Florian Mayer

Adaptations of anaerobic archaea to life under extreme energy limitation

Some anaerobic archaea live on substrates that do not allow the synthesis of 1 mol of ATP per mol of substrate. Energy conservation in these cases is only possible by a chemiosmotic mechanism that involves the generation of an electrochemical ion gradient across the cytoplasmatic membrane that then drives ATP synthesis via an A1AO ATP synthase. The minimal amount of energy required is thus depending on the magnitude of the electrochemical ion gradient, the phosphorylation potential, and the ion/ATP ratio of the ATP synthase. Methanogens, Thermococcus, Pyrococcus, and Ignicoccus have evolved different ways to energize their membranes, such as methyltransferases, H+, or NAD+ reducing electron transport systems fueled by reduced ferredoxin or H2 -dependent sulfur reduction that all operate at the thermodynamic limit of life. The structure and function of the enzymes involved are discussed. Despite the differences in membrane energization, they have in common an A1AO ATP synthase that shows an extraordinary divergence in rotor composition and structural adaptations to life under these conditions. In sum, adaptation of anaerobic archaea to energy-limited substrates involves chemiosmotic energy coupling, often with Na+ as coupling ion and a structurally and functionally highly adapted ATP synthase.

ATP synthases from archaea: the beauty of a molecular motor.

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed.

Molecular Convergence of Bacterial and Eukaryotic Surface Order

Background: Living cells maintain a fluid membrane at their surface. Results: Bacteria and eukaryotes display comparable surface order. Transmembrane proteins order cell membranes in the absence of sterol (Bacteria) and disorder in its presence (Eukarya). Conclusion: Bidirectional ordering may provide a means to achieve similar barrier properties despite compositional differences. Significance: Nature may use different protein/lipid combinations to standardize cell surface order. The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.

Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon

Significance We report here that oxidation of formate to CO2 and H2 that operates close to thermodynamic equilibrium is coupled to vectorial H+ and Na+ transport across the cytoplasmic membrane of the hyperthermophilic archaeon Thermococcus onnurineus NA1. The ion gradient established then drives ATP synthesis via a Na+-ATP synthase. The energy-converting enzyme complex involves a formate dehydrogenase, a membrane-bound hydrogenase with similarity to complex I of the aerobic electron transport chain and a multisubunit Na+/H+ antiporter. Thermococcus onnurineus NA1 is known to grow by the anaerobic oxidation of formate to CO2 and H2, a reaction that operates near thermodynamic equilibrium. Here we demonstrate that this reaction is coupled to ATP synthesis by a transmembrane ion current. Formate oxidation leads to H+ translocation across the cytoplasmic membrane that then drives Na+ translocation. The ion-translocating electron transfer system is rather simple, consisting of only a formate dehydrogenase module, a membrane-bound hydrogenase module, and a multisubunit Na+/H+ antiporter module. The electrochemical Na+ gradient established then drives ATP synthesis. These data give a mechanistic explanation for chemiosmotic energy conservation coupled to formate oxidation to CO2 and H2. Because it is discussed that the membrane-bound hydrogenase with the Na+/H+ antiporter module are ancestors of complex I of mitochondrial and bacterial electron transport these data also shed light on the evolution of ion transport in complex I-like electron transport chains.

A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase IMPLICATIONS FOR c RING FUNCTION AND STRUCTURE

Background: The ATP synthase of Pyrococcus has an unusual gene encoding rotor subunit c. Results: The c ring is made of protomers with one ion-binding site in four transmembrane helices and is highly Na+-specific. Conclusion: Unprecedented subunit c topology and ion configuration in an ATP synthase. Significance: Archaeal ATP synthases are a remnant of primordial bioenergetics. The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na+-DCCD competition experiments revealed only one binding site for DCCD and Na+, indicating that the mature c subunit of this A1AO ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na+-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na+-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na+-specific under in vivo conditions, comparable with the Na+-dependent V1VO ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na+-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A1AO ATP synthases.

AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.

MR-guided biopsies with a newly designed cordless coil in an open low-field system: Initial findings

The purpose of this study was to examine the feasibility and safety of MR-guided biopsies with a newly designed cordless coil in an open low-field magnetic resonance (MR) system. Eleven patients were biopsied using a low-field system (0.2 T, Magnetom Concerto, Siemens) by using the new cordless coil (Siemens). The biopsies were performed in different organ systems [liver (n=7), abdomen (n=1), shoulder (n=1), pelvis (n=1) and hip (n=1)]. The procedures were guided using T1-weighted FLASH (fast low-angle shot) sequences. The lesions were biopsied using the coaxial technique through a 15-gauge puncture needle with a 16-gauge biopsy handy. Coil handling, image quality and complications were evaluated. Imaging quality and visualization of the lesions were optimal up to a penetration depth of 9 cm. In all cases the biopsy procedures were successfully performed with MR guidance without any complications. Pathological findings revealed seven cases of malignant tissue and four cases of non-malignant tissue. The use of the cordless coil allows improved patient access during the biopsy and an improved handling of the coil system. MR-guided biopsy using the novel cordless coil system can be performed safely and precisely with easy handling of the coil. This coil concept, however, is restricted to special indications.

Na+ Transport by the A1AO-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes

Background: The ATP synthase of many archaea is predicted to use Na+ as coupling ion. Results: The enzyme from Thermococcus onnurineus reconstituted in proteoliposomes catalyzed ATP-driven Na+ transport. Conclusion: The enzyme uses Na+ as coupling ion. Significance: First direct proof of ion (Na+) transport by a reconstituted A-ATP synthase. The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-ATP synthases use Na+ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A1AO-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A1AO-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na+ transport. This is the first proof of Na+ transport by an A1AO-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na+ in the bioenergetics of archaea.