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

Characterization of the unique Type IV Nitrogenase Homolog from Endomicrobium proavitum

Anaerobic metabolism

Nitrogenases are a group of complex metalloenzymes that convert molecular nitrogen (N₂) to its bioavailable form ammonia (NH₃). Thus, they play a critical role in biological nitrogen fixation. In general, there are three nitrogenase isoforms in diazotrophic microorganisms. The most common/prevalent nitrogenase is the molybdenum (Mo) nitrogenase and the less abundant vanadium (V) and iron (Fe) nitrogenases, named after the metal composition of the active site cofactor. All three nitrogenase isoforms are known as bona fide nitrogenases. However, there are several nitrogen fixation-like (Nfl)-enzymes that share sequence similarities and conserved regions but lack the ability to fix dinitrogen. The distinct activities of all those enzymes are attributed to differences in their catalytic metal center. The activities of those Nfl-like enzymes range from bacteriochlorophyll synthesis (dark-operative protochlorophyllide oxidoreductase and chlorophyllide a oxidoreductase, reducing carbon-carbon double bonds of the tetrapyrrole rings) (Moser & Layer, Methods Mol Biol, 2019), F430 biosynthesis (Ni2+-sirohydrochlorin a,c-diamide reductase) (Vazquez Ramos et al., FEBS J, 2024 / Moore et al., Nature, 2017) and methylated sulfur compounds (North et al., Science, 2020 / Lago-Maciel et al., bioRxiv, 2024).However, in contrast to the type Nfl-enzymes described above, another type IV homolog from Endomicrobium proavitum has recently been shown to reduce N2 to NH₃ in vivo (Zheng et al., Environ. Microbiol., 2016). Following up on this surprising finding, we set out to comprehensively characterize this enzyme. I am currently investigating its atomic structure using cryo-electron microscopy, determining the metal content of its cofactors by inductively coupled plasma optical emission spectroscopy, and exploring its substrate spectrum in vitro. Our exciting findings will provide new insights into the inner workings of Nfl-enzymes and nitrogenases, potentially revealing the fundamental principles of N2-reduction and the different substrate specificity of those enzymes.

Poster

Unraveling the conversion of methoxylated compounds in Tepidanaerobacter acetatoxydans

Methoxylated aromatic compounds (MACs) are components of lignin and are therefore very abundant on Earth. However, their impact on the global carbon cycle is not well understood. To convert these compounds microorganisms use O-demethylase systems that comprise four different enzymes. So far, the O-demethylase systems of some bacteria and one archaeon [1] have been characterized. The anaerobic bacterium Tepidanaerobacter acetatoxydans is known to use different organic compounds as carbon and energy source such as organic acids, alcohols, sugars and amino acids as well as acetate in syntrophy with methanogens [2]. Although the conversion of MACs has not been previously observed, the organism harbors a genomic region with several genes encoding components of O-demethylase systems in its genome. Chemolithotrophic growth and acetogenesis have not yet been described for T. acetatoxydans, which encodes most of the acetogenic key enzymes, but lacks for formate dehydrogenase [3]. The question rises if the O-demethylase systems in T. acetatoxydans are functional and if the organism can produce acetate from MACs. To answer this we studied several O-demethylase systems from T. acetatoxydans in regard to their biochemical properties and phylogeny. Furthermore, we performed growth studies with T. acetatoxydans to investigate it"s still underexplored metabolic potential. We were able to show the growth of T. acetatoxydans on various MACs. Furthermore, we observed that one O-demethylase system from T. acetatoxydans converts next to the MACs syringate and vanillate also 2-MB and TMB, which is different to all yet characterized bacterial O-demethylase systems but similar to the archaeal one [1]. Different O-demethylases encoded by T. acetatoxydans seem to build a yet uncharacterized phylogenetic clade, which could indicate the use of different substrates. This work will lead to a better understanding of MAC conversion and the involved enzyme systems in T. acetatoxydans and resolve the acetogenic metabolism using MACs as carbon source.[1] Kurth JM et al. (2021). ISME J. 15:3549–65.[2] Westerholm M, Roos S, Schnürer A. (2011). Syst Appl Microbiol. 34:260–6.[3] Müller B et al. (2015). PLoS ONE. 10:e0121237.

Prof. Dr.

Prof., Dr.

Thermophilic adaptations in the autotrophic pathways of Ammonifex degensii

Ammonifex degensii is an autotrophic, anaerobic and extremely thermophilic bacterium belonging to the class Clostridia (order Thermoanaerobacterales) (1). This organism is remarkable for its ability to use two different carbon dioxide fixation pathways for autotrophic growth. Depending on the H2 redox potential, A. degensii uses either the ferredoxin-dependent Wood-Ljungdahl pathway or the more ATP-intensive, ferredoxin-independent Calvin-Benson cycle (2). Notably, A. degensii is the most thermophilic organism known to use the Calvin-Benson cycle, with an optimal growth temperature of 70°C, and the cycle shows specific adaptations to operate under thermophilic conditions. The formation of fructose 6-phosphate from triose phosphates and sedoheptulose 7-phosphate from erythrose 4-phosphate and dihydroxyacetone phosphate is catalyzed by an unprecedented promiscuous, bifunctional fructose 1,6-bisphosphate/ sedoheptulose 1,7-bisphosphate (FBP) aldolase/phosphatase encoded in the Calvin-Benson cycle operon. Furthermore, sedoheptulose 7-phosphate can be produced in the Calvin-Benson cycle from erythrose 4-phosphate and fructose 6-phosphate in a transaldolase reaction. Interestingly, A. degensii also encodes a promiscuous class II fructose 1,6-bisphosphate/ sedoheptulose 1,7-bisphosphate aldolase outside the Calvin-Benson cycle operon. As a stand-alone fructose 1,6-bisphosphatase or sedoheptulose 1,7-bisphosphatase is missing in the A. degensii genome, the functional role of sedoheptulose 1,7-bisphosphate aldolase reaction in this bacterium is unknown.

Prof.

Direct electron transfer between the respiratory cytochrome bcc-aa3 supercomplex and nitrate reductase 1 gives spores of Streptomyces coelicolor a selective energetic advantage

Introduction: Streptomyces coelicolor A3(2) is a filamentous actinobacterium that requires oxygen for growth and uses a branched respiratory chain to respire with O2. A menaquinol:cytochrome bcc oxidoreductase and copper-aa3-type cytochrome c oxidase supercomplex forms one branch, while the other includes a menaquinol:cytochrome bd oxidase. The formation of an electron-transferring supercomplex is necessary because S. coelicolor has a membrane-bound c-type cytochrome. Although S. coelicolor can only grow with oxygen, it also encodes three related respiratory nitrate reductases (Nar); however, these do not support growth but are presumed to help maintain a membrane potential when O2 becomes limiting and nitrate is available. The Nar enzymes are synthesized and active in different stages of the complex developmental life cycle of the bacterium. Nar1 is exclusively present and active in spores, Nar2 mainly in exponentially growing mycelium and Nar3 in stationary mycelium. Nar1 activity depends on a functional cytochrome bcc-aa3 supercomplex, but Nar2 activity in mycelium is not totally dependent on the respiratory supercomplex.Objective: We wish to determine the mechanism underlying this differential dependence of Nar activity on the respiratory supercomplex at different developmental stages.Methods: A combination of proteomic analyses, biochemical assays, use of defined nar mutants, physiological studies, including use of respiratory inhibitors, and determination of nitrate and oxygen consumption rates were used.Results: Protein complex purification studies and proteomic analyses demonstrated that the respiratory supercomplexes differ between spores and mycelium. This finding was verified by determination of O2 respiration rates combined with the use of respiratory inhibitors. The influence of nar1 and nar2 operon mutations on O2 and nitrate reduction in spores and mycelium indicate direct electron transfer between Nar1 and the respiratory supercomplex in spores.Conclusions: Our findings suggest that the cytochrome bcc- aa3 supercomplex is differently organized in spores compared with mycelium. Nar1 appears to receive electrons directly from the cytochrome bcc-aa3 supercomplex in spores, while Nar2 can functions as a menaquinol oxidase in mycelium. The ability of spores to switch rapidly between available electron acceptors (oxygen or nitrate) improves the chances of S. coelicolor spore survival.

PD Dr.

Inoculation strategies for a synthetic co-cultures of a engineered Acetobacterium woodii and Clostridium drakei WT

World climate change causes global higher temperatures and increasingly extreme weather phenomena to become more and more frequent. Thus, measures have to be taken to avoid reaching a point of no return. One possible step is to reduce emissions of greenhouse gases (e.g. CO2) from the production of platform chemicals by switching from crude oil to microbial gas fermentation based production (Sanchez et al., 2023, https://www.iea.org/energy-system/industry/chemicals). The acetogenic bacterium Acetobacterium woodii has already been genetically engineered to produce lactate as a secondary product from H2 and CO2 next to acetate (Mook et al., 2022, doi: 10.1007/s00253-022-11770-z). Since A. woodii lacks the ability to produce higher value products through chain elongation, the addition of the bacterium Clostridium drakei in a synthetic co-culture allows the valorisation of lactate in combination with H2 and CO2 to more thought after chemicals such as butyrate or hexanoate. The inoculation strategy for this co-culture needs to be optimised and cell numbers from both species have to be quantifiable. Quantification of cells was done using fluorescence in situ hybridization (FISH) via distinguishable probes targeting both species individually. Two probes were constructed that hybridise to specific fragments of the V4 region of the 16S rDNA in A. woodii and C. drakei and carry differing fluorescent dyes (Pacific Blue for C. drakei, ATTO495 for A. woodii). These probes were then tested to find the optimal hybridisation conditions. Different inoculation strategies were tested for the co-culture by inoculating the strains both simultaneously and sequentially with different cell concentration ratios (A. woodii/C. drakei: 70/30, 50/50, 30/70). Additionally, carriers were added to test for possible improvements by allowing the cells to form stationary biofilms. Cell counts of both strains were quantified by hybridising the previously mentioned probes under the optimised conditions and observing them by both flow cytometry and fluorescent microscopy. The fermentation characteristics were determined by measuring gas consumption through loss in pressure, product spectra and production rates through gas chromatography and high pressure liquid chromatography, and capturing scanning and transmission electron microscopy images.

Investigation of the primary mechanism for entropy export of metal-reducing microorganisms

Introduction Two fundamental principles of life are coupling electron donor oxidation with electron acceptor reduction for energy harvest and exporting entropy into the environment by secretion of small molecules or heat dissipation. Chemotrophic microorganisms evolved to exploit numerous combinations of electron donor and acceptor, resulting in different mechanisms for entropy export ranging from purely entropy-driven to enthalpy-driven growth[1]. Metal-reducing microorganisms represent a unique variation as they couple their metabolism with insoluble electron acceptors by performing extracellular electron transfer (EET). This process allows the microbes to exchange metabolic electrons with metal ores in nature or electrodes in technical systems. Electrons undergo a phase shift from the liquid to the solid state during EET. Thus, the entropy export of metal-reducing microbes is supposed to differ from microbes utilizing soluble electron acceptors that has energetic consequences for the metabolism.Goal Analysis of the entropy export of metal-reducing microorganisms performing EET.Materials&Methods A bioelectrocalorimetric setup was employed to measure the heat flux of Geobacter sulfurreducens and Geobacter enrichment biofilms during EET with different electrode materials. Furthermore, the contribution of the electrolyte to the heat flux was assessed by applying the entropy of transport concept and fundamental equations[2]. The electrodes were microscopically and spectroscopically analyzed to elucidate the impact of EET on the elemental composition of the electrodes and to visualize biofilm formation.Results We show that the heat evolution depends on the electrode material amounting to 27.3±4.7, 22.1±4.5, and 13.2±5.0 kJ e-mol−1 for Geobacter enrichment biofilms at silver, copper, and gold electrodes. For G. sulfurreducens at graphite electrodes, 27.2±8.1 kJ e-mol−1 were measured, validating first experiments[3]. It is shown that Geobacter"s entropy export differs from microorganisms utilizing soluble electron acceptors as it mainly relies on EET and not the secretion of small molecules or dissipation of heat.Summary Bioelectrocalorimetric experiments show that electron transfer facilitates the main share of entropy export for metal-reducing microbes and depends on the electron acceptor material.References [1] von Stockar, U., et al. 1999. Biochim. Biophys. Acta. 1412 [2] Agar, J.N. 1963. Thermogalvanic Cells [3] Korth, B., et al. 2016. Energy Environ. Sci. 9

Evaluation of Eubacterium callanderi for CO2-neutral, recombinant bioplastics production

Rapid climate change and excessive plastics utilization lead to irreversible negative impact on our planet as well as on human society. The main accelerator of climate change is emission of greenhouse gases, which emerge primarily during fossil-based production processes. An alternative and in the best-case CO2-negative process is microbial gas fermentation with acetogens. Acetogens harbor enzymes of the Wood-Ljungdahl pathway, which enables this group of anaerobic bacteria to use gaseous and liquid single carbon molecules. Some recently reclassified Eubacterium callanderi strains are to date not well characterized but indicate high potential for gas fermentation due to a wide substrate spectrum and genetic accessibility (Flaiz et al., Microb Cell Fact). As E. callanderi should serve as a bio catalysator for recombinant production of the bioplastic poly-3-hydroxybutyrate (PHB) the metabolic and transcriptomic characteristics of different E. callanderi strains should be analyzed to build the basis for selection of the best suitable strain for gas fermentation. The metabolic and transcriptomic characteristics of the strains E. callanderi "FD" (DSM 3662), "Marburg" (DSM 3468) and "2A" (DSM 2593) were examined by growth experiments using glucose and different methanol concentrations. For optimization of transformation of E. callanderi the protocol of Leang et al. (Appl Env Microbiol) was varied. Heterotrophic growth experiments of the E. callanderi strains "FD", "Marburg", and "2A" with glucose as carbon source revealed the ability of the acetogen to perform chain elongation via the reverse β-oxidation, which enables the bacterium to produce butyrate, hexanoate and octanoate. Acetogenic growth of all strains using methanol as carbon source resulted in production of butyrate and hexanoate. Furthermore, the product spectrum of E. callanderi includes acetate, ethanol, and isobutyrate as well as butanol. Antibiotic resistance tests elucidated sensitivity of E. callanderi "Marburg" to thiamphenicol and tetracycline, E. callanderi "FD" and "2A" showed natural resistances. Consequently, E. callanderi "Marburg" was selected for recombinant PHB production. In conclusion the acetogenic species E. callanderi shows great potential as bio catalysator for gas fermentation as it has both a wide substrate and product spectrum and is genetically accessible. Due to sensitivity of E. callanderi "Marburg" to antibiotics, it is the best suitable strain for recombinant PHB production.

Exploring the Uncharted: Morphological changes, antibiotic resistance, and future perspectives on autotrophic growth of the strain M5a3

The anaerobic bacterium designated as M5a3 was originally isolated in 1996 from human fecal samples (Bernalier et al., 1996, doi: 10.1007/s002849900081). However, since its initial isolation, M5a3 has received almost no attention in subsequent research despite its unusual property to produce lactate, along with acetate from H₂ + CO₂ by employing the Wood-Ljungdahl pathway. Lactate production is an uncommon metabolic feature within acetogens.Heterotrophic reactivation of the lyophilized cells from the original 1996 stock was followed by amplifying and sequencing the 16S rDNA and the bacterium was identified as Intestinibacter bartlettii. Intestinibacter is often found to be a member of the human gut microbiota community. To further investigate the metabolic capabilities of I. bartlettii, whole-genome sequencing was performed using Illumina technology. Gram-staining was performed with cells obtained in its exponential and stationary growth phase. Gram-staining indicated that the bacterium showed a Gram-positive staining during the exponential phase, whereas in the stationary phase, a mixed staining pattern was observed. This staining variation may suggest Gram variability in this species. Also, a marked decrease in cell size was observed from the exponential phase (approx. 14 µm) to the stationary phase (approx. 3 µm). Furthermore, detailed morphological analysis using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed and respective micrographs recorded. The heterotrophic substrate spectrum was started to be investigated by growth experiments, which demonstrated its inability to utilize lactose and glycerol as carbon sources. In contrast, fructose served as a favorable substrate and enabled the production of 2.4 mM lactate, 17 mM ethanol and 27 mM acetate, which resulted in a pH decrease to 4.9 of the medium. Growth experiments utilizing H₂ + CO₂ as substrates were performed. The natural antibiotic resistance profile was analyzed using a selection of antibiotics, showing natural resistance against spectinomycin, kanamycin and clarithromycin. In contrast, thiamphenicol and tetracycline consistently inhibited bacterial growth.In summary, cells are still viable after being stored for 28 years and it could be shown that the heterotrophic metabolic profile fits the original description. Further analysis will be done to investigate the strains characteristics with elucidate its acetogenic properties.

Pathway and regulation of nitrate reduction in acetogenic Sporomusa ovata

Introduction: Nitrate is known as an alternative electron acceptor in the acetogenic bacterium Sporomusa ovata[1]. Inspection of the genome sequence and biochemical analyses revealed the presence of a cytochrome c-containing periplasmic nitrate reductase. Moreover, transcriptome analyses revealed nitrite reductase and hydroxylamine reductase genes upregulated in nitrate-grown cells[2].Goals: To get insights into the pathway of cytochrome c-dependent nitrate reduction of S. ovata.Materials & Methods: Bioinformatics, growth experiments, metabolite analyses, resting cells experiments, determination of transcript levels, measurement of enzyme activities.Results: Nitrate was reduced to ammonia as predicted from the transcriptome analyses. In the absence of ammonia, nitrate was also used as nitrogen source by S. ovata. Nitrate and nitrite reductase activities were measured in cell-free extract, cytoplasm and membrane fractions of S. ovata. These activities were induced only in cells grown in the presence of nitrate. Nitrate reductase (1.9 ± 0.4 U/mg) and nitrite reductase activity (6.1 ± 0.8 U/mg) were found almost exclusively in the membrane fraction. So far, only reduced methylviologen was used as artificial electron donor for nitrate and nitrite reductase. Both enzymes contain c-type cytochromes and the reduced cytochromes are speculated to be the physiological electron donors.Summary: S. ovata performs an energy-conserving nitrate respiration. Nitrate-induced membrane-bound nitrate and nitrite reductase activities were identified in the membrane fraction of S. ovata. The pathway of nitrate ammonification will be described.[1] Balk, M., Mehboob, F., van Gelder, A. H., Rijpstra, W. I., Damsté, J. S., Stams, A. J. (2010) Appl Microbiol Biotechnol 88: 595-603[2] Waschinger, L. M., Müller, V. (2024) Abstract VAAM 2024 "A cytochrome c-containing periplasmic nitrate reductase in the acetogen Sporomusa ovata"

Respiratory complex I in strictly anaerobic bacteria

Respiratory complex I is essential for cellular metabolism, serving as a primary entry point for catabolically derived electrons into respiratory chains. In canonical respiration, the NADH-dependent reduction of ubiquinone (E° = 100 mV) drives the translocation of four protons across the cytosolic membrane. This contributes to the establishment of a proton gradient that can be used for ATP synthesis. Membranes of strictly anaerobic microorganisms such as Geobacter metallireducens do not contain ubiquinone but menaquinone (MK) with a considerably lower midpoint redox potential (E° = -74 mV). The Gibbs free energy release of a MK-dependent NADH oxidation, however, is insufficient to support the translocation of four protons. This raises fundamental questions about the bioenergetics of these respiratory complex I enzymes.Our in silico analysis of the NADH-binding module of complex I from the strictly anaerobic model organism G. metallireducens revealed homologies to electron-confurcating FeFe-hydrogenases. After enrichment of the hydrophilic, electron-transferring domain of the enzyme, we observed a higher iron content compared to canonical respiratory complex I, suggesting the presence of additional iron-sulfur clusters. Therefore, we propose that respiratory complex I from strictly anaerobic bacteria overcomes its thermodynamic limitation and enables the translocation of more than two protons per two electrons by simultaneous MK reduction using both NAD(P)H and reduced ferredoxin as electron donors by flavin-based electron confurcation.

Monitoring an anaerobic synthetic co-culture of a lactate producing Acetobacterium woodii mutant and Clostridium drakei FP for the production of butyrate and hexanoate

Climate change is escalating into a global climate disaster. Rising greenhouse gas emissions (e.g. CO2) are fuelling this trend. However, the use of fossil fuels such as crude oil is still increasing (Ripple et al., 2024) and to reverse this trend climate-friendly alternatives are needed. Microbial gas fermentation is investigated to produce platform chemicals such as hexanoate and butyrate. Our approach is using the acetogens A. woodii and C. drakei in a synthetic co-culture. A. woodii is capable of autotrophic growth utilising efficiently H2 + CO2, while producing natively acetate. By plasmid-borne expression of a D-lactate dehydrogenase (ldhD) from Leuconostoc mesenteroides and knocking out A. woodii´s native genes encoding the bifurcating lactate dehydrogenase complex, a strain was constructed, capable of producing lactate as a second product (Mook et al., 2022). C. drakei is capable of producing butyrate and hexanoate via reverse-β-oxidation using H2 + CO2 as well as lactate as a substrate. One challenge during co-cultivation is the monitoring of two different species over the cultivation time. Therefor quantitative PCR (qPCR) is an option to quantify the respective cells ampilification of unique genes. The idea is to define a correlation between respective cq values and cell counts or OD600 values. Primers were designed to amplify the gene encoding the formate dehydrogenase from A. woodii and the gene encoding the phosphate butyryltransferase from C. drakei. To determine a strong correlation between cell counts and OD600 values several measurements were done over the course of heterotrophic growth experiments. Furthermore, dilution series of each specie obtained at the highest OD600 were also prepared and subsequently analysed. A correlation (R²=0.96) was found between OD600 values and the logarhithm of the cell counts for C. drakei during growth. qPCR has been performed with this isolated genomic DNA from the dilution row and the results are currently evaluated. Another method to determine the number of cells in this co-culture is by labelling the cells with species specific fluorescent labelled DNA probes. Therefor probes binding in the V4 region of 16S rDNA of A. woodii and C. drakei have been designed. The probe binding in A. woodii´s DNA is labelled with the fluorophore ATTO495, while the probe binding in C. drakei´s DNA is labelled with Pacific Blue. The fixed and labelled cells will be analysed according to an established hybridization strategy.

Anaerobic metabolism 1

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