The acetogenic bacterium Acetobacterium woodii is able to grow by the oxidation of diols, such as 1,2-propanediol, 2,3-butanediol, or ethylene glycol. Recent analyses demonstrated fundamentally different ways for oxidation of 1,2-propanediol and 2,3-butanediol. Here, we analyzed the metabolism of ethylene glycol. Our data demonstrate that ethylene glycol is dehydrated to acetaldehyde, which is then disproportionated to ethanol and acetyl coenzyme A (acetyl-CoA). The latter is further converted to acetate, and this pathway is coupled to ATP formation by substrate-level phosphorylation. Apparently, the product ethanol is in part further oxidized and the reducing equivalents are recycled by reduction of CO 2 to acetate in the Wood-Ljungdahl pathway. Biochemical data as well as the results of protein synthesis analysis are consistent with the hypothesis that the propane diol dehydratase (PduCDE) and CoA-dependent propionaldehyde dehydrogenase (PduP) proteins, encoded by the pdu gene cluster, also catalyze ethylene glycol dehydration to acetaldehyde and its CoA-dependent oxidation to acetyl-CoA. Moreover, genes encoding bacterial microcompartments as part of the pdu gene cluster are also expressed during growth on ethylene glycol, arguing for a dual function of the Pdu microcompartment system. IMPORTANCEAcetogenic bacteria are characterized by their ability to use CO 2 as a terminal electron acceptor by a specific pathway, the WoodLjungdahl pathway, enabling in most acetogens chemolithoautotrophic growth with H 2 and CO 2 . However, acetogens are very versatile and can use a wide variety of different substrates for growth. Here we report on the elucidation of the pathway for utilization of ethylene glycol by the model acetogen Acetobacterium woodii. This diol is degraded by dehydration to acetaldehyde followed by a disproportionation to acetate and ethanol. We present evidence that this pathway is catalyzed by the same enzyme system recently described for the utilization of 1,2-propanediol. The enzymes for ethylene glycol utilization seem to be encapsulated in protein compartments, known as bacterial microcompartments. In the absence of other energetically more favorable electron acceptors, CO 2 is the only electron acceptor used for energy conservation in anoxic ecosystems. Only two groups of organisms are able to utilize CO 2 as a terminal electron acceptor, namely, methanogenic archaea and acetogenic bacteria. Acetogenic bacteria are a specialized group of strictly anaerobic bacteria that, in most cases, can use molecular hydrogen as an electron donor for CO 2 reduction and can thus grow autotrophically with H 2 and CO 2 . Therefore, they reduce two molecules of CO 2 to acetyl coenzyme A (acetyl-CoA) via the reductive acetyl-CoA pathway (the WoodLjungdahl pathway [WLP]) (1-3). This pathway of CO 2 fixation also provides energy for growth by a chemiosmotic mechanism: an electrochemical ion gradient is established in the course of CO 2 reduction (4-6) and can be used by an ATP synthase to drive the phosphory...
rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na + pump. These studies provide the proof that the Rnf complex is indeed an ion (Na + ) translocating, respiratory enzyme. Together with a Na + -F 1 F O ATP synthase it builds a simple, two-limb respiratory chain in T. maritima . The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.
The acetogenic bacterium Acetobacterium woodii is able to reduce CO 2 to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD ؉ -dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO 2 to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.
The Na+translocating F1FO ATP synthase from Acetobacterium woodii shows a subunit stoichiometry of α3:β3:γ:δ:ε:a:b2:(c2/3)9:c1 and reveals an evolutionary path between synthases and pumps involving adaptations in the rotor c‐ring, which is composed of F‐ and vacuolar‐type c subunits in a stoichiometry of 9 : 1. This hybrid turbine couples rotation with Na+ translocation in the FO part and rotation of the central stalk subunits γ‐ε to drive ATP synthesis in the catalytic α3:β3 headpiece. Here, we isolated a highly pure recombinant A. woodii F‐ATP synthase and present the first projected structure of this hybrid engine as determined by negative‐stain electron microscopy and single‐particle analysis. The uniqueness of the A. woodii F‐ATP synthase is also reflected by an extra 17 amino acid residues loop (195TSGKVKITEETKEEKSK211) in subunit γ. Deleting the loop‐encoding DNA sequence (γΔ195–211) and purifying the recombinant F‐ATP synthase γΔ195–211 mutant provided a platform to study its effect in enzyme stability and activity. The recombinant F‐ATP synthase γΔ195–211 mutant revealed the same subunit composition as the wild‐type enzyme and a minor reduction in ATP hydrolysis. When reconstituted into proteoliposomes ATP synthesis and Na+ transport were diminished, demonstrating the importance of the γ195–211 loop in both enzymatic processes. Based on a structural model, a coupling mechanism for this enzyme is proposed, highlighting the role of the γ‐loop. Finally, the γ195–211 loop of A. woodii is discussed in comparison with the extra γ‐loops of mycobacterial and chloroplasts F‐ATP synthases described to be involved in species‐specific regulatory mechanisms.
Summary More than 2 million tons of glycerol are produced during industrial processes each year and, therefore, glycerol is an inexpensive feedstock to produce biocommodities by bacterial fermentation. Acetogenic bacteria are interesting production platforms and there have been few reports in the literature on glycerol utilization by this ecophysiologically important group of strictly anaerobic bacteria. Here, we show that the model acetogen Acetobacterium woodii DSM1030 is able to grow on glycerol, but contrary to expectations, only for 2–3 transfers. Transcriptome analysis revealed the expression of the pdu operon encoding a propanediol dehydratase along with genes encoding bacterial microcompartments. Deletion of pduAB led to a stable growth of A. woodii on glycerol, consistent with the hypothesis that the propanediol dehydratase also acts on glycerol leading to a toxic end‐product. Glycerol is oxidized to acetate and the reducing equivalents are reoxidized by reducing CO2 in the Wood–Ljungdahl pathway, leading to an additional acetate. The possible oxidation product of glycerol, dihydroxyacetone (DHA), also served as carbon and energy source for A. woodii and growth was stably maintained on that compound. DHA oxidation was also coupled to CO2 reduction. Based on transcriptome data and enzymatic analysis we present the first metabolic and bioenergetic schemes for glycerol and DHA utilization in A. woodii.
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