Recombinant Geobacter sp. ATP synthase subunit delta (atpH)

What is the structural organization of the ATP synthase operon in Geobacter sp. compared to other bacteria?

The ATP synthase genes in bacteria are typically organized in operons with specific arrangements that may vary between species. While Geobacter-specific data is still emerging, comparative analysis with other bacteria shows distinct patterns:

• In Rhodobacter capsulatus, the atpHAGDC operon contains five genes coding for the F₁ sector, with genes for the F₀ sector located elsewhere in the genome

• Clostridium pasteurianum possesses a nine-gene atp operon arranged as atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ε)

• Most bacterial ATP synthase operons follow similar organizational principles, with some variations in gene order and operon structure

For Geobacter sp. research, understanding these comparative structures can guide genomic analysis and cloning strategies. Primer design for the atpH gene should consider potentially conserved regions based on phylogenetically related organisms.

What expression systems are most suitable for producing recombinant Geobacter sp. ATP synthase subunit delta?

The choice of expression system significantly impacts the yield and functionality of recombinant ATP synthase subunits:

• E. coli-based expression systems have been successfully used for ATP synthase subunits from various bacterial sources, including Rhodobacter and Clostridium species

• For Geobacter sp. atpH expression, consider the following methodological approach:

  • Clone the atpH gene into a vector with an inducible promoter (T7 or araBAD)

  • Transform into E. coli strains optimized for recombinant protein expression (BL21(DE3) or derivatives)

  • Optimize induction conditions (temperature, inducer concentration, and duration)

  • Include affinity tags (His₆ or GST) to facilitate purification

When expressing ATP synthase subunits, lower induction temperatures (16-25°C) often improve protein solubility compared to standard 37°C induction protocols.

How can one assess the functional integrity of recombinant Geobacter sp. delta subunit in reconstitution studies?

Functional assessment of recombinant ATP synthase subunits requires specialized assays:

• ATP hydrolysis assays can measure ATPase activity using colorimetric detection of released phosphate:

  • Prepare reaction mixtures containing 100 mM Tris-HCl (pH 8.0), 5 mM MgCl₂

  • Use 2 mM Na-ATP or Mg-ATP as substrate

  • Quantify released phosphate using established colorimetric methods

• For reconstitution studies, purified subunits can be assembled with partner proteins:

  • Solubilize F₁ components with appropriate detergents (n-dodecyl-β-maltoside at 1% has been effective for bacterial ATP synthases)

  • Mix purified components in appropriate stoichiometric ratios

  • Verify assembly by size-exclusion chromatography and/or native gel electrophoresis

  • Assess functionality through ATP synthesis/hydrolysis assays

Comparative ATP hydrolysis rates between intact complexes and delta-deficient complexes can reveal the functional contribution of the delta subunit.

What structural techniques are most informative for characterizing the delta subunit's interactions within the ATP synthase complex?

Multiple structural approaches provide complementary insights:

• Cryo-electron microscopy (cryo-EM) has proven valuable for ATP synthase structural studies:

  • Prepare highly purified samples (>95% purity)

  • Apply contrast transfer function (CTF) correction for optimal resolution

  • Collect data using contemporary cryo-EM instruments

  • Process using standard image analysis pipelines

• Nuclear Magnetic Resonance (NMR) is particularly useful for studying dynamic regions and specific interactions:

  • Express isotopically labeled protein (¹⁵N, ¹³C)

  • Perform solution NMR studies to analyze structural elements

  • This approach has been informative for studying ATP synthase subunit interactions in mycobacterial systems

• Cross-linking mass spectrometry can map specific interaction interfaces:

  • Use bifunctional cross-linkers with varying spacer lengths

  • Digest cross-linked complexes and analyze by LC-MS/MS

  • Identify cross-linked peptides to map interaction surfaces

How do mutations in conserved residues of the delta subunit affect ATP synthase assembly and function?

Site-directed mutagenesis studies provide insights into structure-function relationships:

• For Geobacter sp. delta subunit research, focus on these methodological approaches:

  • Identify conserved residues through multiple sequence alignment with well-characterized bacterial ATP synthases

  • Generate single, double, or multiple mutations using PCR-based methods

  • Express and purify mutant proteins following established protocols

  • Assess structural integrity through circular dichroism or thermal stability assays

  • Evaluate functional impact through ATP synthesis/hydrolysis assays

• From studies on other bacterial ATP synthases, N-terminal deletions can significantly impact function:

  • Deletion of just four amino acids at the N-terminus of mycobacterial epsilon subunit resulted in eightfold ATP hydrolysis increase

  • Similar targeted deletions in delta subunit could reveal functional domains

What are the specific challenges in expressing and purifying the delta subunit compared to other ATP synthase components?

The delta subunit presents unique challenges:

• Solubility issues may arise due to hydrophobic interfaces that normally interact with other subunits

  • Consider fusion tags that enhance solubility (MBP, SUMO)

  • Optimize buffer conditions (pH, salt concentration, additives like glycerol)

  • Co-expression with interacting partners may improve stability

• Structural integrity assessment:

  • Circular dichroism to confirm secondary structure content

  • Size-exclusion chromatography to verify monomeric state

  • Limited proteolysis to identify stable domains

• Functional verification requires:

  • Binding assays with partner subunits (α, β, γ)

  • Activity assays in reconstituted systems

  • Structural studies to confirm native conformation

How can gene transfer agent (GTA) systems be utilized for studying essential ATP synthase genes in Geobacter species?

Gene transfer agent (GTA) approaches offer advantages for manipulating essential genes:

• In Rhodobacter capsulatus, researchers demonstrated that ATP synthase genes are essential, making direct deletion challenging

• A methodology combining GTA transduction with conjugation was developed to study essential ATP synthase genes:

  • Create a complementation construct carrying the wild-type gene

  • Introduce the construct into the recipient strain

  • Use GTA to transfer the deletion mutation

  • Select for double recombinants

This approach is particularly valuable for Geobacter sp. research when:

• Direct knockouts are lethal

• Conditional expression systems are inefficient

• Precise genetic manipulation is required for functional studies

What membrane preparation methods are optimal for isolating ATP synthase complexes from Geobacter species?

Effective membrane preparation is crucial for downstream applications:

• Modified lysozyme methods have proven effective for ATP synthase isolation:

  • Suspend cell paste in lysis buffer (100 mM Tris-HCl [pH 8.0], 0.5 M sucrose)

  • Add lysozyme (2.0 mg/ml) and incubate at 37°C for 1 hour

  • Collect protoplasts by centrifugation at 10,000 × g for 10 minutes

  • Resuspend in buffer containing protease inhibitors (0.5 mM PMSF) and nucleases

  • Disrupt using French press

  • Ultracentrifuge at 100,000 × g for 1 hour to collect membranes

• Membrane quality assessment:

  • Measure protein content using Bradford or BCA assays

  • Determine ATPase activity in membrane preparations

  • Assess purity by SDS-PAGE analysis

What reconstitution approaches best preserve the native function of ATP synthase complexes containing recombinant delta subunit?

Reconstitution into proteoliposomes enables functional studies:

• Detergent-mediated reconstitution protocol:

  • Solubilize purified ATP synthase components with appropriate detergents

  • Mix with preformed liposomes (typically E. coli lipids or synthetic mixtures)

  • Remove detergent using Bio-Beads or gradual dilution

  • Verify reconstitution by freeze-fracture electron microscopy or dynamic light scattering

• Functional assessment of reconstituted complexes:

  • Measure ATP synthesis driven by artificial proton gradients

  • Assess ATP-driven proton pumping using pH-sensitive fluorescent dyes

  • Compare activities of complexes with wild-type versus recombinant delta subunit

Research with mycobacterial ATP synthase has demonstrated the value of reconstituted systems for inhibitor studies and functional characterization .

How does the delta subunit of Geobacter sp. ATP synthase compare structurally with those from other bacterial species?

Comparative analysis reveals evolutionary relationships and functional conservation:

The table is based on comparative analysis from the provided search results and extrapolated predictions for Geobacter sp. Actual sequence analysis would be required to confirm these values.

What insights can be gained from studying ATP synthase delta subunit across diverse bacterial taxa?

Cross-species comparative studies reveal:

• Conservation of core functional domains despite sequence divergence

• Species-specific adaptations that may relate to environmental niches

• Potential targets for species-selective inhibitors

Methodological approach for comparative studies:

• Perform multiple sequence alignment of delta subunits across diverse bacteria

• Identify highly conserved residues likely essential for function

• Map conservation onto available structural models

• Design experiments to test the functional significance of conserved versus variable regions

How do the regulatory mechanisms of ATP synthesis/hydrolysis vary between Geobacter sp. and other bacterial ATP synthases?

Bacterial ATP synthases exhibit diverse regulatory mechanisms:

• Mycobacterial ATP synthases show suppressed ATP hydrolysis activity, mediated by:

  • The C-terminal region of the α subunit (residues 533-545)

  • The ε subunit, particularly its N-terminal residues

• For Geobacter sp. research, consider investigating:

  • Whether the delta subunit participates in regulating ATPase activity

  • If Geobacter exhibits similar latent ATPase mechanisms as observed in mycobacteria

  • How these regulatory mechanisms might relate to Geobacter's unique environmental adaptations

Methodological approach:

• Generate truncated or mutated versions of delta subunit

• Assess their impact on ATP synthesis versus hydrolysis activities

• Perform structural studies to identify potential regulatory interfaces

What emerging techniques show promise for studying dynamic interactions of the delta subunit during ATP synthase catalysis?

Several cutting-edge approaches could advance our understanding:

• Single-molecule FRET studies:

  • Introduce fluorescent labels at strategic positions

  • Monitor distance changes during catalytic cycles

  • Correlate structural dynamics with functional states

• Cryo-electron tomography of membrane-embedded complexes:

  • Visualize ATP synthase in near-native membrane environments

  • Capture different rotational states

  • Identify conformational changes in the delta subunit during catalysis

• Time-resolved structural methods:

  • Pump-probe X-ray techniques at synchrotron facilities

  • Time-resolved cryo-EM with millisecond mixing devices

  • Correlation of structural snapshots with kinetic measurements

How might the unique properties of Geobacter sp. ATP synthase inform bioenergetic applications?

Geobacter species possess unique bioenergetic capabilities:

• As electrogenic bacteria, they can transfer electrons to external acceptors

• Their ATP synthase may have adapted to function under these specialized conditions

Research applications could include:

• Bioelectrochemical systems utilizing Geobacter sp. ATP synthase properties

• Comparative studies to understand how ATP synthases adapt to diverse energy-harvesting mechanisms

• Structure-guided engineering of ATP synthases with enhanced efficiency for biotechnological applications