Recombinant Geobacter sulfurreducens ATP synthase subunit beta (atpD)

What is the ATP synthase subunit beta (atpD) in Geobacter sulfurreducens?

ATP synthase subunit beta (atpD) is a critical component of the F1F0 ATP synthase complex in Geobacter sulfurreducens. This protein (UniProt ID: Q74GY0) functions as part of the catalytic core of the F1 sector, directly participating in ATP synthesis. The full-length protein consists of 470 amino acids and has EC number 3.6.3.14. In G. sulfurreducens, ATP synthase plays a central role in energy metabolism, particularly in the context of the organism's unique electron transfer capabilities .

How does the structure of G. sulfurreducens atpD compare to other bacterial ATP synthase beta subunits?

The G. sulfurreducens atpD protein shares structural similarities with other bacterial ATP synthase beta subunits, with conserved domains for nucleotide binding and catalysis. Its amino acid sequence (MSQNFGKISQVIGAVIDVEFEPGKLPPIYNALRVTNPAIDDKEYNLVLEVAQHLGENAVRTIAMDSTDGLVRGQAVLDTGKQISVPVGRKTLGRILNVIGEPVDEMGPVNAEKEYGIHREAPAFVDQSTKVEAFTTGIKVVDLLAPYARGGKIGLFGGAGVGKTVLIMELINNIAKQHGGFSVFAGVGERTREGNDLWMEMKESGVLDKAALVYGQMNEPPGARARVALS ALSIAEYFRDEE...) contains the characteristic Walker A and B motifs found in ATP-binding proteins . While the core structure is conserved, G. sulfurreducens atpD has evolved specific adaptations that likely enable it to function optimally in anaerobic environments where the bacterium uses extracellular electron acceptors for respiration.

What is the role of ATP synthase in G. sulfurreducens metabolism?

ATP synthase in G. sulfurreducens functions as the central ATP-producing machinery, converting the proton-motive force generated during electron transport into chemical energy as ATP. G. sulfurreducens has a unique energy metabolism that allows it to couple the oxidation of organic compounds (primarily acetate) to the reduction of extracellular electron acceptors like Fe(III), fumarate, or electrode surfaces . During this process, protons are pumped across the membrane, generating a proton gradient that drives ATP synthesis via the F1F0 ATP synthase complex. The beta subunit (atpD) is directly involved in the catalytic conversion of ADP to ATP within this complex. Research has demonstrated that manipulating ATP demand can significantly impact respiration rates in G. sulfurreducens, highlighting the central role of ATP synthase in regulating the organism's energy metabolism .

What are the optimal conditions for handling recombinant G. sulfurreducens atpD protein?

Recombinant G. sulfurreducens atpD protein requires careful handling to maintain its stability and activity. Based on established protocols, the following conditions are recommended:

• Storage: Store at -20°C/-80°C, with -80°C preferred for long-term storage

• Buffer composition: Tris-based buffer with 50% glycerol, optimized for protein stability

• Aliquoting: Prepare small working aliquots to avoid repeated freeze-thaw cycles

• Working temperature: Maintain working aliquots at 4°C for up to one week

• Reconstitution: When using lyophilized protein, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with added glycerol (5-50% final concentration) for stability
  Avoid repeated freeze-thaw cycles as these significantly reduce protein stability and activity. For experimental work requiring active enzyme, freshly thawed aliquots should be used.

What expression systems are most effective for producing recombinant G. sulfurreducens atpD?

Based on research protocols, yeast expression systems have proven effective for producing functional recombinant G. sulfurreducens atpD . E. coli expression systems have also been successfully used for other G. sulfurreducens proteins . When designing an expression strategy, consider the following factors:

• Codon optimization: Adapt the G. sulfurreducens atpD sequence to the codon usage of the host organism

• Purification tags: N-terminal His-tag allows for efficient purification via affinity chromatography

• Expression conditions: For E. coli systems, lower temperatures (16-25°C) and reduced IPTG concentrations often improve soluble protein yield

• Protein solubility: Addition of solubility-enhancing fusion partners (like MBP or SUMO) may improve yield of correctly folded protein

• Quality control: Verify protein integrity using SDS-PAGE (aim for >85% purity) and functional assays
  The choice between prokaryotic and eukaryotic expression systems should be guided by the intended application and required post-translational modifications.

How can researchers verify the functional activity of recombinant G. sulfurreducens atpD?

Verifying the functional activity of recombinant G. sulfurreducens atpD requires assessing its ATP hydrolysis and synthesis capabilities. Recommended approaches include:

• ATP hydrolysis assay: Measure ATPase activity by quantifying inorganic phosphate release using malachite green or similar colorimetric methods

• Coupled enzyme assays: Link ATP hydrolysis to NADH oxidation using pyruvate kinase and lactate dehydrogenase, monitoring absorbance at 340 nm

• Reconstitution experiments: Incorporate purified atpD into liposomes or nanodiscs with other ATP synthase subunits to assess complex formation and activity

• Thermal stability analysis: Use differential scanning fluorimetry to assess protein folding and stability

• Nucleotide binding assays: Measure binding affinities for ATP, ADP, and other nucleotides using isothermal titration calorimetry or fluorescence-based methods
  These assays should be performed under anaerobic conditions when possible to better mimic the native environment of G. sulfurreducens proteins.

How can recombinant G. sulfurreducens atpD be used in studies of extracellular electron transfer?

Recombinant G. sulfurreducens atpD can serve as a valuable tool for investigating the relationship between energy conservation and extracellular electron transfer (EET) mechanisms:

• Protein-protein interaction studies: Use purified atpD to identify interactions with components of the electron transfer chains, particularly those involved in establishing proton gradients

• Reconstitution experiments: Incorporate atpD into artificial membrane systems alongside electron transfer proteins to study coupling mechanisms

• Mutational analysis: Engineer variants of atpD with altered catalytic properties to investigate how ATP synthesis rates affect electron transfer to external acceptors

• Inhibitor studies: Use recombinant atpD to screen for specific inhibitors that can help dissect the relationship between ATP synthesis and electron transfer rates

• Structural studies: Employ the recombinant protein for crystallography or cryo-EM studies to understand structural adaptations that may be unique to G. sulfurreducens
  Research has demonstrated that G. sulfurreducens utilizes c-type cytochromes, pili, and outer membrane proteins for EET , and understanding how these processes couple to ATP synthesis via atpD is a frontier area of research.

What role does atpD play in the metabolic modeling of G. sulfurreducens?

ATP synthase and specifically the atpD subunit play central roles in genome-scale metabolic models of G. sulfurreducens:

• Energy balance calculations: atpD activity determines the ATP yield from electron transfer chains, which is critical for accurate growth predictions in metabolic models

• Flux balance analysis: ATP synthesis rates constrain other metabolic fluxes in the model, particularly in central carbon metabolism

• Redundant pathway analysis: Models have identified several redundant pathways in G. sulfurreducens metabolism that involve different energetic demands and ATP yields

• Growth yield predictions: Accurate parameterization of ATP synthase efficiency is essential for predicting growth yields under different electron acceptor conditions

• Engineering applications: Metabolic models incorporate atpD function to predict how manipulating ATP demand affects respiration rates and substrate utilization
  Research has shown that increasing ATP demand in G. sulfurreducens by expressing the hydrolytic F1 portion of ATP synthase leads to higher respiration rates and altered metabolic fluxes, as predicted by genome-scale models . This demonstrates the central role of atpD in controlling energy metabolism and the value of incorporating ATP synthase parameters in metabolic models.

How does atpD function relate to the engineered increase in respiration rates in G. sulfurreducens?

Research has demonstrated that manipulating ATP demand through expression of ATP synthase components can significantly alter respiration rates in G. sulfurreducens :

• ATP drain mechanism: Expression of the hydrolytic F1 portion of ATP synthase creates an ATP drain, decreasing cellular ATP content by more than half

• Metabolic consequences: Cells with increased ATP demand exhibit higher respiration rates, slower growth, and lower cell yields

• Transcriptional response: Induction of ATP drain increases expression of genes involved in energy metabolism, particularly TCA cycle enzymes and NADH dehydrogenase

• Electron transfer enhancement: The heightened respiratory activity correlates with increased expression of redox-active proteins, including c-type cytochromes

• Practical applications: This approach provides a genetic engineering strategy to increase the rate of electron transfer to extracellular acceptors, with potential applications in bioremediation and microbial fuel cells
  This research highlights how atpD and ATP synthase function can be leveraged as control points for engineering desired metabolic phenotypes in G. sulfurreducens .

What approaches are available for genetic manipulation of atpD in G. sulfurreducens?

Several genetic approaches have been developed for manipulating atpD and other genes in G. sulfurreducens:

• Single-step gene replacement: Target-specific sequences can be disrupted using suicide vectors carrying antibiotic resistance markers

• Expression vectors: Both IncQ and pBBR1 broad-host-range vectors have been shown to replicate in G. sulfurreducens, with IncQ plasmid pCD342 being particularly suitable as an expression vector

• Electroporation protocols: Optimized conditions for introducing foreign DNA into G. sulfurreducens have been established, with specific parameters for voltage, resistance, and cell preparation

• Complementation strategies: Genes can be expressed in trans to confirm phenotypes of deletion mutants, as demonstrated with the nifD gene

• CRISPR-Cas9 approaches: More recent studies have applied CRISPR-based genome editing to G. sulfurreducens for precise genetic modifications
  When working specifically with atpD, researchers should consider the essential nature of this gene and may need to employ conditional expression systems or partial deletions to study its function without completely disrupting cell viability.

How can researchers study the expression and regulation of atpD in G. sulfurreducens?

Several molecular approaches are available for studying atpD expression and regulation:

What is known about the role of atpD in syntrophic relationships involving G. sulfurreducens?

G. sulfurreducens forms syntrophic relationships with other microorganisms that involve complex energy and electron exchanges, in which atpD likely plays a critical role:

• Direct interspecies electron transfer (DIET): G. sulfurreducens can establish DIET with other bacteria, where ATP synthesis coupled to electron transfer may be a key mechanism

• Syntrophic growth: G. sulfurreducens can grow syntrophically with denitrifying communities, accelerating denitrification rates

• Metabolic shifts: During syntrophic growth with Clostridium pasteurianum, G. sulfurreducens influences the fermentation product profile, suggesting altered electron and energy flows that would involve ATP synthase

• Bioremediation applications: In uranium-contaminated environments, G. sulfurreducens competes with other metal-reducing bacteria, with energy efficiency (which depends on ATP synthase function) being a key determinant of competitive success

• Microbial fuel cells: In electrode-based systems, ATP synthesis rates in G. sulfurreducens likely influence current production and biofilm formation
  Research has demonstrated that syntrophic growth of G. sulfurreducens with denitrifying communities eliminates lag phases and improves denitrification rates by 13-51% , suggesting that energy coupling mechanisms involving ATP synthase may be optimized during these interactions.

What are the challenges in studying ATP synthase function in the context of G. sulfurreducens' unique electron transfer capabilities?

Researchers face several challenges when investigating ATP synthase function in relation to G. sulfurreducens' electron transfer mechanisms:

• Anaerobic requirements: Maintaining strict anaerobic conditions for protein purification and functional studies presents technical challenges

• Membrane protein complexes: The multi-subunit nature of ATP synthase makes it difficult to study in isolation while maintaining native interactions

• Coupling mechanisms: Understanding how electron transfer to extracellular acceptors generates a proton gradient that drives ATP synthase requires specialized experimental setups

• Redundant pathways: The presence of multiple redundant metabolic pathways in G. sulfurreducens complicates the interpretation of mutant phenotypes

• In vivo measurements: Quantifying ATP synthesis rates in living cells during electron transfer to different extracellular acceptors requires sophisticated approaches
  To address these challenges, researchers can employ techniques like anaerobic protein purification, reconstitution into liposomes, electrode-based experimental systems, and in vivo ATP biosensors.

How can contradictions in experimental data regarding atpD function be resolved?

When researchers encounter contradictory results regarding atpD function in G. sulfurreducens, several approaches can help resolve these discrepancies:

• Strain variation analysis: Verify that all experiments use the same strain of G. sulfurreducens, as genomic differences between laboratory strains may exist

• Growth condition standardization: Ensure consistent growth conditions, as small variations in media composition, temperature, or redox potential can significantly impact energy metabolism

• Genetic background characterization: Check for potential compensatory mutations that may arise when manipulating essential genes like atpD

• Methodology validation: Cross-validate results using multiple independent techniques for measuring the same parameters

• Systems biology approaches: Integrate transcriptomic, proteomic, and metabolomic data to develop a more comprehensive understanding of atpD function in different contexts
  The metabolic flexibility of G. sulfurreducens, including its redundant pathways , may explain apparently contradictory results under different experimental conditions.

What future research directions could advance our understanding of G. sulfurreducens atpD?

Several promising research directions could significantly advance our understanding of atpD function in G. sulfurreducens:

• Structure-function studies: Obtain high-resolution structures of G. sulfurreducens ATP synthase to identify unique adaptations for functioning in anaerobic, metal-reducing environments

• Single-molecule approaches: Apply techniques like single-molecule FRET or high-speed AFM to study the dynamics of ATP synthase during catalysis

• In situ characterization: Develop approaches to study ATP synthase function in intact G. sulfurreducens biofilms on electrodes or mineral surfaces

• Synthetic biology applications: Engineer chimeric ATP synthases with components from G. sulfurreducens and other organisms to understand specialized adaptations

• Evolutionary analyses: Compare ATP synthase sequences and functions across Geobacteraceae to understand how this enzyme complex has evolved to support extracellular electron transfer
  Additionally, investigating how atpD function is integrated with the recently discovered oxidative stress response pathways in G. sulfurreducens could reveal important insights into the organism's energy metabolism and survival strategies.

How can recombinant G. sulfurreducens atpD be used in bioremediation research?

Recombinant atpD can advance bioremediation research in several ways:

• Biomarker development: atpD activity or expression levels could serve as biomarkers for monitoring the metabolic activity of Geobacter species during bioremediation processes

• Metabolic engineering: Modifying atpD expression or structure could enhance G. sulfurreducens' ability to reduce contaminants like uranium, by altering the coupling between energy conservation and electron transfer

• Environmental sensors: Developing biosensors based on atpD function could help monitor bioremediation progress in the field

• Community interaction studies: Using recombinant atpD in protein-protein interaction studies could reveal how G. sulfurreducens interacts energetically with other microorganisms in mixed communities during bioremediation

• Inhibitor screening: Identifying compounds that specifically target atpD could help control microbial community composition during bioremediation
  Research has shown that G. sulfurreducens plays a key role in uranium bioremediation , and understanding how ATP synthesis couples to this process could lead to improved bioremediation strategies.

What are the methodological considerations for using G. sulfurreducens atpD in microbial fuel cell research?

Researchers using atpD in microbial fuel cell (MFC) studies should consider:

• Expression analysis: Quantifying atpD expression levels can provide insights into the energy conservation efficiency during electron transfer to electrodes

• Genetic engineering approaches: Modifying atpD expression could potentially increase power output in MFCs by altering the balance between growth and respiration

• Biofilm formation: Understanding how ATP synthesis rates affect biofilm development on electrodes is crucial, as robust biofilms are essential for efficient MFC operation

• Protein-protein interactions: Studying interactions between atpD and electron transfer components could reveal bottlenecks in electron flow to electrodes

• Metabolic modeling: Incorporating ATP synthase parameters into metabolic models can help predict how different operational conditions will affect MFC performance
  The finding that engineered increases in ATP demand can enhance respiration rates in G. sulfurreducens provides a promising approach for improving MFC performance through targeted modification of energy conservation pathways.

What experimental protocols are recommended for studying atpD in the context of direct interspecies electron transfer?

To investigate atpD's role in direct interspecies electron transfer (DIET), researchers should consider the following protocols:

• Co-culture systems: Establish defined co-cultures of G. sulfurreducens with partners like G. metallireducens or denitrifying bacteria

• Gene expression analysis: Use qRT-PCR to quantify atpD expression levels during syntrophic growth compared to pure cultures

• Protein localization: Apply immunofluorescence or other localization techniques to determine whether ATP synthase distribution changes during DIET

• Metabolic flux analysis: Use isotope labeling to track carbon and electron flows during syntrophic growth, correlating these with ATP synthesis

• Genetic manipulation: Create atpD variants with altered catalytic properties to test how ATP synthesis rates affect DIET efficiency
  Research has shown that G. sulfurreducens forms spherical aggregates with denitrifying partners during syntrophic growth , suggesting specific cellular arrangements that may optimize energy exchange during DIET. Understanding atpD's role in these processes could provide insights into the mechanisms of interspecies cooperation.