Reprogramming the metabolism of an acetogenic bacterium to homoformatogenesis.

Methyl groups are abundant in anoxic environments and their utilization as carbon and energy sources by microorganisms involves oxidation of the methyl groups to CO2, followed by transfer of the electrons to an acceptor. In acetogenic bacteria, the electron acceptor is CO2 that is reduced to enzyme bound carbon monoxide, the precursor of the carboxyl group in acetate. Here, we describe the generation of a mutant of the acetogen Acetobacterium woodii in which the last step in methyl group oxidation, formate oxidation to CO2 catalyzed by the HDCR enzyme, has been genetically deleted. The mutant grew on glycine betaine as methyl group donor, and in contrast to the wild type, formed formate alongside acetate, in a 1:2 ratio, demonstrating that methyl group oxidation stopped at the level of formate and reduced electron carriers were reoxidized by CO2 reduction to acetate. In the presence of the alternative electron acceptor caffeate, CO2 was no longer reduced to acetate, formate was the only product and all the carbon went to formate. Apparently, acetogenesis was not required to sustain formatogenic growth. This is the first demonstration of a genetic reprogramming of an acetogen into a formatogen that grows by homoformatogenesis from methyl groups. Formate production from methyl groups is not only of biotechnological interest but also for the mechanism of electron transfer in syntrophic interactions in anoxic environments.

Anaerobic Biohydrogenation of Isoprene by Acetobacterium wieringae Strain Y.

Isoprene is a ubiquitously distributed, biogenic, and climate-active organic compound. Microbial isoprene degradation in oxic environments is fairly well understood; however, studies exploring anaerobic isoprene metabolism remain scarce, with no isolates for study available. Here, we obtained an acetogenic isolate, designated Acetobacterium wieringae strain Y, which hydrogenated isoprene to a mixture of methyl-1-butenes at an overall rate of 288.8 ± 20.9 µM day-1 with concomitant acetate production at a rate of 478.4 ± 5.6 µM day-1. Physiological characterization demonstrated that isoprene was not utilized in a respiratory process; rather, isoprene promoted acetogenesis kinetically. Bioinformatic analysis and proteomics experiments revealed the expression of candidate ene-reductases responsible for isoprene biohydrogenation. Notably, the addition of isoprene to strain Y cultures stimulated the expression of proteins associated with the Wood-Ljungdahl pathway, indicating unresolved impacts of isoprene on carbon cycling and microbial ecology in anoxic environments (e.g., promoting CO2 plus H2 reductive acetogenesis while inhibiting methanogenesis). Our new findings advance understanding of microbial transformation of isoprene under anoxic conditions and suggest that anoxic environments are isoprene sinks. IMPORTANCE Isoprene is the most abundant, biologically generated, volatile organic compound on Earth, with estimated emissions in the same magnitude as methane. Nonetheless, a comprehensive knowledge of isoprene turnover in the environment is lacking, impacting global isoprene flux models and our understanding of the environmental fate and longevity of isoprene. A critical knowledge gap that has remained largely unexplored until recently is the microbiology and associated molecular mechanisms involved in the anaerobic biotransformation of isoprene. By integrating culture-dependent approaches with omics techniques, we isolated an acetogen, Acetobacterium wieringae strain Y, capable of anaerobic biohydrogenation of isoprene. We obtained the complete genome of strain Y, and proteomic experiments identified candidate ene-reductases for catalyzing the asymmetric reduction of the electronically activated carbon-carbon double bond of isoprene. We also demonstrated that isoprene biohydrogenation stimulates the expression of Wood-Ljungdahl pathway enzymes. This study emphasizes the ecological roles of specialized Acetobacterium on the natural cycling of isoprene in anoxic environments and the potential effects of isoprene biohydrogenation on acetogens and methanogens, which have implications for global climate change and bioenergy production.

A clean in-frame knockout system for gene deletion in Acetobacterium woodii.

Acetogenic bacteria produce acetate following the fixation of CO2 via the Wood-Ljungdahl pathway. As such, they represent excellent process organisms for the production of novel chemicals and fuels from this waste greenhouse gas. Acetobacterium woodii is the model acetogen and numerous studies have been conducted investigating its biochemistry, gas consumption and use as a production chassis. However, there are a dearth of available tools for A. woodii gene modification which limits the research options available for genetic studies. Here, the previously proposed Clostridia Roadmap is implemented in A. woodii leading to the derivation of a knockout system for the generation of clean, in-frame deletions. The replicon of the Gram-positive plasmid pCD6 that originated in Clostridioides difficile was identified as being replication-defective in A. woodii, a property that was exploited to construct a pseudo-suicide knockout plasmid which was used to generate an auxotrophic, pyrE mutant. This allowed the subsequent use of a heterologous pyrE gene (from Clostridium acetobutylicum) as a counter selection marker and the deletion of a number of genes by allelic exchange. Specific mutants generated were affected in growth on glucose, fructose and ethanol as a consequence of deletion of fruA, pstG and adhE, respectively.

One substrate, many fates: different ways of methanol utilization in the acetogen Acetobacterium woodii.

Acetogenic bacteria such as Acetobacterium woodii use the Wood-Ljungdahl pathway (WLP) for fixation of CO2 and energy conservation. This pathway enables conversion of diverse substrates to the main product of acetogenesis, acetate. Methyl group containing substrates such as methanol or methylated compounds, derived from pectin, are abundant in the environment and a source for CO2 . Methyl groups enter the WLP at the level of methyltetrahydrofolic acid (methyl-THF). For methyl transfer from methanol to THF a substrate-specific methyltransferase system is required. In this study, we used genetic methods to identify mtaBC2A (Awo_c22760-Awo_c22740) as the methanol-specific methyltransferase system of A. woodii. After methyl transfer, methyl-THF serves as carbon and/or electron source and the respiratory Rnf complex is required for redox homeostasis if methanol + CO2 is the substrate. Resting cells fed with methanol + CO2 , indeed converted methanol to acetate in a 4:3 stoichiometry. When methanol was fed in combination with other electron sources such as H2  + CO2 or CO, methanol was converted Rnf-independently and the methyl group was condensed with CO to build acetate. When fed in combination with alternative electron sinks such as caffeate methanol was oxidized only and resulting electrons were used for non-acetogenic growth. These different pathways for the conversion of methyl-group containing substrates enable acetogens to adapt to various ecological niches and to syntrophic communities.

Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii.

Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major contribution to reducing greenhouse gas. Acetobacterium woodii is one of the very few acetogens that can utilize methanol to produce acetate as sole product. Since A. woodii is genetically tractable, it is an interesting candidate to introduce recombinant pathways for production of bio-commodities from methanol. In this study, we introduced the butyrate production operon from a related acetogen, Eubacterium callanderi KIST612, into A. woodii and show a stable production of butyrate from methanol. This study also reveals how butyrate production by recombinant A. woodii strains can be enhanced with addition of electrons in the form of carbon monoxide. Our results not only show a stable expression system of non-native enzymes in A. woodii but also increase in the product spectrum of A. woodii to compounds with higher economic value.

Physiology and genetics of ethanologenesis in the acetogenic bacterium Acetobacterium woodii.

The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2  + CO2 , formate, pyruvate, lactate or alanine. In the absence of Na+ , the Wood-Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.

Propionate Production from Carbon Monoxide by Synthetic Cocultures of Acetobacterium wieringae and Propionigenic Bacteria.

Gas fermentation is a promising way to convert CO-rich gases to chemicals. We studied the use of synthetic cocultures composed of carboxydotrophic and propionigenic bacteria to convert CO to propionate. So far, isolated carboxydotrophs cannot directly ferment CO to propionate, and therefore, this cocultivation approach was investigated. Four distinct synthetic cocultures were constructed, consisting of Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T), Ac. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T), Ac. wieringae strain JM and P. propionicus (DSM 2379T), and Ac. wieringae strain JM and An. neopropionicum (DSM 3847T). Propionate was produced by all the cocultures, with the highest titer (∼24 mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum, which also produced isovalerate (∼4 mM), butyrate (∼1 mM), and isobutyrate (0.3 mM). This coculture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during cocultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during cocultivation to fully understand coculture behavior. IMPORTANCE Syngas fermentation has great potential for the sustainable production of chemicals from wastes (via prior gasification) and flue gases containing CO/CO2. Research efforts need to be directed toward expanding the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol. This study provides the basis for a microbial process to produce propionate from CO using synthetic cocultures composed of acetogenic and propionigenic bacteria and elucidates the metabolic pathways involved. Furthermore, based on proteomics results, we hypothesize that the two bacterial species engage in an interaction that results in amino acid exchange, which subsequently promotes isovalerate and isobutyrate production. These findings provide a new understanding of gas fermentation and a coculturing strategy for expanding the product spectrum of microbial conversion of CO/CO2.

Flavodoxin hydroquinone provides electrons for the ATP-dependent reactivation of protein-bound corrinoid cofactors.

Corrinoid-dependent enzyme systems rely on the super-reduced state of the protein-bound corrinoid cofactor to be functional, for example, in methyl transfer reactions. Due to the low redox potential of the [CoII ]/[CoI ] couple, autoxidation of the corrinoid cofactor occurs and leads to the formation of the inactive [CoII ]-state. For the reactivation, which is an energy-demanding process, electrons have to be transferred from a physiological donor to the corrinoid cofactor by the help of a reductive activator protein. In this study, we identified reduced flavodoxin as electron donor for the ATP-dependent reduction of protein-bound corrinoid cofactors of bacterial O-demethylase enzyme systems. Reduced flavodoxin was generated enzymatically using pyruvate:ferredoxin/flavodoxin oxidoreductase rather than hydrogenase. Two of the four flavodoxins identified in Acetobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were functional in supplying electrons for corrinoid reduction. They exhibited a midpoint potential of about -400 mV (ESHE , pH 7.5) for the semiquinone/hydroquinone transition. Reduced flavodoxin could be replaced by reduced clostridial ferredoxin. It was shown that the low-potential electrons of reduced flavodoxin are first transferred to the iron-sulfur cluster of the reductive activator and finally to the protein-bound corrinoid cofactor. This study further highlights the importance of reduced flavodoxin, which allows maintaining a variety of enzymatic reaction cycles by delivering low-potential electrons.

It does not always take two to tango: "Syntrophy" via hydrogen cycling in one bacterial cell.

Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."

Growth of the acetogenic bacterium Acetobacterium woodii by dismutation of acetaldehyde to acetate and ethanol.

Acetogenic bacteria are a group of strictly anaerobic bacteria that may have been first life forms on Earth since they employ an ancient pathway for CO2 fixation into acetyl-CoA that is coupled to the synthesis of ATP, the Wood-Ljungdahl pathway. Electrons for CO2 reduction are derived from oxidation of H2 or CO and thus, these bacteria can grow lithotrophically on gases present on early Earth. Among the organic molecules present on early Earth is acetaldehyde, a highly volatile C2 compound. Here, we demonstrate that the acetogenic model bacterium Acetobacterium woodii grows on acetaldehyde. Acetaldehyde is dismutated to ethanol and acetyl-CoA, most likely by the bifunctional alcohol dehydrogenase AdhE. Acetyl-CoA is converted to acetate by two subsequent enzymes, phosphotransacetylase and acetate kinase, accompanied by the synthesis of ATP by substrate-level phosphorylation. Apparently, growth on acetaldehyde does not employ the Wood-Ljungdahl pathway. Our finding opens the possibility of a simple and ancient metabolic pathway with only three enzymes that allows for biomass (acetyl-CoA) and ATP formation on early Earth.

Microbiome for the Electrosynthesis of Chemicals from Carbon Dioxide.

The price for renewable electricity is rapidly decreasing, and the availability of such energy is expected to increase in the coming years. This is a welcomed outcome considering that mitigation of climate disruption due to the use of fossil carbon is reaching a critical stage. However, the economy will remain dependent on carbon-based chemicals and the problem of electricity storage persists. Therefore, the development of electrosynthetic processes that convert electricity and CO2 into chemicals and energy dense fuels, perhaps even food, would be desirable. Electrochemistry has been applied to the manufacture of many valuable products and at a large industrial scale, but it is difficult to produce multicarbon chemicals from CO2 by chemistry alone. Being that the biological world possesses expertise at the construction of C-C bonds, it is being examined in conjunction with electrochemistry to discover new ways of synthesizing chemicals from electricity and CO2. One approach is microbial electrosynthesis. This Account describes the development of a microbial electrosynthesis system by the authors. A biocathode consisting of a carbon-based electrode and a microbial community produced short chain fatty acids, primarily acetate. The device works by electrolysis of water, but microbes facilitate electron transfer from the cathode while reducing CO2 by the Wood-Ljungdahl pathway possessed by an Acetobacterium sp. While this acetogenic microorganism dominates the microbiome growing on the cathode surface, 13 total species of microbes overall were ecologically selected on the cathode and genomes for each have been assembled. The combined species may contribute to the stability of the microbiome, a common feature of naturally selected microbial communities. The microbial electrosynthesis system was demonstrated to operate continuously at a cathode for more than 2 years and could also be used with intermittent power, thus demonstrating the stability of the microbiome living at the cathode. In addition to the description of reactor design and startup procedures, the possible mechanisms of electron transfer are described in this Account. While mysteries remain to be solved, much evidence indicates that the microbiome may facilitate electron transfer by supplying catalyst(s) external to the bacterial cells and onto the cathode surface. This may be in the form of a hydrogen-producing catalyst that enhances hydrogen generation by an inert carbon-based electrode. Through the enrichment of the electrosynthetic microbiome along with several modifications in reactor design and operation, the productivity and efficiency were improved. In addition to the intrinsic value of the current products, coupling the process with a secondary stage might be used to produce more valuable products from the acetic acid stream such as lipids, biocrude oil, or higher value food supplements. Alternatively, additional work on the mechanism of electron transfer, reactor design/operation, and modification of the microbes through synthetic biology, particularly to enhance carbon efficiency into higher value chemicals, are the needed next steps to advance microbial electrosynthesis so that it may be used to transform renewable electrons and CO2 directly into products and help solve the problem of climate disruption.

Insights into the genome structure of four acetogenic bacteria with specific reference to the Wood-Ljungdahl pathway.

Acetogenic bacteria are obligate anaerobes with the ability of converting carbon dioxide and other one-carbon substrates into acetate through the Wood-Ljungdahl (WL) pathway. These substrates are becoming increasingly important feedstock in industrial microbiology. The main potential industrial application of acetogenic bacteria is the production of metabolites that constitute renewable energy sources (biofuel); such bacteria are of particular interest for this purpose thanks to their low energy requirements for large-scale cultivation. Here, we report new genome sequences for four species, three of them are reported for the first time, namely Acetobacterium paludosum DSM 8237, Acetobacterium tundrae DSM 917, Acetobacterium bakii DSM 8239, and Alkalibaculum bacchi DSM 221123. We performed a comparative genomic analysis focused on the WL pathway's genes and their encoded proteins, using Acetobacterium woodii as a reference genome. The Average Nucleotide Identity (ANI) values ranged from 70% to 95% over an alignment length of 5.4-6.5 Mbp. The core genome consisted of 363 genes, whereas the number of unique genes in a single genome ranged from 486 in A. tundrae to 2360 in A.bacchi. No significant rearrangements were detected in the gene order for the Wood-Ljungdahl pathway however, two species showed variations in genes involved in formate metabolism: A. paludosum harbor two copies of fhs1, and A. bakii a truncated fdhF1. The analysis of protein networks highlighted the expansion of protein orthologues in A. woodii compared to A. bacchi, whereas protein networks involved in the WL pathway were more conserved. This study has increased our understanding on the evolution of the WL pathway in acetogenic bacteria.

New Horizons in Acetogenic Conversion of One-Carbon Substrates and Biological Hydrogen Storage.

Strictly anaerobic, acetogenic (acetate-forming) bacteria are characterized by a reductive pathway in which two mol of CO2 are reduced to one mol of acetyl coenzyme A (acetyl-CoA) and then further to acetate, ethanol, or butyrate. Therefore, they have come into focus for an alternative, CO2-based bioeconomy. Other one-carbon (C1) substrates, such as formic acid or methanol, are promising feedstocks for an alternative bioeconomy using acetogens as biocatalysts that have been somewhat overlooked. In addition, acetogens, such as Acetobacterium woodii and Thermoanaerobacter kivui, have a unique enzyme system capable of reducing CO2 to formate with H2 as reductant that is superior over any chemical catalyst for CO2-based hydrogen storage. Therefore, acetogens are also promising candidates in the hydrogen economy as potential catalysts for hydrogen storage or production.

Reductive metabolism of the important atmospheric gas isoprene by homoacetogens.

Isoprene is the most abundant biogenic volatile organic compound (BVOC) in the Earth's atmosphere and plays important roles in atmospheric chemistry. Despite this, little is known about microbiological processes serving as a terrestrial sink for isoprene. While aerobic isoprene degrading bacteria have been identified, there are no known anaerobic, isoprene-metabolizing organisms. In this study an H2-consuming homoacetogenic enrichment was shown to utilize 1.6 µmoles isoprene h-1 as an electron acceptor in addition to HCO3-. The isoprene-reducing community was dominated by Acetobacterium spp. and isoprene was shown to be stoichiometrically reduced to three methylbutene isomers (2-methyl-1-butene (>97%), 3-methyl-1-butene (≤2%), 2-methyl-2-butene (≤1%). In the presence of isoprene, 40% less acetate was formed suggesting that isoprene reduction is coupled to energy conservation in Acetobacterium spp. This study improves our understanding of linkages and feedbacks between biogeochemistry and terrestrial microbial activity.

Enhancing hydrogen-dependent growth of and carbon dioxide fixation by Clostridium ljungdahlii through nitrate supplementation.

Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of CO2 and renewable production of fuels and chemicals. However, the pathway operates near the thermodynamic limit of life, resulting in minimal adenosine triphosphate (ATP) production and long doubling times. This calls into question the feasibility of producing high-energy compounds at industrially relevant levels. In this study, we investigated the possibility of co-utilizing nitrate as an inexpensive additional electron acceptor to enhance ATP production during H2 -dependent growth of Clostridium ljungdahlii, Moorella thermoacetica, and Acetobacterium woodii. In contrast to other acetogens tested, growth rate and final biomass titer were improved for C. ljungdahlii growing on a mixture of H2 and CO2 when supplemented with nitrate. Transcriptomic analysis, 13CO2 labeling, and an electron balance were used to understand how electron flux was partitioned between CO2 and nitrate. We further show that, with nitrate supplementation, the ATP/adenosine diphosphate (ADP) ratio and acetyl-CoA pools were increased by fivefold and threefold, respectively, suggesting that this strategy could be useful for the production of ATP-intensive heterologous products from acetyl-CoA. Finally, we propose a pathway for enhanced ATP production from nitrate and use this as a basis to calculate theoretical yields for a variety of products. This study demonstrates a viable strategy for the decoupling of ATP production from carbon dioxide fixation, which will serve to significantly improve the CO2 fixation rate and the production metrics of other chemicals from CO2 and H2 in this host.

Photosensitization of electro-active microbes for solar assisted carbon dioxide transformation.

Tandem bio-inorganic platform by combining efficient light harvesting properties of nano-inorganic semiconductor cadmium sulfide (CdS) with biocatalytic ability of electro-active bacteria (EAB) towards carbon dioxide (CO2) conversion is reported. Sulfur was obtained from either cysteine (EAB-Cys-CdS) or hydrogen sulfide (EAB-H2S-CdS) and experiments were carried out under similar conditions. Anchoring of the nano CdS cluster on the microbe surface was confirmed using electronic microscope. Bio-inorganic hybrid system was able to produce single and multi-carbon compounds from CO2 in visible spectrum (λ > 400 nm). Though, acetic acid was dominant (EAB-Cys-CdS, 1.46 g/l and EAB-H2S-CdS, 1.55 g/l) in both the microbe-CdS hybrids, its concentration as well as product slate varied significantly. EAB-H2S-CdS produced hexanoic acid and less methanol fraction, while the EAB-Cys-CdS produced no hexanoic acid along with almost double the concentration of methanol. Due to easy harvesting process, this bio-inorganic hybrid represents unique sustainable approach for solar-to-chemical production via CO2 transformation.

An Acetobacterium strain isolated with metallic iron as electron donor enhances iron corrosion by a similar mechanism as Sporomusa sphaeroides.

Sporomusa sphaeroides related strains are to date the only homoacetogens known to increase metallic iron corrosion. The goal of this work was to isolate additional homoacetogenic bacteria capable of using Fe(0) as electron donor and to explore their extracellular electron transfer mechanism. Enrichments were started from anoxic corrosion products and yielded Acetobacterium as main homoacetogenic genus. Isolations were performed with a new procedure using plates with a Fe(0) powder top layer. An Acetobacterium strain, closely related to A. malicum and A. wieringae, was isolated, in addition to a S. sphaeroides strain. The Acetobacterium isolate significantly increased Fe(0) corrosion ((1.44 ± 0.16)-fold) compared to abiotic controls. The increase of corrosion by type strains ranged from (1.28 ± 0.13)-fold for A. woodii to (2.03 ± 0.22)-fold for S. sphaeroides. Hydrogen mediated the electron uptake from Fe(0) by the acetogenic isolates and tested type strains. Exchange of the medium and SEM imaging suggested that cells were attached to Fe(0). The corrosion enhancement mechanism is for all tested strains likely related to free extracellular components catalyzing hydrogen formation on the Fe(0) surface, or to the maintenance of low hydrogen concentrations on the Fe(0) surface by attached cells thereby thermodynamically favoring hydrogen formation.

Alanine, a Novel Growth Substrate for the Acetogenic Bacterium Acetobacterium woodii.

Acetogenic bacteria are an ecophysiologically important group of strictly anaerobic bacteria that grow lithotrophically on H2 plus CO2 or on CO or heterotrophically on different substrates such as sugars, alcohols, aldehydes, or acids. Amino acids are rarely used. Here, we describe that the model acetogen Acetobacterium woodii can use alanine as the sole carbon and energy source, which is in contrast to the description of the type strain. The alanine degradation genes have been identified and characterized. A key to alanine degradation is an alanine dehydrogenase which has been characterized biochemically. The resulting pyruvate is further degraded to acetate by the known pathways involving the Wood-Ljungdahl pathway. Our studies culminate in a metabolic and bioenergetic scheme for alanine-dependent acetogenesis in A. woodiiIMPORTANCE Peptides and amino acids are widespread in nature, but there are only a few reports that demonstrated use of amino acids as carbon and energy sources by acetogenic bacteria, a central and important group in the anaerobic food web. Our finding that A. woodii can perform alanine oxidation coupled to reduction of carbon dioxide not only increases the number of substrates that can be used by this model acetogen but also raises the possibility that other acetogens may also be able to use alanine. Indeed, the alanine genes are also present in at least two more acetogens, for which growth on alanine has not been reported so far. Alanine may be a promising substrate for industrial fermentations, since acid formation goes along with the production of a base (NH3) and pH regulation is a minor issue.

Regulation of lactate metabolism in the acetogenic bacterium Acetobacterium woodii.

Acetogenic bacteria compete in an energy-limited environment by coupling different metabolic routes to their central metabolism of CO2 fixation. The underlying regulatory mechanisms are often still not understood. In this work, we analysed how lactate metabolism is regulated in the model acetogen Acetobacterium woodii. Construction of a ΔlctCDEF mutant and growth analyses demonstrated that the genes are essential for growth on lactate. Subsequent bridging PCR and quantitative PCR analyses revealed that the lctBCDEF genes form an operon that was expressed only during lactate metabolism. The lctA gene was cloned, expressed in Escherichia coli and purified. LctA bound to the intergenic DNA region between lctA and the lct operon in electromobility shift assays, and binding was revoked in the presence of lactate. Further restriction site protection analyses consolidated the lactate-dependent binding of LctA and identified the binding site within the DNA. Cells grew mixotrophically on lactate and another energy source and showed no diauxic growth. From these data, we conclude that the catabolic lactate metabolism is encoded by the lct operon and its expression is negatively regulated by the DNA-binding repressor LctA.

Glycine betaine metabolism in the acetogenic bacterium Acetobacterium woodii.

The quarternary, trimethylated amine glycine betaine (GB) is widespread in nature but its fate under anoxic conditions remains elusive. It can be used by some acetogenic bacteria as carbon and energy source but the pathway of GB metabolism has not been elucidated. We have identified a gene cluster involved in GB metabolism and studied acetogenesis from GB in the model acetogen Acetobacterium woodii. GB is taken up by a secondary active, Na+ coupled transporter of the betaine-choline-carnitine (BCC) family. GB is demethylated to dimethylglycine, the end product of the reaction, by a methyltransferase system. Further conversion of the methyl group requires CO2 as well as Na+ indicating that GB metabolism involves the Wood-Ljungdahl pathway. These studies culminate in a model for the path of carbon and electrons during acetogenensis from GB and a model for the bioenergetics of acetogenesis from GB.