Structural basis for sequence-specific recognition of guide and target strands by the Archaeoglobus fulgidus Argonaute protein.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like 'long' and truncated 'short' pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a truncated long-B Argonaute protein encoded by an archaeon Archaeoglobus fulgidus. We demonstrate that AfAgo associates with small RNA molecules carrying 5'-terminal AUU nucleotides in vivo, and characterize its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.

Self-assembling ferritin-dendrimer nanoparticles for targeted delivery of nucleic acids to myeloid leukemia cells.

BACKGROUND: In recent years, the use of ferritins as nano-vehicles for drug delivery is taking center stage. Compared to other similar nanocarriers, Archaeoglobus fulgidus ferritin is particularly interesting due to its unique ability to assemble-disassemble under very mild conditions. Recently this ferritin was engineered to get a chimeric protein targeted to human CD71 receptor, typically overexpressed in cancer cells. RESULTS: Archaeoglobus fulgidus chimeric ferritin was used to generate a self-assembling hybrid nanoparticle hosting an aminic dendrimer together with a small nucleic acid. The positively charged dendrimer can indeed establish electrostatic interactions with the chimeric ferritin internal surface, allowing the formation of a protein-dendrimer binary system. The 4 large triangular openings on the ferritin shell represent a gate for negatively charged small RNAs, which access the internal cavity attracted by the dense positive charge of the dendrimer. This ternary protein-dendrimer-RNA system is efficiently uptaken by acute myeloid leukemia cells, typically difficult to transfect. As a proof of concept, we used a microRNA whose cellular delivery and induced phenotypic effects can be easily detected. In this article we have demonstrated that this hybrid nanoparticle successfully delivers a pre-miRNA to leukemia cells. Once delivered, the nucleic acid is released into the cytosol and processed to mature miRNA, thus eliciting phenotypic effects and morphological changes similar to the initial stages of granulocyte differentiation. CONCLUSION: The results here presented pave the way for the design of a new family of protein-based transfecting agents that can specifically target a wide range of diseased cells.

Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal ß-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

DNA and RNA editing without sequence limitation using the flap endonuclease 1 guided by hairpin DNA probes.

Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage.

Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.

Bacteria protect themselves from infections using a system called CRISPR-Cas, which helps the cells to detect and destroy invading threats. The type III CRISPR-Cas system, in particular, is one of the most widespread and efficient at killing viruses. When a bacterium is infected, the CRISPR-Cas system takes a fragment of the genetic material of the virus, and copies it into a molecule. These molecular 'police mugshots' are then loaded into a complex of Cas proteins that patrol the cell, looking for a match and destroying any virus that can be identified. Some Cas proteins also produce alarm signals, called cyclic oligoadenylates (cOAs), which can trigger additional defences. However, this process can damage the genetic material of the bacterium, harming or even killing the cell. Enzymes known as ring nucleases can promptly degrade cOAs and turn off this defence system before it causes harm. However, ring nucleases have only been found in a few species to date; how most bacteria deal with cOA toxicity has remained unknown. Here, Athukoralage et al. set out to determine whether a widespread enzyme known as Csx3, which is often associated with type III CRISPR-Cas systems, could be an alternative off switch for cOA triggered defences. Initial 'test tube' experiments with purified Csx3 proteins confirmed that the enzyme could indeed break down cOAs. A careful dissection of Csx3's molecular structure, using biochemical and biophysical techniques, revealed that it worked by 'sandwiching' a cOA molecule between two co-operating portions of the enzyme. As a final test, Csx3 was introduced into strains of bacteria genetically engineered to have a fully functional Type III CRISPR-Cas system. In these cells, Csx3 successfully turned off the Type III immune response. These results reveal a new way that bacteria avoid the toxic side effects of their own immune defences. Ultimately, this could pave the way for the development of anti-bacterial drugs that work by blocking Csx3 or similar proteins.

Experimental characterization of two archaeal inosine 5'-monophosphate cyclohydrolases.

There is variability as to how archaea catalyze the final step of de novo purine biosynthesis to form inosine 5'-monophosphate (IMP) from 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR). Although non-archaea almost uniformly use the bifunctional PurH protein, which has an N-terminal IMP cyclohydrolase (PurH2) fused to a C-terminal folate-dependent aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (PurH1) domain, a survey of the genomes of archaea reveals use of PurH2 (with or without fusion to PurH1), the "euryarchaeal signature protein" PurO, or an unidentified crenarchaeal IMP cyclohydrolase. In this report, we present the cloning and functional characterization of two representatives of the known IMP cyclohydrolase families. The locus TK0430 in Thermococcus kodakarensis encodes a PurO-type IMP cyclohydrolase with demonstrated activity despite its position in a cluster of apparently redundant biosynthetic genes, the first functional characterization of a PurO from a non-methanogen. Kinetic characterization reveals a Km for FAICAR of 1.56 ± 0.39 µM and a kcat of 0.48 ± 0.04 s-1. The locus AF1811 from Archaeoglobus fulgidus encodes a PurH2-type IMP cyclohydrolase. This Archaeoglobus fulgidus PurH2 has a Km of 7.8 ± 1.8 µM and kcat of 1.32 ± 0.14 s-1, representing the first characterization of an archaeal PurH2 and the first characterization of PurH2 that naturally occurs unfused to an AICAR formyltransferase domain. Each of these two characterized IMP cyclohydrolases converts FAICAR to IMP in vitro, and each cloned gene allows the growth on purine-deficient media of an E. coli purine auxotroph lacking the purH2 gene.

Application of crossflow ultrafiltration for scaling up the purification of a recombinant ferritin.

Ferritin proteins are taking center stage as smart nanocarriers for drug delivery due to their hollow cage-like structures and their unique 24-meric assembly. Among all ferritins, the chimeric Archaeoglobus ferritin (HumFt) is able assemble/disassemble varying the ionic strength of the medium while recognizing human TfR1 receptor overexpressed in cancer cells. In this paper we present a highly efficient, large scale purification protocol mainly based on crossflow ultrafiltration, starting from fermented bacterial paste. This procedure allows one to obtain about 2 g of purified protein starting from 100 g of fermented bacterial paste. The current procedure can easily remove contaminant proteins as well as DNA molecules in the absence of expensive and time consuming chromatographic steps.

The Different Effects of Substrates and Nucleotides on the Complex Formation of ABC Transporters.

The molybdate importer (ModBC-A of Archaeoglobus fulgidus) and the vitamin B12 importer (BtuCD-F of Escherichia coli) are members of the type I and type II ABC importer families. Here we study the influence of substrate and nucleotide binding on complex formation and stability. Using native mass spectrometry we show that the interaction between the periplasmic substrate-binding protein (SBP) ModA and the transporter ModBC is dependent upon binding of molybdate. By contrast, vitamin B12 disrupts interactions between the transporter BtuCD and the SBP BtuF. Moreover, while ATP binds cooperatively to BtuCD-F, and acts synergistically with vitamin B12 to destabilize the BtuCD-F complex, no effect is observed for ATP binding on the stability of ModBC-A. These observations not only highlight the ability of mass spectrometry to capture these importer-SBP complexes but allow us to add molecular detail to proposed transport mechanisms.

The role of RNA structure in translational regulation by L7Ae protein in archaea.

A recent study has shown that archaeal L7Ae binds to a putative k-turn structure in the 5'-leader of the mRNA of its structural gene to regulate translation. To function as a regulator, the RNA should be unstructured in the absence of protein, but it should adopt a k-turn-containing stem-loop on binding L7Ae. Sequence analysis of UTR sequences indicates that their k-turn elements will be unable to fold in the absence of L7Ae, and we have demonstrated this experimentally in solution using FRET for the Archaeoglobus fulgidus sequence. We have solved the X-ray crystal structure of the complex of the A. fulgidus RNA bound to its cognate L7Ae protein. The RNA adopts a standard k-turn conformation that is specifically recognized by the L7Ae protein, so stabilizing the stem-loop. In-line probing of the natural-sequence UTR shows that the RNA is unstructured in the absence of L7Ae binding, but folds on binding the protein such that the ribosome binding site is occluded. Thus, L7Ae regulates its own translation by switching the conformation of the RNA to alter accessibility.

Efficient molecular evolution to generate enantioselective enzymes using a dual-channel microfluidic droplet screening platform.

Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~107 enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.

Thermostable exoshells fold and stabilize recombinant proteins.

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.

Sequence determinants of the folding properties of box C/D kink-turns in RNA.

Folding properties differ markedly between kink-turns (k-turns) that have different biological functions. While ribosomal and riboswitch k-turns generally fold into their kinked conformation on addition of metal ions, box C/D snoRNP k-turns remain completely unfolded under these conditions, although they fold on addition of L7Ae protein. Sequence elements have been systematically exchanged between a standard ribosomal k-turn (Kt-7) that folds on addition of metal ions, and a box C/D k-turn. Folding was studied using fluorescence resonance energy transfer and gel electrophoresis. Three sequence elements each contribute in an approximately additive manner to the different folding properties of Kt-7 and box C/D k-turns from archaea. Bioinformatic analysis indicates that k-turn sequences evolve sequences that suit their folding properties to their biological function. The majority of ribosomal and riboswitch k-turns have sequences allowing unassisted folding in response to the presence of metal ions. In contrast, box C/D k-turns have sequences that require the binding of proteins to drive folding into the kinked conformation, consistent with their role in the assembly of the box C/D snoRNP apparatus. The rules governing the influence of sequence on folding properties can be applied to other standard k-turns to predict their folding characteristics.

Thermophilic Ferritin 24mer Assembly and Nanoparticle Encapsulation Modulated by Interdimer Electrostatic Repulsion.

Protein cage self-assembly enables encapsulation and sequestration of small molecules, macromolecules, and nanomaterials for many applications in bionanotechnology. Notably, wild-type thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) exists as a stable dimer of four-helix bundle proteins at a low ionic strength, and the protein forms a hollow assembly of 24 protomers at a high ionic strength (∼800 mM NaCl). This assembly process can also be initiated by highly charged gold nanoparticles (AuNPs) in solution, leading to encapsulation. These data suggest that salt solutions or charged AuNPs can shield unfavorable electrostatic interactions at AfFtn dimer-dimer interfaces, but specific "hot-spot" residues controlling assembly have not been identified. To investigate this further, we computationally designed three AfFtn mutants (E65R, D138K, and A127R) that introduce a single positive charge at sites along the dimer-dimer interface. These proteins exhibited different assembly kinetics and thermodynamics, which were ranked in order of increasing 24mer propensity: A127R < wild type < D138K ≪ E65R. E65R assembled into the 24mer across a wide range of ionic strengths (0-800 mM NaCl), and the dissociation temperature for the 24mer was 98 °C. X-ray crystal structure analysis of the E65R mutant identified a more compact, closed-pore cage geometry. A127R and D138K mutants exhibited wild-type ability to encapsulate and stabilize 5 nm AuNPs, whereas E65R did not encapsulate AuNPs at the same high yields. This work illustrates designed protein cages with distinct assembly and encapsulation properties.

Tethering an N-Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex.

Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where X is not proline. A sequon was tethered to an archaeal OST enzyme via a disulfide bond. The positions of the cysteine residues in the OST protein and the sequon-containing acceptor peptide were selected by reference to the eubacterial OST structure in a noncovalent complex with an acceptor peptide. We determined the crystal structure of the cross-linked OST-sequon complex. The Ser/Thr-binding pocket recognizes the Thr residue in the sequon, and the catalytic structure termed the "carboxylate dyad" interacted with the Asn residue. Thus, the recognition and the catalytic mechanism of the sequon are conserved between the archaeal and eubacterial OSTs. We found that the tethered peptides in the complex were efficiently glycosylated in the presence of the oligosaccharide donor. The stringent requirements are greatly relaxed in the cross-linked state. The two conserved acidic residues in the catalytic structure were each dispensable, although the double mutation abolished the activity. A Gln residue at the Asn position in the sequon functioned as an acceptor, and the hydroxy group at position +2 was not required. In the standard assay using short free peptides, strong amino acid preferences were observed at the X position, but the preferences, except for Pro, completely disappeared in the cross-linked state. By skipping the initial binding process and stabilizing the complex state, the catalytically competent cross-linked complex offers a unique system for studying the oligosaccharyl transfer reaction.

Probing bulky ligand entry in engineered archaeal ferritins.

BACKGROUND: A set of engineered ferritin mutants from Archaeoglobus fulgidus (Af-Ft) and Pyrococcus furiosus (Pf-Ft) bearing cysteine thiols in selected topological positions inside or outside the ferritin shell have been obtained. The two apo-proteins were taken as model systems for ferritin internal cavity accessibility in that Af-Ft is characterized by the presence of a 45Å wide aperture on the protein surface whereas Pf-Ft displays canonical (threefold) channels. METHODS: Thiol reactivity has been probed in kinetic experiments in order to assess the protein matrix permeation properties towards the bulky thiol reactive DTNB (5,5'-dithiobis-2-nitrobenzoic acid) molecule. RESULTS: Reaction of DTNB with thiols was observed in all ferritin mutants, including those bearing free cysteine thiols inside the ferritin cavity. As expected, a ferritin mutant from Pf-Ft, in which the cysteine thiol is on the outer surface displays the fastest binding kinetics. In turn, also the Pf-Ft mutant in which the cysteine thiol is placed within the internal cavity, is still capable of full stoichiometric DTNB binding albeit with an almost 200-fold slower rate. The behaviour of Af-Ft bearing a cysteine thiol in a topologically equivalent position in the internal cavity was intermediate among the two Pf-Ft mutants. CONCLUSIONS AND GENERAL SIGNIFICANCE: The data thus obtained indicate clearly that the protein matrix in archaea ferritins does not provide a significant barrier against bulky, negatively charged ligands such as DTNB, a finding of relevance in view of the multiple biotechnological applications of these ferritins that envisage ligand encapsulation within the internal cavity.

Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.

The 20 aminoacyl tRNA synthetases (aaRSs) couple each amino acid to their cognate tRNAs. During evolution, 19 aaRSs expanded by acquiring novel noncatalytic appended domains, which are absent from bacteria and many lower eukaryotes but confer extracellular and nuclear functions in higher organisms. AlaRS is the single exception, with an appended C-terminal domain (C-Ala) that is conserved from prokaryotes to humans but with a wide sequence divergence. In human cells, C-Ala is also a splice variant of AlaRS. Crystal structures of two forms of human C-Ala, and small-angle X-ray scattering of AlaRS, showed that the large sequence divergence of human C-Ala reshaped C-Ala in a way that changed the global architecture of AlaRS. This reshaping removes the role of C-Ala in prokaryotes for docking tRNA and instead repurposes it to form a dimer interface presenting a DNA-binding groove. This groove cannot form with the bacterial ortholog. Direct DNA binding by human C-Ala, but not by bacterial C-Ala, was demonstrated. Thus, instead of acquiring a novel appended domain like other human aaRSs, which engendered novel functions, a new AlaRS architecture was created by diversifying a preexisting appended domain.

Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.

Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L.

The Balancing Act of Ribonucleotides in DNA.

The abundance of ribonucleotides in DNA remained undetected until recently because they are efficiently removed by the ribonucleotide excision repair (RER) pathway, a process similar to Okazaki fragment (OF) processing after incision by Ribonuclease H2 (RNase H2). All DNA polymerases incorporate ribonucleotides during DNA synthesis. How many, when, and why they are incorporated has been the focus of intense work during recent years by many labs. In this review, we discuss recent advances in ribonucleotide incorporation by eukaryotic DNA polymerases that suggest an evolutionarily conserved role for ribonucleotides in DNA. We also review the data that indicate that removal of ribonucleotides has an important role in maintaining genome stability.

Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity.

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.

Assessment of the Carbon Monoxide Metabolism of the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus VC-16 by Comparative Transcriptome Analyses.

The hyperthermophilic, sulfate-reducing archaeon, Archaeoglobus fulgidus, utilizes CO as an energy source and it is resistant to the toxic effects of high CO concentrations. Herein, transcription profiles were obtained from A. fulgidus during growth with CO and sulfate or thiosulfate, or without an electron acceptor. This provided a basis for a model of the CO metabolism of A. fulgidus. The model suggests proton translocation by "Mitchell-type" loops facilitated by Fqo catalyzing a Fd(red):menaquinone oxidoreductase reaction, as the major mode of energy conservation, rather than formate or H2 cycling during respiratory growth. The bifunctional CODH (cdhAB-2) is predicted to play an ubiquitous role in the metabolism of CO, and a novel nitrate reductase-associated respiratory complex was induced specifically in the presence of sulfate. A potential role of this complex in relation to Fd(red) and APS reduction is discussed. Multiple membrane-bound heterodisulfide reductase (DsrMK) could promote both energy-conserving and non-energy-conserving menaquinol oxidation. Finally, the FqoF subunit may catalyze a Fd(red):F420 oxidoreductase reaction. In the absence of electron acceptor, downregulation of F420H2 dependent steps of the acetyl-CoA pathway is linked to transient formate generation. Overall, carboxidotrophic growth seems as an intrinsic capacity of A. fulgidus with little need for novel resistance or respiratory complexes.