Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_2352 (AF_2352)

What is Archaeoglobus fulgidus and why is it significant for protein research?

Archaeoglobus fulgidus is a hyperthermophilic archaeon belonging to the phylum Euryarchaeota. It grows optimally at 83°C under strict anaerobic conditions . A. fulgidus is significant for protein research due to its extremophilic nature, which makes its proteins particularly stable under harsh conditions. Its genome has been fully sequenced (approximately 2,178,000 base pairs) , revealing numerous uncharacterized proteins with potential novel functions adapted to extreme environments. The organism's proteins often exhibit exceptional thermostability, making them valuable for both basic research and biotechnological applications requiring robust enzymatic activities.

What are the general approaches to studying an uncharacterized protein like AF_2352?

Studying an uncharacterized protein typically involves a multi-faceted approach. Begin with bioinformatic analysis to identify conserved domains, potential homologs, and predicted functions based on sequence similarity. Express the recombinant protein using systems such as E. coli, as demonstrated with other A. fulgidus proteins . Purify using appropriate columns (e.g., StrepTactin or Histrap columns) . Characterize basic biochemical properties including molecular weight, oligomeric state, thermal stability, pH optima, and potential cofactor requirements. Employ structural biology techniques such as X-ray crystallography or cryo-EM, similar to approaches used for other A. fulgidus proteins . Finally, conduct functional assays based on predicted activities from sequence analysis. For hyperthermophilic proteins like those from A. fulgidus, ensure all functional assays are performed at elevated temperatures (60-95°C) to match native conditions .

How should I design expression systems for recombinant AF_2352 production?

Design your expression system by first codon-optimizing the AF_2352 sequence for your host organism, typically E. coli. Based on successful approaches with other A. fulgidus proteins, construct dual-plasmid systems such as pBAD with TwinStrep tags and pCDFDuet with His tags to facilitate purification and allow for co-expression of interacting partners . Induction conditions should be carefully optimized; for other A. fulgidus proteins, expression at 37°C in LB medium with 0.2% w/v l-arabinose for pBAD vectors or 0.5 mM IPTG and 0.2% l-arabinose for pBAD + pCDF vectors has been effective . Given the thermophilic origin of AF_2352, consider expression in cold-shock conditions followed by heat treatment of the lysate to exploit the protein's expected thermostability as a first purification step. Incorporate appropriate protease inhibitors like PMSF (2 mM) in your lysis buffer, along with reducing agents such as 2-mercaptoethanol (5 mM) to maintain protein stability .

What purification methods are most effective for recombinant AF_2352?

For effective purification of recombinant AF_2352, implement a multi-step chromatography approach. First, apply affinity chromatography using either StrepTactin or Histrap columns depending on your chosen tag system (TwinStrep or His-tag) . Maintain moderate salt concentration (100 mM NaCl) throughout all buffer solutions to prevent protein aggregation . For thermostable proteins from A. fulgidus, incorporate a heat treatment step (65-75°C for 15-20 minutes) prior to chromatography to eliminate less stable E. coli proteins. Follow affinity chromatography with size exclusion chromatography to achieve higher purity and assess oligomeric state. Ion exchange chromatography may serve as an additional step if needed. Throughout purification, monitor protein stability and activity at elevated temperatures reflective of A. fulgidus' optimal growth temperature (83°C) . For storage, test protein stability in various buffer compositions with and without reducing agents and glycerol at different temperatures (4°C, -20°C, and -80°C).

What crystallization approaches should be considered for structural determination of AF_2352?

For crystallization of AF_2352, employ a systematic approach considering the hyperthermophilic nature of the protein. Begin with commercial sparse matrix screens at both room temperature and elevated temperatures (37-45°C) to mimic the protein's native thermal environment. Use highly purified protein (>95% by SDS-PAGE) at multiple concentrations (typically 5-15 mg/ml). Based on approaches used for other A. fulgidus proteins, consider incorporating ligands or cofactors predicted from sequence analysis to potentially stabilize the protein structure . Test both vapor diffusion (hanging and sitting drop) and microbatch methods. For data collection, prepare crystals with appropriate cryoprotection solutions before cryo-cooling . Process diffraction data using software packages such as XDS, POINTLESS, AIMLESS, and TRUNCATE as used for other A. fulgidus proteins . If molecular replacement is challenging due to low sequence homology, consider preparation of selenomethionine-substituted protein for experimental phasing.

How can I overcome challenges in obtaining soluble recombinant AF_2352?

To overcome solubility challenges with recombinant AF_2352, implement a multi-faceted strategy. First, explore different fusion tags beyond standard His or Strep tags, such as MBP, SUMO, or thioredoxin, which can enhance solubility. Consider co-expression with molecular chaperones like GroEL/ES or DnaK/J-GrpE to assist in proper folding. Based on findings with other A. fulgidus proteins that form complexes with partner proteins , identify potential interaction partners through genomic context analysis and consider co-expression of these partners to enhance solubility. Optimize expression conditions by varying temperature (15-30°C), induction strength (IPTG/arabinose concentration), and duration. Test different E. coli expression strains (BL21(DE3), Rosetta, Arctic Express) that may better accommodate the codon usage and folding requirements of archaeal proteins. If necessary, explore refolding protocols from inclusion bodies using gradual dialysis with decreasing denaturant concentrations. Additionally, supplement growth media and lysis buffers with potential cofactors or ligands that might stabilize the protein structure.

What bioinformatic approaches can predict the potential function of AF_2352?

To predict potential functions of AF_2352, employ a comprehensive bioinformatic workflow. Begin with sequence analysis using PSI-BLAST and HHpred to identify remote homologs beyond standard BLAST searches. Analyze the protein using multiple structure prediction tools including AlphaFold2 and RoseTTAFold to generate reliable structural models, which may reveal functional sites not apparent from sequence alone. Examine genomic context analysis similar to the approach used for AfAgo , analyzing genes located within the same operon or adjacent to AF_2352, as proteins in the same operon often participate in related functions. Apply tools like WebFlags to analyze gene neighborhoods (three genes upstream and downstream) . Utilize domain prediction tools such as InterPro, Pfam, and SMART to identify conserved domains and motifs. Perform phylogenetic analysis to place AF_2352 within evolutionary context, which may provide functional insights based on characterized relatives. Consider specialized tools for archetypal functional elements such as DNA/RNA binding prediction, transmembrane region prediction, and enzyme active site prediction. Employ protein-protein interaction prediction tools to identify potential binding partners that might illuminate function within cellular pathways.

How should enzymatic assays for AF_2352 be designed considering A. fulgidus' hyperthermophilic nature?

When designing enzymatic assays for AF_2352, account for A. fulgidus' hyperthermophilic nature with the following methodological considerations. Conduct all assays at elevated temperatures (70-85°C) to match A. fulgidus' optimal growth temperature of 83°C , using heat blocks, water baths, or thermocyclers with heated lids to prevent evaporation. Select thermostable buffers that maintain pH at high temperatures, such as phosphate or PIPES, avoiding Tris which has a high temperature coefficient. Utilize thermostable detection systems and substrates that remain stable at elevated temperatures, or implement sampling approaches where reaction aliquots are cooled and analyzed at room temperature. Based on A. fulgidus' anaerobic lifestyle , conduct assays under oxygen-limited conditions using sealed vessels or anaerobic chambers when appropriate. Consider incorporating components from A. fulgidus' natural environment, such as high salt concentrations or specific metal ions that might be crucial for activity. Design control experiments at lower temperatures (30-40°C) to distinguish temperature-dependent activity from spontaneous substrate degradation at high temperatures. For kinetic measurements, factor in increased reaction rates at elevated temperatures by utilizing more frequent sampling or continuous monitoring technologies adapted for high-temperature use.

What methods can determine if AF_2352 interacts with nucleic acids, given findings with other A. fulgidus proteins?

To determine if AF_2352 interacts with nucleic acids, particularly relevant given findings with other A. fulgidus proteins like AfAgo , implement multiple complementary approaches. Conduct electrophoretic mobility shift assays (EMSAs) using radiolabeled or fluorescently labeled DNA and RNA substrates at varying temperatures (room temperature to 83°C), testing different sequence compositions and structures (single-stranded, double-stranded, specific motifs). Utilize fluorescence anisotropy or microscale thermophoresis (MST) to quantitatively measure binding affinities and kinetics. Perform filter binding assays as an alternative approach for quantitative binding assessment, particularly useful for thermophilic proteins. Based on the methodology used for AfAgo , consider co-crystallization of AF_2352 with potential nucleic acid ligands followed by X-ray crystallography or cryo-EM to determine complex structures. Employ UV crosslinking and immunoprecipitation approaches, adapted for thermophilic conditions, to identify binding sites. Use differential scanning fluorimetry to detect thermal stability shifts upon nucleic acid binding. Consider systematic evolution of ligands by exponential enrichment (SELEX) to identify specific nucleic acid sequences or structures preferentially bound by AF_2352. Test the effects of buffer conditions, including salt concentration and pH, to optimize detection of nucleic acid interactions.

How might AF_2352 function in the context of A. fulgidus' adaptation to extreme environments?

AF_2352's potential role in A. fulgidus' adaptation to extreme environments should be analyzed through an integrative approach. Compare expression levels of AF_2352 under varied stress conditions (temperature shifts, pH changes, oxidative stress) using RNA-Seq or qRT-PCR to determine if it's stress-responsive. Given A. fulgidus' ability to form biofilms under environmental stresses (extreme pH, temperature, metals, antibiotics, xenobiotics, or oxygen) , investigate AF_2352's potential contribution to biofilm formation through knockout/knockdown studies and biofilm component analysis. Consider AF_2352's role in DNA repair mechanisms, particularly relevant as A. fulgidus possesses base excision repair (BER) systems to handle potential DNA damage from high temperatures . Examine potential contributions to thermal stability of cellular components using thermal proteome profiling. Analyze AF_2352's potential involvement in sulfate reduction pathways, central to A. fulgidus' energy metabolism . Create temperature-sensitive mutants through site-directed mutagenesis to identify residues critical for thermostability. Utilize immunolocalization to determine AF_2352's cellular localization under different stress conditions. Through these methodologies, establish whether AF_2352 plays a structural, enzymatic, or regulatory role in extremophilic adaptation.

What are the approaches to study potential post-translational modifications of AF_2352?

To comprehensively study potential post-translational modifications (PTMs) of AF_2352, employ both mass spectrometry-based and biochemical approaches. Perform high-resolution mass spectrometry analysis using both bottom-up (protein digestion followed by peptide analysis) and top-down (intact protein analysis) approaches to identify and map PTMs. Use enrichment strategies specific to common PTMs (phosphorylation, methylation, acetylation) prior to MS analysis to increase detection sensitivity. Employ multiple protease digestion strategies to ensure comprehensive sequence coverage. Apply targeted multiple reaction monitoring (MRM) to quantify specific modifications across different growth conditions. Investigate PTMs using specific antibodies against common modifications through Western blotting. Consider the unique high-temperature environment of A. fulgidus (optimal growth at 83°C) when analyzing PTMs, as this may lead to unusual or thermostable modifications. Use site-directed mutagenesis to mutate potential modification sites and assess functional consequences. Apply differential scanning calorimetry to determine if PTMs affect thermal stability. Examine if modifications change in response to environmental stresses relevant to A. fulgidus' natural habitat. Create a quantitative PTM profile across different growth phases to understand temporal regulation of modifications.

How can in vivo studies of AF_2352 be designed given the challenges of working with A. fulgidus?

Designing in vivo studies for AF_2352 requires specialized approaches considering A. fulgidus' hyperthermophilic, anaerobic growth requirements . Establish reliable transformation protocols for A. fulgidus or closely related species, optimizing DNA introduction methods such as electroporation or liposome-mediated transfer for these archaeal cells. Develop selectable markers functional at high temperatures (83°C) for genetic manipulation. Design expression constructs with native A. fulgidus promoters and codon usage to ensure proper expression. Create gene deletion or CRISPR-Cas9 systems adapted for high-temperature functioning to generate knockout mutants. Implement complementation assays to confirm phenotypes are specifically due to AF_2352 manipulation. For phenotypic analysis, assess growth rates under various conditions (different carbon sources, sulfate concentrations, stress conditions) to identify conditional phenotypes. Use RNA-Seq to analyze transcriptome changes in AF_2352 knockout/overexpression strains. Develop fluorescent protein variants stable at high temperatures for subcellular localization studies. Create translational fusions with epitope tags for co-immunoprecipitation to identify interaction partners. If direct genetic manipulation proves challenging, consider heterologous expression in more genetically tractable thermophiles like Thermus thermophilus, followed by functional complementation tests. Alternatively, develop cell-free systems using A. fulgidus extracts to study AF_2352 function in a near-native context.

How should homology modeling of AF_2352 be approached using related archaeal proteins?

For effective homology modeling of AF_2352, implement a systematic workflow that accounts for archaeal protein peculiarities. Begin by identifying suitable templates through iterative searches using PSI-BLAST, HHpred, and FFAS against the PDB database, paying special attention to characterized proteins from related archaeal species. Given the molecular signatures showing relatedness between Archaeoglobus, methanogens, and Thermococci , consider proteins from these groups as potential templates. Use multiple sequence alignment tools like MAFFT with accuracy-oriented modes (L-INS-i) to align AF_2352 with identified templates . Consider secondary structure predictions to guide alignment in regions of low sequence similarity. Generate multiple models using software suites like MODELLER, Rosetta, or Swiss-Model, and evaluate them using structure validation tools (PROCHECK, VERIFY3D, ProSA). Refine models through energy minimization and molecular dynamics simulations at high temperatures (70-85°C) to reflect native conditions. Validate models through comparison with experimental data if available (limited proteolysis, chemical cross-linking coupled with mass spectrometry). As demonstrated with other A. fulgidus proteins , complement homology modeling with modern deep learning approaches like AlphaFold2 or RoseTTAFold, especially useful for proteins with limited homology to characterized structures. Compare models generated through different approaches to identify consistent structural features with higher confidence.

What phylogenetic approaches can place AF_2352 in evolutionary context within Archaea?

To place AF_2352 in its proper evolutionary context within Archaea, implement a multi-faceted phylogenetic approach. Collect homologs through BLAST searches against comprehensive databases, reducing redundancy through sequence clustering with tools like MMseqs2 (90% identity threshold) . Construct high-quality multiple sequence alignments using accuracy-oriented MAFFT modes (L-INS-i) , followed by alignment curation using trimAl to remove columns with excessive gap content . Infer phylogenetic trees using maximum likelihood methods (RAxML, IQ-TREE) or Bayesian approaches (MrBayes), with appropriate amino acid substitution models selected through model testing. Root the resulting tree using midpoint rooting or outgroup methods, and annotate using visualization tools like iToL . Perform gene context analysis examining genes neighboring AF_2352, as implemented for other A. fulgidus proteins , investigating three genes upstream and downstream to identify conserved operonic structures across archaeal lineages. Couple standard phylogenetics with gene content analysis to detect horizontal gene transfer events. Implement reconciliation analysis comparing the AF_2352 gene tree with established archaeal species trees. Calculate evolutionary rates to identify conserved functional regions versus rapidly evolving sites. Construct presence-absence matrices of AF_2352 across archaeal genomes to understand its distribution pattern and correlation with ecological niches.

How can heterologous expression in model organisms help understand AF_2352 function?

Heterologous expression in model organisms provides valuable insights into AF_2352 function through multiple methodological approaches. Express AF_2352 in E. coli using codon-optimized sequences with appropriate tags (His, Strep) for purification and detection, as successfully done with other A. fulgidus proteins . Test expression in thermophilic bacteria like Thermus thermophilus to better approximate native temperature conditions. Use yeast complementation assays by expressing AF_2352 in deletion strains of potential homologs, assessing whether the archaeal protein can rescue phenotypes. Employ yeast two-hybrid or bacterial two-hybrid systems to identify interaction partners, providing clues to function. Consider expression in cell-free systems derived from thermophilic organisms to maintain appropriate folding conditions. If targeting subcellular compartments is necessary, incorporate appropriate localization signals. For potential enzyme activity, develop high-throughput substrate screening assays adapted for thermostable proteins. Create fusion proteins with thermostable reporters (modified GFP variants) for localization and expression studies. Implement synthetic biology approaches by incorporating AF_2352 into minimal circuits to test specific hypothetical functions. Monitor effects on host physiology when AF_2352 is expressed under different conditions, which may reveal toxic or beneficial effects providing functional clues. Design these heterologous systems to operate at elevated temperatures when possible to account for the protein's adaptation to A. fulgidus' optimal growth temperature of 83°C .

What proteomics approaches can identify interaction partners of AF_2352 in A. fulgidus?

To identify interaction partners of AF_2352 in A. fulgidus, employ complementary proteomics approaches adapted for hyperthermophilic conditions. Implement affinity purification-mass spectrometry (AP-MS) using tagged recombinant AF_2352 as bait under physiologically relevant conditions (anaerobic, 83°C) . Apply chemical crosslinking followed by MS analysis (XL-MS) using thermostable crosslinkers that maintain reactivity at high temperatures to capture transient interactions. Perform proximity-dependent biotin identification (BioID) or APEX2 approaches with thermostable biotin ligase or peroxidase variants fused to AF_2352. Consider co-immunoprecipitation using antibodies raised against recombinant AF_2352, combined with MS identification of pulled-down proteins. Validate interactions through reciprocal pulldowns and orthogonal methods such as bacterial/archaeal two-hybrid systems adapted for thermophiles. Implement protein correlation profiling across different fractionation methods (size exclusion, ion exchange) to identify proteins with similar elution profiles. Apply quantitative interaction proteomics comparing wild-type and AF_2352 deletion/overexpression strains to identify differentially associated proteins. Use structural information from cross-linking experiments to guide docking studies of potential complexes. As demonstrated with other A. fulgidus proteins like AfAgo that forms heterodimeric complexes , consider whether AF_2352 may functionally interact with proteins encoded in proximity on the genome, particularly those in the same operon.

How can cryo-EM be utilized for structural characterization of AF_2352 complexes?

For cryo-EM structural characterization of AF_2352 complexes, implement a methodical workflow adapted from approaches used with other A. fulgidus proteins . Begin with biochemical characterization to ensure sample homogeneity, using techniques such as size exclusion chromatography combined with multi-angle light scattering to verify complex formation and stability. Optimize buffer conditions through systematic screening to enhance particle distribution and prevent aggregation. Apply GraFix (gradient fixation) to stabilize transient complexes if necessary. For sample preparation, test multiple grid types and sample application methods, including graphene oxide or ultrathin carbon support films to prevent preferred orientation issues. Perform preliminary negative stain EM to assess sample quality before proceeding to cryo-EM. During data collection, implement automated acquisition software with dose fractionation to minimize radiation damage. Process data using standard software packages (RELION, cryoSPARC) for 2D classification, 3D reconstruction, and refinement. Apply focused refinement approaches to resolve heterogeneous or flexible regions. Integrate cryo-EM data with complementary structural information from X-ray crystallography or crosslinking-MS, as demonstrated for other A. fulgidus proteins . Validate structures through independent half-dataset processing, local resolution estimation, and model-to-map correlation analysis. Consider time-resolved cryo-EM if studying dynamic complexes involving AF_2352. The resulting structures should be analyzed in context of A. fulgidus' hyperthermophilic lifestyle to identify structural adaptations that enable function at elevated temperatures.

What computational approaches can model the effect of high temperatures on AF_2352 structure and dynamics?

To model high-temperature effects on AF_2352 structure and dynamics, implement specialized computational methodologies. Perform molecular dynamics (MD) simulations at elevated temperatures (70-90°C) using force fields optimized for high-temperature conditions. Extend simulation timescales to microseconds to capture relevant conformational changes, employing enhanced sampling techniques such as replica exchange MD or metadynamics to overcome energy barriers. Calculate root mean square fluctuations (RMSF) and principal component analysis to identify regions with increased flexibility at high temperatures. Model disulfide bond formation and salt bridge networks, which are often critical for thermostability in archaeal proteins. Apply Machine Learning approaches to predict temperature-dependent stability changes across the protein structure. Implement Quantum Mechanics/Molecular Mechanics (QM/MM) simulations for regions involving catalytic residues to accurately model electronic changes at high temperatures. Calculate free energy landscapes at different temperatures to identify stabilizing interactions unique to thermophilic conditions. Conduct in silico mutagenesis to identify residues critical for thermostability. Compare simulations of AF_2352 with mesophilic homologs to identify thermoadaptation features. Model potential ligand binding using molecular docking and MD simulations at elevated temperatures to account for binding pocket dynamics unique to thermophilic conditions. Integrate computational predictions with experimental thermal stability data when available. These approaches should provide insights into the molecular adaptations that allow AF_2352 to function in A. fulgidus' optimal growth temperature of 83°C .

How might CRISPR-based technologies be adapted for studying AF_2352 in A. fulgidus?

Adapting CRISPR-based technologies for studying AF_2352 in A. fulgidus requires specialized approaches for hyperthermophilic archaea. Identify thermostable Cas proteins, potentially from other hyperthermophiles or engineered for stability at A. fulgidus' growth temperature (83°C) . Design guide RNAs with higher GC content and thermally stable modifications to prevent degradation at elevated temperatures. Optimize transformation protocols specifically for A. fulgidus, considering its unique cell envelope structure. Develop selectable markers functional at high temperatures for screening transformants. Implement inducible expression systems responsive under anaerobic, high-temperature conditions to control Cas9 expression. Design homology-directed repair templates with extended homology arms to improve recombination efficiency. Create gene knock-in systems to introduce epitope tags or fluorescent proteins for tracking AF_2352. Consider CRISPR interference (CRISPRi) using catalytically inactive Cas9 for gene repression rather than deletion. Employ base editing or prime editing systems adapted for high temperatures to introduce specific mutations without double-strand breaks. Validate editing efficiency using deep sequencing approaches. If direct transformation proves challenging, consider developing in vitro CRISPR systems using cell extracts to study AF_2352 function. Alternatively, implement CRISPR-based strategies in heterologous systems expressing AF_2352, which may provide functional insights while technical challenges in A. fulgidus are being addressed.

What high-throughput approaches can screen for potential enzymatic activities of AF_2352?

To screen for potential enzymatic activities of AF_2352, implement diverse high-throughput methodologies adapted for thermostable proteins. Develop microplate-based assays with thermostable fluorogenic or chromogenic substrates spanning major enzyme classes (hydrolases, transferases, oxidoreductases), conducted at elevated temperatures (70-85°C). Apply substrate profiling arrays to simultaneously test hundreds of potential substrates under thermophilic conditions. Implement metabolite profiling using LC-MS/MS to detect changes in metabolite pools when AF_2352 is incubated with cellular extracts. Develop activity-based protein profiling using thermostable chemical probes to identify catalytic mechanisms. Design thermal shift assays to screen for ligands or substrates that stabilize AF_2352, particularly relevant given its thermophilic nature. Consider computational docking of virtual compound libraries to identify potential substrates based on binding poses. Implement differential scanning fluorimetry in 96-well format to rapidly screen condition effects (pH, salt, metal ions) on protein stability. Use functional metagenomics approaches where AF_2352 is expressed in libraries screened for specific phenotypes. Develop cell-free systems derived from thermophilic organisms to test enzymatic activity in near-native conditions. Apply multiplexed assays using micro-droplet technology to increase throughput. Consider directed evolution approaches to amplify weak native activities to detectable levels. These approaches should be conducted at temperatures reflective of A. fulgidus' optimal growth (83°C) to identify activities that might only manifest under thermophilic conditions.

How can systems biology approaches integrate AF_2352 into the broader metabolic network of A. fulgidus?

Integrating AF_2352 into A. fulgidus' metabolic network requires comprehensive systems biology approaches. Develop genome-scale metabolic models of A. fulgidus, incorporating known pathways for sulfate reduction, carbon metabolism, and energy production . Perform differential transcriptomics (RNA-Seq) comparing wild-type and AF_2352 knockout strains under various conditions to identify affected pathways. Implement proteomics to quantify protein expression changes resulting from AF_2352 manipulation, using isobaric tagging for quantification. Apply metabolomics to identify metabolite profile changes associated with AF_2352 activity, focusing on thermostable metabolites relevant to A. fulgidus' lifestyle. Conduct flux balance analysis to predict how AF_2352 deletion would affect metabolic flux distributions. Utilize protein-protein interaction networks, constructed from co-immunoprecipitation or crosslinking studies, to place AF_2352 within cellular interaction networks. Perform comparative genomics across Archaeoglobus species to identify conserved genomic neighborhoods and potential functional associations. Implement constraint-based modeling to predict phenotypic consequences of AF_2352 perturbation. Apply machine learning approaches to integrate multi-omics datasets and predict AF_2352 function based on network positions. Create visualization tools to map AF_2352 interactions within the broader cellular network. Consider evolutionary systems biology approaches to understand how AF_2352's role may have evolved within archaeal metabolism. These integrated approaches should account for A. fulgidus' unique metabolism, including its sulfate-reducing capabilities and the presence of nearly complete sets of genes for methanogenesis .