Acetogenic Bacterium Acetobacterium Woodii Research Articles

Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria

BackgroundLignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock for producing chemicals by microbial cell factories. Because of the heterogeneous nature of the lignin feedstocks, the biological upgrading of lignin relying on the metabolic routes of aerobic bacteria is currently considered as the most promising approach. However, the limited substrate range and the inefficient catabolism of the production hosts hinder the upgrading of lignin-related aromatics. Particularly, the aerobic O-demethylation of the methoxyl groups in aromatic substrates is energy-limited, inhibits growth, and results in carbon loss in the form of CO2.ResultsIn this study, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic O-demethylation; in the process, the carbon from the methoxyl groups is fixed together with CO2 to form acetate, while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. By utilizing this cocultivation approach, we demonstrated an upgrading of guaiacol, an abundant but inaccessible substrate to most microbes, into a plastic precursor muconate, with a nearly equimolar yields (0.9 mol/mol in a small-scale cultivation and 1.0 mol/mol in a one-pot bioreactor cultivation). The process required only a minor genetic engineering, namely a single gene knock-out. Noticeably, by employing a metabolic integration of the two bacteria, it was possible to produce biomass and muconate by utilizing only CO2 and guaiacol as carbon sources.ConclusionsBy the novel approach, we were able to overcome the issues related to aerobic O-demethylation of methoxylated aromatic substrates and demonstrated carbon-wise conversion of lignin-related aromatics to products with yields unattainable by aerobic processes. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

Integrated biorefinery strategy for poly(3-hydroxybutyrate) accumulation in Cupriavidus necator DSM 545 using a sugar rich syrup from cereal waste and acetate from gas fermentation

Poly(3-hydroxybutyrate) (PHB) is one of the most well-known biodegradable and biocompatible biopolymers produced by prokaryotic microorganisms. It belongs to the family of polyhydroxyalkanoates (PHAs), and it has gained significant attention in recent years due to its potential as a sustainable alternative to traditional petroleum-based plastics. Cupriavidus necator has been identified as a potential producer of PHB for industrial applications due to its ability to produce high amounts of the polymer under controlled conditions, using a wide range of waste substrates. In this study, the ability of Cupriavidus necator DSM 545 strain to produce PHB was tested in a fed-batch strategy providing two different organic substrates. The first is a sugar-based syrup (SBS), derived from cereal waste. The second is an acetate-rich medium obtained through CO2 -H2 fermentation by the acetogenic bacterium Acetobacterium woodii. The carbon sources were tested to improve the accumulation of PHB in the strain. C. necator DSM 545 proved to be able to grow and to perform high accumulation of biopolymer on waste substrates containing glucose, fructose, and acetate, reaching about 10 g/L of PHB, 83% of biopolymer accumulation in cell dry mass, in 48 h of fed-batch fermentation in 0.6 L working volume in a bioreactor. Moreover, a Life Cycle Assessment analysis was performed to evaluate the environmental impact of the process converting the sugar syrup alone and the integrated one. It demonstrated that the integrated process is more sustainable and that the most impactful step is the PHB production, followed by the polymer extraction.

A new metabolic trait in an acetogen: Mixed acid fermentation of fructose in a methylene-tetrahydrofolate reductase mutant of Acetobacterium woodii.

To inactivate the Wood-Ljungdahl pathway in the acetogenic model bacterium Acetobacterium woodii, the genes metVF encoding two of the subunits of the methylene-tetrahydrofolate reductase were deleted. As expected, the mutant did not grow on C1 compounds and also not on lactate, ethanol or butanediol. In contrast to a mutant in which the first enzyme of the pathway (hydrogen-dependent CO2 reductase) had been genetically deleted, cells were able to grow on fructose, albeit with lower rates and yields than the wild-type. Growth was restored by addition of an external electron sink, glycine betaine + CO2 or caffeate. Resting cells pre-grown on fructose plus an external electron acceptor fermented fructose to two acetate and four hydrogen, that is, performed hydrogenogenesis. Cells pre-grown under fermentative conditions on fructose alone redirected carbon and electrons to form lactate, formate, ethanol as well as hydrogen. Apparently, growth on fructose alone induced enzymes for mixed acid fermentation (MAF). Transcriptome analyses revealed enzymes potentially involved in MAF and a quantitative model for MAF from fructose in A. woodii is presented.

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.

Exploring Bacterial Microcompartments in the Acetogenic Bacterium Acetobacterium woodii.

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.

The Rnf complex from the acetogenic bacterium Acetobacterium woodii: Purification and characterization of RnfC and RnfB

rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na+, H+) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex.

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.

Efficient whole cell biocatalyst for formate-based hydrogen production

BackgroundMolecular 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.ResultsWe 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.ConclusionsAcetobacterium 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.

Acetone production with metabolically engineered strains of Acetobacterium woodii

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_actthlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters PthlA and Ppta-ack. Batch fermentations using A. woodii [pMTL84151_actthlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high Km value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_actthlA] was increased from 1.2mgL−1h−1 in bottle fermentation to 26.4mgL−1h−1 in continuous gas fermentation.

Occurrence of ferredoxin:NAD(+) oxidoreductase activity and its ion specificity in several Gram-positive and Gram-negative bacteria.

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.

A novel route for ethanol oxidation in the acetogenic bacterium Acetobacterium woodii: the acetaldehyde/ethanol dehydrogenase pathway.

Ethanol is a common substrate for anaerobic microorganisms despite its high redox potential (E0' etha- nol/acetaldehyde = -190mV), which does not allow for NAD(+) reduction. How this thermodynamic barrier is overcome is largely unknown. The acetogenic bacterium Acetobacterium woodii can also grow on ethanol. The genome harbours 11 genes encoding putative alcohol dehydrogenases, but only one, adhE, was upregulated during growth on ethanol. The bifunctional acetaldehyde/ethanol dehydrogenase (AdhE) was purified from ethanol-grown cells. It catalysed the NAD(+) - and CoA-dependent oxidation of ethanol via acetaldehyde to acetyl-CoA. The enzyme was regulated by free coenzyme A: in the absence of coenzyme A, the Km value for ethanol was shifted from 3.4 to 40 mM. The alcohol dehydrogenase domain could also oxidize 1-propanol and 1-butanol; however, the aldehyde dehydrogenase domain was highly specific for acetaldehyde as substrate. Apparently, the bifunctional AdhE allows for NAD(+) reduction by lowering the concentration of acetaldehyde, which makes the first oxidation reaction thermodynamically feasible.

A low phosphorylation potential in the acetogen Acetobacterium woodii reflects its lifestyle at the thermodynamic edge of life.

The anaerobic, acetogenic bacterium Acetobacterium woodii grows on hydrogen and carbon dioxide and uses the Wood-Ljungdahl pathway to fix carbon but also to synthesize ATP. The free energy change of acetogenesis from H2+CO2 allows for synthesis of only a fraction of an ATP under environmental conditions, and A. woodii is clearly a paradigm for microbial life under extreme energy limitation. However, it was unknown how much energy is required to make ATP under these conditions. In the present study, we determined the phosphorylation potential in cells metabolizing three different acetogenic substrates. It accounts to 37.9±1.3kJ/mol ATP during acetogenesis from fructose, 32.1±0.3kJ/mol ATP during acetogenesis from H2+CO2 and 30.2±0.9kJ/mol ATP during acetogenesis from CO, the lowest phosphorylation potential ever described. The physiological consequences in terms of energy conservation under extreme energy limitation are discussed.

A novel mode of lactate metabolism in strictly anaerobic bacteria.

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0 ' = -190 mV excludes direct NAD(+) reduction (E0 ' = -320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron-transferring flavoprotein (Etf) that exhibited NAD(+) reduction only when reduced ferredoxin (Fd(2-) ) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD(+) as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0 ' ≈ -500 mV) to NAD(+) according to: lactate + Fd(2-) + 2 NAD(+) → pyruvate + Fd + 2 NADH. The reduced Fd(2-) is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical ( Δ μ ˜ Na + ) and finally redox energy (Fd(2-) from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.

The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na(+)-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD(+) with the generation of a primary electrochemical Na(+) potential across its cytoplasmic membrane. In previous assays in which Ti(3+) was used to reduce ferredoxin, Na(+) transport was observed, but not a Na(+) dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD(+) reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na(+) dependence of this electron transfer reaction. The Km was 0.2 mm. Na(+) could be partly substituted by Li(+). Na(+) dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na(+) as a coupling ion. Electron transport from reduced ferredoxin to NAD(+) was coupled to electrogenic Na(+) transport, indicating the generation of ΔμNa(+). Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of ΔμNa(+), and was accompanied by Na(+) efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na(+) gradient. The physiological significance of this finding is discussed.

An Electron-bifurcating Caffeyl-CoA Reductase

A low potential electron carrier ferredoxin (E0' ≈ -500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD(+) oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA.

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) hybrid rotor that contains nine copies of a F(O)-like c subunit and one copy of a V(O)-like c(1) subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its β subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na(+) F(1)F(O) ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.

A Bacterial Electron-bifurcating Hydrogenase

The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.

An Ancient Pathway Combining Carbon Dioxide Fixation with the Generation and Utilization of a Sodium Ion Gradient for ATP Synthesis

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.

A Na+-translocating Pyrophosphatase in the Acetogenic Bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii.

An intermediate step in the evolution of ATPases – a hybrid F0–V0 rotor in a bacterial Na+ F1F0 ATP synthase

The Na(+) F(1)F(0) ATP synthase operon of the anaerobic, acetogenic bacterium Acetobacterium woodii is unique because it encodes two types of c subunits, two identical 8 kDa bacterial F(0)-like c subunits (c(2) and c(3)), with two transmembrane helices, and a 18 kDa eukaryal V(0)-like (c(1)) c subunit, with four transmembrane helices but only one binding site. To determine whether both types of rotor subunits are present in the same c ring, we have isolated and studied the composition of the c ring. High-resolution atomic force microscopy of 2D crystals revealed 11 domains, each corresponding to two transmembrane helices. A projection map derived from electron micrographs, calculated to 5 A resolution, revealed that each c ring contains two concentric, slightly staggered, packed rings, each composed of 11 densities, representing 22 transmembrane helices. The inner and outer diameters of the rings, measured at the density borders, are approximately 17 and 50 A. Mass determination by laser-induced liquid beam ion desorption provided evidence that the c rings contain both types of c subunits. The stoichiometry for c(2)/c(3) : c(1) was 9 : 1. Furthermore, this stoichiometry was independent of the carbon source of the growth medium. These analyses clearly demonstrate, for the first time, an F(0)-V(0) hybrid motor in an ATP synthase.