GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

IMPORTANCE: New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

Structural Elements Involved in ATP Hydrolysis Inhibition and ATP Synthesis of Tuberculosis and Nontuberculous Mycobacterial F-ATP Synthase Decipher New Targets for Inhibitors.

The F1FO-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 F1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (αCTD) of subunit α is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.

Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii / Biosíntesis de butirato a partir de metanol y monóxido de carbono por Acetobacterium woodii recombinante

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.(AU)

ATP synthesis in an ancient ATP synthase at low driving forces.

Hyperthermophilic archaea are close to the origin of life. Some hyperthermophilic anaerobic archaea live under strong energy limitation and have to make a living near thermodynamic equilibrium. Obviously, this requires adaptations of the energy-conserving machinery to harness small energy increments. Their ATP synthases often have an unusual motor subunit c that is predicted to prevent ATP synthesis. We have purified and reconstituted into liposomes such an archaeal ATP synthase found in a mesophilic bacterium. The enzyme indeed synthesized ATP at physiological membrane potentials, despite its unusual c subunit, but the minimal driving force for ATP synthesis was found to be even lower than in ATP synthases with usual c subunits. These data not only reveal an intermediate in the transition from ATP hydrolases to ATP synthases but also give a rationale for a bioenergetic adaptation of microbial growth near the thermodynamic equilibrium.

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.

Mutational Analysis of Mycobacterial F-ATP Synthase Subunit δ Leads to a Potent δ Enzyme Inhibitor.

While many bacteria are able to bypass the requirement for oxidative phosphorylation when grown on carbohydrates, Mycobacterium tuberculosis is unable to do so. Differences of amino acid composition and structural features of the mycobacterial F-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) compared to its prokaryotic or human counterparts were recently elucidated and paved avenues for the discovery of molecules interfering with various regulative mechanisms of this essential energy converter. In this context, the mycobacterial peripheral stalk subunit δ came into focus, which displays a unique N-terminal 111-amino acid extension. Here, mutants of recombinant mycobacterial subunit δ were characterized, revealing significant reduction in ATP synthesis and demonstrating essentiality of this subunit for effective catalysis. These results provided the basis for the generation of a four-feature model forming a δ receptor-based pharmacophore and to identify a potent subunit δ inhibitor DeMF1 via in silico screening. The successful targeting of the δ subunit demonstrates the potential to advance δ's flexible coupling as a new area for the development of F-ATP synthase inhibitors.

Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ.

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:ß3: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.

Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source.

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 H2 + CO2 . 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 H2 + CO2 using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.

Functional production of an archaeal ATP synthase with a V-type c subunit in Escherichia coli.

ATP synthases are the key elements of cellular bioenergetics and present in any life form and the overall structure and function of this rotary energy converter is conserved in all domains of life. However, ancestral microbes, the archaea, have a unique and huge diversity in the size and number of ion-binding sites in their membrane-embedded rotor subunit c. Due to the harsh conditions for ATP synthesis in these life forms it has never been possible to address the consequences of these unusual c subunits for ATP synthesis. Recently, we have found a Na+-dependent archaeal ATP synthase with a V-type c subunit in a mesophilic bacterium and here, we have cloned and expressed the genes in the ATP synthase-negative strain Escherichia coli DK8. The enzyme was present in membranes of E. coli DK8 and catalyzed ATP hydrolysis with a rate of 35 nmol·min-1·mg protein-1. Inverted membrane vesicles of this strain were then checked for their ability to synthesize ATP. Indeed, ATP was synthesized driven by NADH oxidation despite the V-type c subunit. ATP synthesis was dependent on Na+ and inhibited by ionophores. Most importantly, ATPase activity was inhibited by DCCD and this inhibition was relieved by addition of Na+, indicating a functional coupling of the F1 and FO domains, a prerequisite for studies on structure-function relationship. A first step in this direction was the exchange of a conserved arginine (Arg530) in the FO motor subunit a which led to loss of ATP synthesis whereas ATP hydrolysis was retained.

A Na+ A1 AO ATP synthase with a V-type c subunit in a mesophilic bacterium.

A1 AO 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 A1 AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1 AO 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 A1 AO ATP synthase into liposomes, ATP-dependent primary transport of 22 Na+ as well as ΔµNa+ -driven ATP synthesis could be demonstrated. The Na+ A1 AO 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.

Enantiomer discrimination in ß-phenylalanine degradation by a newly isolated Paraburkholderia strain BS115 and type strain PsJN.

Despite their key role in numerous natural compounds, ß-amino acids have rarely been studied as substrates for microbial degradation. Fermentation of the newly isolated Paraburkholderia strain BS115 and the type strain P. phytofirmans PsJN with ß-phenylalanine (ß-PA) as sole nitrogen source revealed (S)-selective transamination of ß-PA to the corresponding ß-keto acid by both strains, accompanied by substantial formation of acetophenone (AP) from spontaneous decarboxylation of the emerging ß-keto acid. While the PsJN culture became stationary after entire (S)-ß-PA consumption, BS115 showed further growth at a considerably slower rate, consuming (R)-ß-PA without generation of AP which points to a different degradation mechanism for this enantiomer. This is the first report on degradation of both enantiomers of any ß-amino acid by one single bacterial strain.