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 H 2 + CO 2. 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 H 2 + CO 2 using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.
Summary 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.
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.
The F 1 F O -ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F 1 -ATPase and the F 1 F O -ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis.
A 1 A O 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 A 1 A O ATP synthase with a V-type c subunit in a mesophilic organism revealed an A 1 A O 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 UÁmg À1 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 0 -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 A 1 A O ATP synthase into liposomes, ATP-dependent primary transport of 22 Na + as well as DµNa + -driven ATP synthesis could be demonstrated. The Na + A 1 A O 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. AbbreviationsDµNa + , electrochemical sodium ion potential; DpNa, sodium ion potential; DΨ, membrane potential; DCCD, N,N 0 -dicyclohexylcarbodiimide; DDM, n-dodecyl-b-maltoside; ETH 2120, N,N,N,N 0 -tetra-cyclo-hexyl-1,2-phenylenedioxydiacetamide; MALDI, matrix-assisted laser desorption/ ionization; TCS, 3,3 0 ,4 0 ,5-tetrachlorosalicylanilide.
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