Refine
Year of publication
Has Fulltext
- yes (63)
Is part of the Bibliography
- no (63)
Keywords
- Acetogenesis (4)
- Acetogen (3)
- Bioenergetics (3)
- Biohydrogen (3)
- Hydrogenase (3)
- Thermoanaerobacter kivui (3)
- Wood-Ljungdahl pathway (3)
- Wood–Ljungdahl pathway (3)
- compatible solutes (3)
- hydrogen storage (3)
Institute
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.
The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+, in osmostress response. Growth of A. baumannii was K+-dependent and the K+-dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+. After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.
Species of the genus Blautia are typical inhabitants of the human gut and considered as beneficial gut microbes. However, their role in the gut microbiome and their metabolic features are poorly understood. Blautia schinkii was described as an acetogenic bacterium, characterized by a functional Wood–Ljungdahl pathway (WLP) of acetogenesis from H2 + CO2. Here we report that two relatives, Blautia luti and Blautia wexlerae do not grow on H2 + CO2. Inspection of the genome sequence revealed all genes of the WLP except genes encoding a formate dehydrogenase and an electron-bifurcating hydrogenase. Enzyme assays confirmed this prediction. Accordingly, resting cells neither converted H2 + CO2 nor H2 + HCOOH + CO2 to acetate. Carbon monoxide is an intermediate of the WLP and substrate for many acetogens. Blautia luti and B. wexlerae had an active CO dehydrogenase and resting cells performed acetogenesis from HCOOH + CO2 + CO, demonstrating a functional WLP. Bioinformatic analyses revealed that many Blautia strains as well as other gut acetogens lack formate dehydrogenases and hydrogenases. Thus, the use of formate instead of H2 + CO2 as an interspecies hydrogen and electron carrier seems to be more common in the gut microbiome.
BACKGROUND: Acetogenic bacteria are able to use CO2 as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H2. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H2+CO2 and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO2 to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD+ with the export of Na+ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui.
RESULTS: The genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G+C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H2+CO2 was dependent on Na+ and acetate formation was inhibited by a protonophore, indicating that H+ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F1FO ATP synthase does not have the conserved Na+ binding motif. A search for potential H+-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech).
CONCLUSIONS: The thermophilic acetogen T. kivui does not use Na+ but H+ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H2+CO2 make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.
A1AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg−1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+)-binding site. Indeed, ATP hydrolysis was strictly Na+-dependent. N,N′-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+. Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1AO ATP synthase into liposomes, ATP-dependent primary transport of 22Na+ as well as ΔµNa+-driven ATP synthesis could be demonstrated. The Na+ A1AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.
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 Mg2+. In addition, activity was strictly dependent on Na+ with a Km of 1.1 mm. Hydrolysis of pyrophosphate was accompanied by 22Na+ transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that 22Na+ 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.
Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N′-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.
Acetobacterium woodii utilizes the Wood‐Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non‐acetogenic growth on 1,2‐propanediol (1,2‐PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2‐PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC‐encapsulated propanol‐generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH‐dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2‐PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.
Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.
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.