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Abstract Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to products such as acetate and biofuels. The model acetogen Acetobacterium woodii can grow on CO2, formate or methanol, but not on carbon monoxide, an important industrial waste product. Since hydrogenases are targets of CO inhibition, here, we genetically delete the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. We show that the ∆hydBA/hydA2 mutant indeed grows on CO and produces acetate, but only after a long adaptation period. SNP analyzes of CO-adapted cells reveal a mutation in the HycB2 subunit of the HydA2/HydB2/HydB3/Fdh-containing hydrogen-dependent CO2 reductase (HDCR). We observe an increase in ferredoxin-dependent CO2 reduction and vice versa by the HDCR in the absence of the HydA2 module and speculate that this is caused by the mutation in HycB2. In addition, the CO-adapted ∆hydBA/hydA2 mutant growing on formate has a final biomass twice of that of the wild type.

Executive summary Microbes are all pervasive in their distribution and influence on the functioning and well‐being of humans, life in general and the planet. Microbially‐based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision‐makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative–the IMiLI–is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence‐based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner‐centric, not academic microbiology‐centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators–learners. As such, they will collect and analyse feedback from the end‐users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators–learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships–a global societally relevant microbiology education ecosystem–in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. Abstract The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well‐being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us–individuals/communities/nations/the human world–and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. Here, we present our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision‐making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner‐centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091–1111). Importantly, although the primary target of the freely available (CC BY‐NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity‐driven, web‐based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

ABSTRACTThermoanaerobacter kivui is the thermophilic acetogenic bacterium with the highest temperature optimum (66°C) and with high growth rates on hydrogen (H2) plus carbon dioxide (CO2). The bioenergetic model suggests that its redox and energy metabolism depends on energy-converting hydrogenases (Ech). Its genome encodes two Echs, Ech1 and Ech2, as sole coupling sites for energy conservation during growth on H2 + CO2. During growth on other substrates, its redox activity, the (proton-gradient-coupled) oxidation of H2 may be essential to provide reduced ferredoxin (Fd) to the cell. While Ech activity has been demonstrated biochemically, the physiological function of both Ech’s is unclear. Toward that, we deleted the complete gene cluster encoding Ech2. Surprisingly, the ech2 mutant grew as fast as the wild type on sugar substrates and H2 + CO2. Hence, Ech1 may be the essential enzyme for energy conservation, and either Ech1 or another enzyme may substitute for H2-dependent Fd reduction during growth on sugar substrates, putatively the H2-dependent CO2 reductase (HDCR). Growth on pyruvate and CO, substrates that are oxidized by Fd-dependent enzymes, was significantly impaired, but to a different extent. While ∆ech2 grew well on pyruvate after four transfers, ∆ech2 did not adapt to CO. Cell suspensions of ∆ech2 converted pyruvate to acetate, but no acetate was produced from CO. We analyzed the genome of five T. kivui strains adapted to CO. Strikingly, all strains carried mutations in the hycB3 subunit of HDCR. These mutations are obviously essential for the growth on CO but may inhibit its ability to utilize Fd as substrate.IMPORTANCEAcetogens thrive by converting H2+CO2 to acetate. Under environmental conditions, this allows for only very little energy to be conserved (∆G′

Abstract Background Enzymes from thermophiles are of great interest for research and bioengineering due to their stability and efficiency. Thermophilic expression hosts such as Thermus thermophilus [T. thermophilus] can overcome specific challenges experienced with protein production in mesophilic expression hosts, such as leading to better folding, increased protein stability, solubility, and enzymatic activity. However, available inducible promoters for efficient protein production in T. thermophilus HB27 are limited. Results In this study, we characterized the pilA4 promoter region and evaluated its potential as a tool for production of thermostable enzymes in T. thermophilus HB27. Reporter gene analysis using a promoterless β-glucosidase gene revealed that the pilA4 promoter is highly active under optimal growth conditions at 68 °C and downregulated during growth at 80 °C. Furthermore, growth in minimal medium led to significantly increased promoter activity in comparison to growth in complex medium. Finally, we proved the suitability of the pilA4 promoter for heterologous production of thermostable enzymes in T. thermophilus by producing a fully active soluble mannitol-1-phosphate dehydrogenase from Thermoanaerobacter kivui [T. kivui], which is used in degradation of brown algae that are rich in mannitol. Conclusions Our results show that the pilA4 promoter is an efficient tool for gene expression in T. thermophilus with a high potential for use in biotechnology and synthetic biology applications.

Abstract The challenge of endergonic reduction of NADP+ using NADH is overcome by ferredoxin-dependent transhydrogenases that employ electron bifurcation for electron carrier adjustments in the ancient Wood-Ljungdahl pathway. Recently, an electron-bifurcating transhydrogenase with subunit compositions distinct from the well-characterized Nfn-type transhydrogenase was described: the Stn complex. Here, we present the single-particle cryo-EM structure of the Stn family transhydrogenase from the acetogenic bacterium Sporomusa ovata and functionally dissect its electron transfer pathway. Stn forms a tetramer consisting of functional heterotrimeric StnABC complexes. Our findings demonstrate that the StnAB subunits assume the structural and functional role of a bifurcating module, homologous to the HydBC core of the electron-bifurcating HydABC complex. Moreover, StnC contains a NuoG-like domain and a GltD-like NADPH binding domain that resembles the NfnB subunit of the NfnAB complex. However, in contrast to NfnB, StnC lost the ability to bifurcate electrons. Structural comparison allows us to describe how the same fold on one hand evolved bifurcation activity on its own while on the other hand combined with an associated bifurcating module, exemplifying modular evolution in anaerobic metabolism to produce activities critical for survival at the thermodynamic limit of life.

ABSTRACT The Mycobacterium tuberculosis (Mtb) F-ATP synthase generates most of the biological energy currency ATP. Previously, we identified the mycobacterium-specific loop of the F-ATP synthase subunit γ as a new anti-tuberculosis target and discovered the novel diaminopyrimidine GaMF1, whose potency was improved by structure-activity relationship studies leading to the analog GaMF1.39. Here, we report that GaMF1.39 depletes cellular ATP formation by targeting the mycobacterial F-ATP synthase without affecting proton coupling or oxygen consumption. The antimycobacterial compound is bactericidal and potent against Mtb in macrophages without inducing phenotypic changes in biofilm formation, planktonic bacteria, or being toxic to zebrafish larvae. Combining GaMF1.39 with the NADH dehydrogenase inhibitor clofazimine, the cyt-bcc:aa3 inhibitor Telacebec, or the F-ATP synthase inhibitor TBAJ-876 showed enhanced whole ATP synthesis inhibition and anti-tuberculosis activity. These results suggest that GaMF1.39 may add value to a compound combination targeting oxidative phosphorylation for tuberculosis treatment. IMPORTANCE New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

ABSTRACT The viable but non-culturable (VBNC) state is a persistence strategy adopted by bacteria to withstand long-lasting periods of unfavorable conditions. VBNC cells evade classical detection methods and are therefore easily transmitted in the hospital causing relapsing infections. The opportunistic human pathogen Acinetobacter baumannii has become a major threat in health care institutions and the food industry due to multiple antibiotic resistances and its ability to quickly adapt to very different ecological niches. Here, we report an additional, novel survival strategy of A. baumannii. Upon prolonged incubation in high-salt media, cells became unculturable. However, LIVE/DEAD staining followed by flow cytometry, respiratory activity assays, and resuscitation experiments revealed that these cells were viable but non-culturable. VBNC cells underwent large morphological changes. Entry into the VBNC state was also induced by pH and temperature stress, as well as by desiccation and anaerobiosis. The VBNC state was found in several strains of A. baumannii. Genome-wide expression profiling revealed a plethora of genes differentially regulated upon entry into the VBNC state. In summary, this study presents unequivocal evidence for a dormancy state in A. baumannii that has important consequences for detection of this pathogen and recurrent outbreaks. IMPORTANCE Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.

Abstract DNA uptake is widespread among microorganisms and considered a strategy for rapid adaptation to new conditions. While both DNA uptake and adaptation are referred to in the context of natural environments, they are often studied in laboratories under defined conditions. For example, a strain of the thermophile Thermoanaerobacter kivui had been adapted to growth on high concentrations of carbon monoxide (CO). Unusual phenotypes of the CO-adapted strain prompted us to examine it more closely, revealing a horizontal gene transfer (HGT) event from another thermophile, Thermoanaerobacter sp. strain X514, being cultured in the same laboratory. The transferred genes conferred on T. kivui the ability to utilize trehalose, a trace component of the yeast-extract added to the media during CO-adaptation. This same HGT event simultaneously deleted a native operon for thiamine biosynthesis, which likely explains why the CO-adapted strain grows poorly without added vitamins. Attempts to replicate this HGT by providing T. kivui with genomic DNA from Thermoanaerobacter sp. strain X514 revealed that it is easily reproducible in the lab. This subtle form of “genome contamination” is difficult to detect, since the genome remains predominantly T. kivui, and no living cells from the original contamination remain. Unexpected HGT between two microorganisms as well as simultaneous adaptation to several conditions may occur often and unrecognized in laboratory environments, requiring caution and careful monitoring of phenotype and genotype of microorganisms that are naturally-competent for DNA uptake.

Summary Eubacterium limosum KIST612 is one of the few acetogenic bacteria that has the genes encoding for butyrate synthesis from acetyl‐CoA, and indeed, E. limosum KIST612 is known to produce butyrate from CO but not from H2 + CO2. Butyrate production from CO was only seen in bioreactors with cell recycling or in batch cultures with addition of acetate. Here, we present detailed study on growth of E. limosum KIST612 on different carbon and energy sources with the goal, to find other substrates that lead to butyrate formation. Batch fermentations in serum bottles revealed that acetate was the major product under all conditions investigated. Butyrate formation from the C1 compounds carbon dioxide and hydrogen, carbon monoxide or formate was not observed. However, growth on glucose led to butyrate formation, but only in the stationary growth phase. A maximum of 4.3 mM butyrate was observed, corresponding to a butyrate:glucose ratio of 0.21:1 and a butyrate:acetate ratio of 0.14:1. Interestingly, growth on the C1 substrate methanol also led to butyrate formation in the stationary growth phase with a butyrate:methanol ratio of 0.17:1 and a butyrate:acetate ratio of 0.33:1. Since methanol can be produced chemically from carbon dioxide, this offers the possibility for a combined chemical‐biochemical production of butyrate from H2 + CO2 using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.

The upcoming European emission standards for light as well as heavy-duty road vehicles in the 2025/26 timeframe will include a remote monitoring mandate as a key element of the regulation to ensure continuous ultra-low real world emission behavior. Real-time data regarding tailpipe emission performance for multiple pollutant species will be recorded and compared with the legal limits, summarized under heading On-Board Monitoring (OBM) for defined pollutant species. In addition, engine and exhaust gas aftertreatment operating conditions are required to be collected via an electronic control unit to complete the monitoring status and analysis in the necessary post-processing routines. The EU legal authorities within the rule making sections are on track to define an EU-wide standard that extends the responsibility of the OEMs from emission compliance in pre-defined operating profiles to full in-use emission behavior that is essentially independent from boundaries such as environmental and operating conditions.As the emission-related certification process for EU-7/-VII applications is entirely based on PEMS-measurements in all kinds of real-world driving cycles, the intended OBM aspect is also entirely related to PEMS-based emission criteria, marginally reduced by physical constraints like realizing reliable operating conditions for installed sensors, like e.g., warm-up, linearization or dew point protection [1].Under the anticipated Euro 7/VII standards, vehicle emissions are considered as being generated by a single, holistic system, with tailpipe emissions generally understood to be below the limit, regardless of what technology package the powertrain contains. With a multitude of factors determining the actual emissions behavior of the application as well as the measuring accuracy, meeting the intended continuous emissions monitoring represents even in comparison to the strict, ultra-low emission limits, the dominating challenge in meeting the upcoming EU-7 applications. This is due to the extreme complexity of the requirement and the very sensitive trade-off between the adherence to the formulated legal OBM limits and the realization of undesired and unnecessary consequences of the monitoring system, impacting the robust all-day usability of the vehicle over the complete service.Facing these extreme challenges, the paper in hand aims to provide a clear starting point resp. an aligned base for solution finding and delivers already some initial thoughts for solution finding and initial descriptions of the mandatory steps forward for realization and implementation.

ABSTRACT Acetogenic bacteria are a group of strictly anaerobic bacteria that make a living from acetate formation from two molecules of CO2 via the Wood-Ljungdahl pathway (WLP). The free energy change of this reaction is very small and allows the synthesis of only a fraction of an ATP. How this pathway is coupled to energy conservation has been an enigma since its discovery ~90 years ago. Here, we describe an electron transport chain in the cytochrome- and quinone-containing acetogen Sporomusa ovata that leads from molecular hydrogen as an electron donor to an intermediate of the WLP, methylenetetrahydrofolate (methylene-tetrahydrofolate [THF]), as an electron acceptor. The catalytic site of the hydrogenase is periplasmic and likely linked cytochrome b to the membrane. We provide evidence that the MetVF-type methylenetetrahydrofolate reductase is linked proteins MvhD and HdrCBA to the cytoplasmic membrane. Membrane preparations catalyzed the H2-dependent reduction of methylene-THF to methyl-THF. In our model, a transmembrane electrochemical H+ gradient is established by both scalar and vectorial protons that leads to the synthesis of 0.5 mol ATP/mol methylene-THF by a H+-F1Fo ATP synthase. This H2- and methylene-THF-dependent electron transport chain may be present in other cytochrome-containing acetogens as well and represents a third way of chemiosmotic energy conservation in acetogens, but only in addition to the well-established respiratory enzymes Rnf and Ech. IMPORTANCE Acetogenic bacteria grow by making acetate from CO2 and are considered the first life forms on Earth since they couple CO2 reduction to the conservation of energy. How this is achieved has been an enigma ever since. Recently, two respiratory enzymes, a ferredoxin:NAD+ oxidoreductase (Rnf) and a ferredoxin:H+ oxidoreductase (Ech), have been found in cytochrome-free acetogenic model bacteria. However, some acetogens contain cytochromes in addition, and there has been a long-standing assumption of a cytochrome-containing electron transport chain in those acetogens. Here, we provide evidence for a respiratory chain in Sporomusa ovata that has a cytochrome-containing hydrogenase as the electron donor and a methylenetetrahydrofolate reductase as the terminal electron acceptor. This is the third way of chemiosmotic energy conservation found in acetogens.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low‐potential ferredoxin (Fd2−) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg−1) and ferredoxin (Fd; 27.2 U·mg−1) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein−1, consistent with the presence of three predicted [4Fe–4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His‐tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron‐bifurcating enzymes, to keep a constant level of reduced Fd.

Lactate oxidation with NAD+ as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB)2 complex of Acetobacterium woodii at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD+ binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed.

Abstract Background Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study. Results We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis. Discussion We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.

Summary Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar‐based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2‐dependent CO2 reductase and furthermore belong to different classes of ‘Rnf’‐ and ‘Ech‐acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g−1 h−1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO‐based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.

Abstract Background In times of global climate change, the conversion and capturing of inorganic CO2 have gained increased attention because of its great potential as sustainable feedstock in the production of biofuels and biochemicals. CO2 is not only the substrate for the production of value-added chemicals in CO2-based bioprocesses, it can also be directly hydrated to formic acid, a so-called liquid organic hydrogen carrier (LOHC), by chemical and biological catalysts. Recently, a new group of enzymes were discovered in the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which catalyze the direct hydrogenation of CO2 to formic acid with exceptional high rates, the hydrogen-dependent CO2 reductases (HDCRs). Since these enzymes are promising biocatalysts for the capturing of CO2 and the storage of molecular hydrogen in form of formic acid, we designed a whole-cell approach for T. kivui to take advantage of using whole cells from a thermophilic organism as H2/CO2 storage platform. Additionally, T. kivui cells were used as microbial cell factories for the production of formic acid from syngas. Results This study demonstrates the efficient whole-cell biocatalysis for the conversion of H2 + CO2 to formic acid in the presence of bicarbonate by T. kivui. Interestingly, the addition of KHCO3 not only stimulated formate formation dramatically but it also completely abolished unwanted side product formation (acetate) under these conditions and bicarbonate was shown to inhibit the membrane-bound ATP synthase. Cell suspensions reached specific formate production rates of 234 mmol g protein −1 h−1 (152 mmol g CDW −1 h−1), the highest rates ever reported in closed-batch conditions. The volumetric formate production rate was 270 mmol L−1 h−1 at 4 mg mL−1. Additionally, this study is the first demonstration that syngas can be converted exclusively to formate using an acetogenic bacterium and high titers up to 130 mM of formate were reached. Conclusions The thermophilic acetogenic bacterium T. kivui is an efficient biocatalyst which makes this organism a promising candidate for future biotechnological applications in hydrogen storage, CO2 capturing and syngas conversion to formate.

ABSTRACT CsrA is a global regulator widespread in bacteria and known to be involved in different physiological processes, including pathogenicity. Deletion of csrA of Acinetobacter baumannii strain ATCC 19606 resulted in a mutant that was unable to utilize a broad range of carbon and energy sources, including amino acids. This defect in amino acid metabolism was most likely responsible for the growth inhibition of the ΔcsrA mutant in human urine, where amino acids are the most abundant carbon source for A. baumannii. Recent studies revealed that deletion of csrA in the A. baumannii strains AB09-003 and ATCC 17961 resulted in an increase in hyperosmotic stress resistance. However, the molecular basis for this observation remained unknown. This study aimed to investigate the role of CsrA in compatible solute synthesis. We observed striking differences in the ability of different A. baumannii strains to cope with hyperosmotic stress. Strains AB09-003 and ATCC 17961 were strongly impaired in hyperosmotic stress resistance in comparison to strain ATCC 19606. These differences were abolished by deletion of csrA and are in line with the ability to synthesize compatible solutes. In the salt-sensitive strains AB09-003 and ATCC 17961, compatible solute synthesis was repressed by CsrA. This impairment is mediated via CsrA and could be overcome by deletion of csrA from the genome. IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii has become one of the leading causes of nosocomial infections around the world due to the increasing prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. Recently, it was found that CsrA, a global mRNA binding posttranscriptional regulator, plays a role in osmotic stress adaptation, virulence, and growth on amino acids of A. baumannii AB09-003 and ATCC 17961. Here, we report that this is also the case for A. baumannii ATCC 19606. However, we observed significant differences in the ΔcsrA mutants with respect to osmostress resistance, such as the AB09-003 and 17961 mutants being enhanced in osmostress resistance whereas the ATCC 19606 mutant was not. This suggests that the role of CsrA in osmotic stress adaptation is strain specific. Furthermore, we provide clear evidence that CsrA is essential for growth in human urine and at high temperatures.

ABSTRACT Acetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity. IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions worldwide. The success of A. baumannii is based on the rise of multiple antibiotic resistances and its outstanding potential to persist in the human host and under conditions of low water activity in hospital environments. Combating low water activities involves osmoprotective measures such as uptake of compatible solutes and K+. To address the role of K+ uptake in the physiology of A. baumannii we have identified K+ transporter encoding genes in the genome of A. baumannii ATCC 19606. The corresponding genes (kup, trk, kdp) were deleted and the phenotype of the mutants was studied. The triple mutant was defective in K+ uptake which resulted in a pronounced growth defect at high osmolarities (300 mM NaCl). Additionally, mannitol and glutamate synthesis were strongly reduced in the mutant. To mimic host conditions and to study its role as an uropathogen, we performed growth studies with the K+ transporter deletion mutants in human urine. Both, the double (ΔkupΔtrk) and the triple mutant were significantly impaired in growth. This could be explained by the inability of ΔkupΔtrkΔkdp to metabolize various amino acids properly. Moreover, the reactive oxygen species resistance of the triple mutant was significantly reduced in comparison to the wild type, making it susceptible to one essential part of the innate immune response. Finally, the triple and the double mutant were strongly impaired in Galleria mellonella killing giving first insights in the importance of K+ uptake in virulence.

Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.

The strictly anaerobic acetogenic bacterium Acetobacterium woodii is metabolically diverse and grows on variety of substrates which includes H2 + CO2, sugars, alcohols and diols. It is unique in producing bacterial microcompartments (BMC) during growth on different substrates such as 1,2-propanediol, 2,3-butanediol, ethanol or fructose. In this study, we analyzed the genetic organization and expression of the BMC genes within the A. woodii genome, the previously described 18 gene pdu cluster as well as four other cluster potentially encoding one or two shell proteins. Expression analysis of respective gene clusters revealed that the pdu gene cluster is highly expressed during growth on 1,2-PD, 2,3-BD, ethanol and ethylene glycol. The promoter region upstream of the pduA gene was identified and used to establish a reporter gene assay based on chloramphenicol acetyl transferase as a reporter protein. The reporter gene assay confirmed the qPCR data and demonstrated that 1,2-PD is superior over ethanol and ethylene glycol as inducer. BMCs were enriched from cells grown on 2,3- BD and 1,2-PD and shown to have typical structure in electron micrographs. Biochemical analyses revealed several of the protein encoded by the pdu cluster to be part of the isolated BMCs. These data demonstrate a very unique situation in A. woodii in which apparently one BMC gene cluster in expressed during growth on different substrates.

Acetogenic microorganisms utilize organic substrates such as sugars in addition to hydrogen (H2) + carbon dioxide (CO2). Recently, we reported that the thermophilic acetogenic microorganism Thermoanaerobacter kivui is among the few acetogens that utilize the sugar alcohol mannitol, dependent on a gene cluster encoding mannitol uptake, phosphorylation and oxidation of mannitol-1-phosphate to fructose-6-phosphate. Here, we studied mannitol metabolism with resting cells of T. kivui; and found that mannitol was “fermented” in a homoacetogenic manner, i.e., acetate was the sole product if HCO3– was present. We found an acetate:mannitol ratio higher than 3, indicating the requirement of external CO2, and the involvement of the WLP as terminal electron accepting pathway. In the absence of CO2 (or bicarbonate, HCO3–), however, the cells still converted mannitol to acetate, but slowly and with stoichiometric amounts of H2 formed in addition, resulting in a “mixed” fermentation. This showed that–in addition to the WLP–the cells used an additional electron sink–protons, making up for the “missing” CO2 as electron sink. Growth was 2.5-fold slower in the absence of external CO2, while the addition of formate completely restored the growth rate. A model for mannitol metabolism is presented, involving the major three hydrogenases, to explain how [H] make their way from glycolysis into the products acetate or acetate + H2.

The hydrogen-dependent carbon dioxide reductase is a soluble enzyme complex that directly utilizes hydrogen (H2) for the reduction of carbon dioxide (CO2) to formate in the first step of the acetyl-coenzyme A- or Wood-Ljungdahl pathway (WLP). HDCR consists of 2 catalytic subunits, a hydrogenase and a formate dehydrogenase (FDH) and two small subunits carrying iron-sulfur clusters. The enzyme complex has been purified and characterized from two acetogenic bacteria, from the mesophile Acetobacterium woodii and, recently, from the thermophile Thermoanaerobacter kivui. Physiological studies toward the importance of the HDCR for growth and formate metabolism in acetogens have not been carried out yet, due to the lack of genetic tools. Here, we deleted the genes encoding HDCR in T. kivui taking advantage of the recently developed genetic system. As expected, the deletion mutant (strain TKV_MB013) did not grow with formate as single substrate or under autotrophic conditions with H2 + CO2. Surprisingly, the strain did also not grow on any other substrate (sugars, mannitol or pyruvate), except for when formate was added. Concentrated cell suspensions quickly consumed formate in the presence of glucose only. In conclusion, HDCR provides formate which was essential for growth of the T. kivui mutant. Alternatively, extracellularly added formate served as terminal electron acceptor in addition to CO2, complementing the growth deficiency. The results show a tight coupling of multi-carbon substrate oxidation to the WLP. The metabolism in the mutant can be viewed as a coupled formate + CO2 respiration, which may be an ancient metabolic trait.

Abstract Background Replacing fossil fuels as energy carrier requires alternatives that combine sustainable production, high volumetric energy density, easy and fast refueling for mobile applications, and preferably low risk of hazard. Molecular hydrogen (H2) has been considered as promising alternative; however, practical application is struggling because of the low volumetric energy density and the explosion hazard when stored in large amounts. One way to overcome these limitations is the transient conversion of H2 into other chemicals with increased volumetric energy density and lower risk hazard, for example so-called liquid organic hydrogen carriers such as formic acid/formate that is obtained by hydrogenation of CO2. Many homogenous and heterogenous chemical catalysts have been described in the past years, however, often requiring high pressures and temperatures. Recently, the first biocatalyst for this reaction has been described opening the route to a biotechnological alternative for this conversion. Results The hydrogen-dependent CO2 reductase (HDCR) is a highly active biocatalyst for storing H2 in the form of formic acid/formate by reversibly catalyzing the hydrogenation of CO2. We report the identification, isolation, and characterization of the first thermostable HDCR operating at temperatures up to 70 °C. The enzyme was isolated from the thermophilic acetogenic bacterium Thermoanaerobacter kivui and displays exceptionally high activities in both reaction directions, substantially exceeding known chemical catalysts. CO2 hydrogenation is catalyzed at mild conditions with a turnover frequency of 9,556,000 h−1 (specific activity of 900 µmol formate min−1 mg−1) and the reverse reaction, H2 + CO2 release from formate, is catalyzed with a turnover frequency of 9,892,000 h−1 (930 µmol H2 min−1 mg−1). The HDCR of T. kivui consists of a [FeFe] hydrogenase subunit putatively coupled to a tungsten-dependent CO2 reductase/formate dehydrogenase subunit by an array of iron–sulfur clusters. Conclusions The discovery of the first thermostable HDCR provides a promising biological alternative for a chemically challenging reaction and might serve as model for the better understanding of catalysts able to efficiently reduce CO2. The catalytic activity for reversible CO2 hydrogenation of this enzyme is the highest activity known for bio- and chemical catalysts and requiring only ambient temperatures and pressures. The thermostability provides more flexibility regarding the process parameters for a biotechnological application.

Abstract Background Molecular hydrogen (H2) is an attractive future energy carrier to replace fossil fuels. Biologically and sustainably produced H2 could contribute significantly to the future energy mix. However, biological H2 production methods are faced with multiple barriers including substrate cost, low production rates, and low yields. The C1 compound formate is a promising substrate for biological H2 production, as it can be produced itself from various sources including electrochemical reduction of CO2 or from synthesis gas. Many microbes that can produce H2 from formate have been isolated; however, in most cases H2 production rates cannot compete with other H2 production methods. Results We established a formate-based H2 production method utilizing the acetogenic bacterium Acetobacterium woodii. This organism can use formate as sole energy and carbon source and possesses a novel enzyme complex, the hydrogen-dependent CO2 reductase that catalyzes oxidation of formate to H2 and CO2. Cell suspensions reached specific formate-dependent H2 production rates of 71 mmol gprotein−1 h−1 (30.5 mmol gCDW−1 h−1) and maximum volumetric H2 evolution rates of 79 mmol L−1 h−1. Using growing cells in a two-step closed batch fermentation, specific H2 production rates reached 66 mmol gCDW−1 h−1 with a volumetric H2 evolution rate of 7.9 mmol L−1 h−1. Acetate was the major side product that decreased the H2 yield. We demonstrate that inhibition of the energy metabolism by addition of a sodium ionophore is suitable to completely abolish acetate formation. Under these conditions, yields up to 1 mol H2 per mol formate were achieved. The same ionophore can be used in cultures utilizing formate as specific switch from a growing phase to a H2 production phase. Conclusions Acetobacterium woodii reached one of the highest formate-dependent specific H2 productivity rates at ambient temperatures reported so far for an organism without genetic modification and converted the substrate exclusively to H2. This makes this organism a very promising candidate for sustainable H2 production and, because of the reversibility of the A. woodii enzyme, also a candidate for reversible H2 storage.

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

Abstract Acinetobacter baumannii is a nosocomial pathogen which can persist in the hospital environment not only due to the acquirement of multiple antibiotic resistances, but also because of its exceptional resistance against disinfectants and desiccation. A suitable desiccation assay was established in which A. baumannii ATCC 19606T survived for ca. 1 month. The growth medium slightly influenced survival after subsequent desiccation. A significant effect could be attributed to the growth phase in which bacteria were dried: In exponential phase, cells were much more desiccation sensitive. The main focus of the present study was the elucidation of the role of compatible solutes, which are known to protect many bacteria under low water activity conditions, in desiccation survival of A. baumannii. Exogenous trehalose was shown to efficiently protect A. baumannii on dry surfaces, in contrast to other compatible solutes tested such as mannitol or glycine betaine. To analyze the importance of intracellularly accumulated solutes, a double mutant lacking biosynthesis pathways for mannitol and trehalose was generated. This mutant accumulated glutamate as sole solute in the presence of high NaCl concentrations and showed severe growth defects under osmotic stress conditions. However, no effect on desiccation tolerance could be seen, neither when cells were dried in water nor in the presence of NaCl.

In times of global warming caused by the extensive use of fossil fuels, the need to capture gaseous carbon compounds is growing bigger. Several groups of microorganisms can fix the greenhouse gas CO2. Out of these, acetogenic bacteria are role models in their ability to reduce CO2 with hydrogen to acetate, which makes acetogens prime candidates for genetic modification towards biotechnological production of value-added compounds from CO2, such as biofuels. However, growth of acetogens on gaseous substrates is strongly energy-limited, and successful metabolic engineering requires a detailed knowledge of the bioenergetics. In 1939, Clostridium aceticum was the first acetogen to be described. A recent genomic study revealed that this organism contains cytochromes and therefore may use a proton gradient in its respiratory chain. We have followed up these studies and will present data that C. aceticum does not use a H+ but a Na+ gradient for ATP synthesis, established by a Na+-Rnf. Experimental data and in silico analyses enabled us to propose the biochemistry and bioenergetics of acetogenesis from H2 + CO2 in C. aceticum.

Hydrogenases are key enzymes of the energy metabolism of many microorganisms. Especially in anoxic habitats where molecular hydrogen (H2) is an important intermediate, these enzymes are used to expel excess reducing power by reducing protons or they are used for the oxidation of H2 as energy and electron source. Despite the fact that hydrogenases catalyze the simplest chemical reaction of reducing two protons with two electrons it turned out that they are often parts of multimeric enzyme complexes catalyzing complex chemical reactions with a multitude of functions in the metabolism. Recent findings revealed multimeric hydrogenases with so far unknown functions particularly in bacteria from the class Clostridia. The discovery of [FeFe] hydrogenases coupled to electron bifurcating subunits solved the enigma of how the otherwise highly endergonic reduction of the electron carrier ferredoxin can be carried out and how H2 production from NADH is possible. Complexes of [FeFe] hydrogenases with formate dehydrogenases revealed a novel enzymatic coupling of the two electron carriers H2 and formate. These novel hydrogenase enzyme complex could also contribute to biotechnological H2 production and H2 storage, both processes essential for an envisaged economy based on H2 as energy carrier.

Some anaerobic archaea and bacteria live on substrates that do not allow the synthesis of one mol of ATP per mol of substrate via substrate level phosphorylation (SLP). Energy conservation in these cases is only possible by a chemiosmotic mechanism that involves the generation of an electrochemical ion gradient across the cytoplasmic membrane that then drives ATP synthesis via an ATP synthase. The minimal amount of energy required for ATP synthesis is thus dependent on the magnitude of the electrochemical ion gradient, the phosphorylation potential in the cell and the ion/ATP ratio of the ATP synthase. It was always thought that the minimum biological energy quantum is defined as the amount of energy required to translocate one ion across the cytoplasmic membrane. We will discuss the thermodynamics of the reactions involved in chemiosmosis and describe the limitations for ion transport and ATP synthesis that led to the proposal that at least −20 kJ/mol are required for ATP synthesis. We will challenge this hypothesis by arguing that the enzyme energizing the membrane may translocate net less than one ion: By using a primary pump connected to an antiporter module a stoichiometry below one can be obtained, implying that the minimum biological energy quantum that sustains life is even lower than assumed to date.

The capability of osmoadaptation is a prerequisite of organisms that live in an environment with changing salinities. Halobacillus halophilus is a moderately halophilic bacterium that grows between 0.4 and 3 M NaCl by accumulating both chloride and compatible solutes as osmolytes. Chloride is absolutely essential for growth and, moreover, was shown to modulate gene expression and activity of enzymes involved in osmoadaptation. The synthesis of different compatible solutes is strictly salinity- and growth phase-dependent. This unique hybrid strategy of H. halophilus will be reviewed here taking into account the recently published genome sequence. Based on identified genes we will speculate about possible scenarios of the synthesis of compatible solutes and the uptake of potassium ion which would complete our knowledge of the fine-tuned osmoregulation and intracellular osmolyte balance in H. halophilus.

A ferredoxin:NAD+ oxidoreductase was recently discovered as a redox-driven ion pump in the anaerobic, acetogenic bacterium Acetobacterium woodii. The enzyme is assumed to be encoded by the rnf genes. Since these genes are present in the genomes of many bacteria, we tested for ferredoxin:NAD+ oxidoreductase activity in cytoplasmic membranes from several different Gram-positive and Gram-negative bacteria that have annotated rnf genes. We found this activity in Clostridium tetanomorphum, Clostridium ljungdahlii, Bacteroides fragilis, and Vibrio cholerae but not in Escherichia coli and Rhodobacter capsulatus. As in A. woodii, the activity was Na+-dependent in C. tetanomorphum and B. fragilis but Na+-independent in C. ljungdahlii and V. cholerae. We deleted the rnf genes from B. fragilis and demonstrated that the mutant has greatly reduced ferredoxin:NAD+ oxidoreductase activity. This is the first genetic proof that the rnf genes indeed encode the reduced ferredoxin:NAD+ oxidoreductase activity.

The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.

The evaluation of a health promotion program of several years for primary school children resulted in two problems. First, qualitative instruments had to be developed for a population (children at the age between 5 and 10), for which standardized procedures would not be suitable. Second, the program was tested in a total of 20 school classes, and longitudinal verbal and pictorial-based data were collected. Thus, over a period of almost four years, an enormous amount of qualitative data were collected. New procedures were developed in order to analyze these qualitative data comprehensibly in a quantitative way. In addition, it had to be taken into account that the qualitative categories used for data analysis had to be elaborated (had to become more differentiated) in the course of time. In order to ensure the longitudinal comparabililty, the earlier codings had to be brought into line with the respective elaborations of the coding scheme. Overall, a constant standard of excellence of qualitative analysis could be achieved. Moreover, the coding scheme could be improved by considering the quantitative results. In this article, these procedures and their efficiency in evaluating the promotion program are presented. URN: urn:nbn:de:0114-fqs0002165