experiments were carried out anaerobically with 1% (w/v) goethite, hematite, and the laterites BaSt and BaC (from a site in Brazil). Iron reduction efficiency was monitored using the Ferrozine assay in all experiments. Fe, Ni and Co concentrations in the leach solution from laterite bioleaching were detected using ICP-OES.The experiments revealed significant differences in iron reduction efficiency depending on the substrate used. Peptone showed the best results followed by S0 with a complete iron reduction after 5 and 7 days. The other substrates showed either a very slow reduction (L-alanine), incomplete reduction (yeast extract) and/or precipitation of iron(III) (yeast extract, glutamic acid). For the other amino acids and D-mannose, iron reduction was also observed in the negative controls. The bioleaching experiments revealed, that laterite and goethite were successfully leached by M. sedula, which will be explored for further application in reductive bioleaching.[1] Stankovic et al. (2022). Effect of mineralogy on Co and Ni extraction from Brazilian limonitic laterites via bioleaching and chemical leaching. Miner. Eng., 184, 107604.[2] Malik, L., & Hedrich, S. (2021). Ferric Iron Reduction in Extreme Acidophiles. Front. Microbiol., 12, 818414

Prof., Dr.

Identifying the missing steps of the autotrophic 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in Nitrosopumilus maritimus

Ammonia-oxidizing archaea (AOA) of the phylum Nitrososphaerota are the predominant nitrifiers in the ocean and in various soils. Most organisms in this group are autotrophs that use a modified 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle. In this cycle, acetyl-CoA is carboxylated to succinyl-CoA, which is then converted to two acetyl-CoA molecules with 4-hydroxybutyrate as the key intermediate. Compared to the HP/HB cycle operating in Thermoproteota, the AOA variant saves two ATP equivalents per turn, making it the most energy-efficient aerobic pathway for inorganic carbon fixation. These two variants of the HP/HB cycle evolved convergently, with a number of enzymes catalyzing the specific reactions of the cycle being non-homologous, as judged by the absence of homologs of the Thermoproteota enzymes in the genomes of autotrophic AOA. Here we identify all the missing enzymes of the cycle in the model AOA Nitrosopumilus maritumus, i.e. malonyl-CoA reductase, succinyl-CoA reductase and succinic semialdehyde reductase, and characterize ketothiolase that catalyzes the final step of the pathway, the formation of acetyl-CoA. The characterization of these enzymes elucidates the properties of the HP/HB cycle operating in Nitrososphaerota and clarifies the function of F420 in this group of archaea.

Fatty acid metabolism in Sulfolobus acidocaldarius

Archaea synthesize membranes using isoprenoid-based ether lipids, while Bacteria and Eukarya use fatty acid-based ester lipids. Despite these differences, some studies have suggested that fatty acids are present in a few Archaea, such as Sulfolobus spp. (1, 2). Furthermore, several archaeal genomes encode complete sets of bacterial-like β oxidation homologues and few archaeal species were found to degrade fatty acids (1, 3). However, no studies to date have been able to fully elucidate the fatty acid metabolism in archaeal species. Here, we address this open question by investigating fatty acid degradation in the archaeal model organisms Sulfolobus acidocaldarius by biochemical characterization and in vitro pathway reconstitution. This work shows that a set of β oxidation homologues present in S. acidocaldarius constitute a functional fatty acid degradation pathway. This pathway involves the sequential activities of an acyl-CoA dehydrogenase (Saci_1123), a bifunctional 3(S)-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase fusion protein (Saci_1109) and β-ketothiolase (Saci_1114) in addition to an unusual fused archaeal electron transfer flavoprotein (Saci_0315). All four proteins were reconstituted in vitro to a complete and fully functional β oxidation cascade that catalyzed the complete degradation of medium chain fatty acid to acetyl-CoA with a pronounced specificity for NAD+ and the (S)-hydroxyacyl-CoA stereoisomers. Importantly, a detailed characterization of these enzymes also supports that several mechanistic, specificity and thermodynamic constraints prevent this pathway from functioning in the reverse direction to support fatty acid synthesis. Instead, we propose the presence of a novel fatty acid synthesis pathway in S. acidocaldarius consisting of a β-ketothiolase (Saci_1121), ketoacyl-CoA reductase (Saci_1104), and hydroxyacyl-CoA dehydratase (Saci_1085) that form a stable assembly mediated by a DUF35 domain protein (Saci_1120), while the final step is catalysed by an NADPH-dependent enoyl-CoA reductase (Saci_1115). In summary our study demonstrates that Archaea are capable of synthesizing and degrading fatty acids and elucidates the molecular mechanisms involved in these processes.[1] D. V. Dibrova et al., Environmental microbiology 16, 907-918 (2014)[2] T. Hamerly et al., Archaea 2015, 9 (2015)[3] K. Wang et al., Nature communications 10, 1542 (2019).

Anaerobic metabolism & Archaea

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