Recombinant Acidobacterium capsulatum ATP synthase subunit delta (atpH)

How is the atpHAGDC operon organized in Acidobacterium capsulatum compared to other bacteria?

While the specific organization of ATP synthase genes in Acidobacterium capsulatum has not been fully characterized in the provided search results, we can draw comparisons to related organisms. In Rhodobacter capsulatus, the atpHAGDC operon contains five genes coding for the F1 sector of ATP synthase, while the F0 sector genes are located elsewhere in the genome . This separation of F0 and F1 operons appears to be a feature shared among several photosynthetic bacteria, including Rhodospirillum rubrum and Rhodopseudomonas blastica .

What growth conditions should be optimized for Acidobacterium capsulatum cultivation prior to atpH expression studies?

For optimal cultivation of Acidobacterium capsulatum:

• pH range: Maintain medium pH between 3.0 and 6.0, with optimal growth typically around pH 4.0-5.0. Growth ceases at pH 6.5 and above .

• Carbon sources: Provide glucose, starch, cellobiose, or maltose as primary carbon sources. A. capsulatum cannot utilize elemental sulfur or ferrous iron as energy sources .

• Temperature: As a mesophilic organism, maintain cultures at 25-30°C.

• Oxygen conditions: While A. capsulatum is facultatively anaerobic, varying oxygen conditions may influence ATP synthase expression levels .

• Media composition: Consider the presence of exopolysaccharides coating the cells, which may affect nutrient uptake and cell harvesting processes .

What expression systems are most effective for producing recombinant Acidobacterium capsulatum ATP synthase subunit delta?

When selecting an expression system for recombinant A. capsulatum atpH, consider the following approaches:

• Escherichia coli-based systems:

  • BL21(DE3) derivatives with pET vectors containing T7 promoters offer high expression levels.

  • Consider codon optimization, as A. capsulatum may have different codon usage patterns compared to E. coli.

  • Expression as a fusion protein with solubility tags (MBP, SUMO, or TrxA) may improve folding and solubility.

• Alternative hosts:

  • Rhodobacter species may provide a more native-like environment for proper folding, especially if post-translational modifications are required .

  • Expression in a related acidophilic bacterium might preserve native structural features.

Based on experiences with other ATP synthase subunits, expression often requires optimization of:

• Induction conditions (temperature, inducer concentration, duration)

• Cell lysis methods to preserve protein integrity

• Inclusion of stabilizing agents (glycerol, specific ions) in purification buffers

Drawing from principles established in the optimization of recombinant protein production in other systems, a systematic approach testing multiple expression constructs is recommended .

How can researchers introduce site-directed mutations in the atpH gene for structure-function studies?

For site-directed mutagenesis studies of A. capsulatum atpH:

• Plasmid-based approach:

  • Clone the atpH gene into a suitable vector

  • Use PCR-based mutagenesis (e.g., QuikChange method or overlap extension PCR)

  • Transform into an expression host for protein production

  • Purify and characterize mutant proteins

• Chromosomal modification approach:
  Drawing from methods developed for Rhodobacter capsulatus :

  • Combine gene transfer agent (GTA) transduction with conjugation for introducing mutations

  • Create a complementation plasmid carrying the wild-type atpH to maintain viability

  • Replace the wild-type gene with the mutated version

A key insight from Rhodobacter research is the essential nature of ATP synthase genes, which necessitates specialized approaches for generating viable mutants . When the ATP synthase is essential, a two-step process might be required:

a) Introduce a complementing wild-type copy
b) Replace the chromosomal copy with the mutant version

Key residues to target for mutagenesis should be identified through sequence alignment with well-characterized ATP synthase delta subunits from other organisms.

What purification strategies yield the highest purity and activity of recombinant atpH?

A systematic purification strategy for recombinant A. capsulatum atpH would include:

• Initial capture:

  • If expressed with an affinity tag: Ni-NTA chromatography (His-tag), amylose resin (MBP-tag), or glutathione sepharose (GST-tag)

  • Without tag: Ion exchange chromatography based on the predicted pI of atpH

• Intermediate purification:

  • Size exclusion chromatography to separate monomeric atpH from aggregates and contaminants

  • Consider hydrophobic interaction chromatography if appropriate

• Polishing step:

  • Second ion exchange step under different pH conditions

  • Hydroxyapatite chromatography

Specific considerations for A. capsulatum atpH:

• Buffer pH may need adjustment considering the acidophilic nature of the source organism

• Include stabilizing agents (glycerol 5-10%) to prevent aggregation

• Test stability in NADH-containing buffers based on potential regulatory mechanisms

How can researchers design experiments to study the interaction between atpH and other ATP synthase subunits in Acidobacterium capsulatum?

To investigate subunit interactions within the ATP synthase complex:

• In vitro reconstitution studies:

  • Express and purify individual subunits (α, β, γ, δ, ε) of the F1 complex

  • Perform stepwise reconstitution experiments

  • Use analytical techniques (native PAGE, analytical ultracentrifugation, light scattering) to verify complex formation

  • Measure ATP hydrolysis/synthesis activities of reconstituted complexes

• Protein-protein interaction analysis:

  • Surface plasmon resonance (SPR) to determine binding kinetics between atpH and other subunits

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Crosslinking studies followed by mass spectrometry to identify interaction interfaces

• Structural studies:

  • X-ray crystallography of the recombinant atpH alone or in complex with interacting partners

  • Cryo-electron microscopy of the assembled F1 or F0F1 complex

  • NMR studies for dynamics information if the protein size permits

• FRET-based approaches:

  • Create fluorescently labeled versions of atpH and potential interaction partners

  • Monitor energy transfer as evidence of proximity and interaction

Drawing from insights on ATP synthase regulation by NADH , consider examining:

• The role of metabolic state (NADH levels) in modulating subunit interactions

• Potential conformational changes in atpH under different redox conditions

What methodological approaches are recommended for studying the effect of pH on recombinant A. capsulatum atpH structure and function?

Given A. capsulatum's adaptation to acidic environments , examining pH effects on atpH is particularly relevant:

• Structural stability across pH range:

  • Circular dichroism spectroscopy to monitor secondary structure changes at pH 3.0-7.0

  • Intrinsic tryptophan fluorescence to detect tertiary structure alterations

  • Differential scanning calorimetry to determine thermal stability at different pH values

• Functional assays at varying pH:

  • ATPase activity measurements using phosphate release assays

  • Binding assays with other subunits at different pH values

  • If possible, proton pumping assays in reconstituted liposomes

• Molecular dynamics simulations:

  • In silico prediction of pH-dependent structural changes

  • Identification of key residues with altered protonation states

Experimental design should include:

• Fine pH gradients (0.5 unit increments) within the range 3.0-7.0

• Extended incubation times to ensure equilibration

• Multiple buffer systems with overlapping pH ranges to control for buffer-specific effects

How should researchers interpret conflicting kinetic data from recombinant atpH versus native ATP synthase complexes?

When facing discrepancies between recombinant atpH and native complex data:

• Systematic comparison approach:

  • Create a comprehensive table of kinetic parameters (Km, Vmax, kcat) from both systems

  • Identify patterns in the discrepancies (e.g., consistently higher/lower values)

  • Test hypotheses about the causes of differences through targeted experiments

• Context interpretation framework:

  • Recombinant atpH alone may lack regulatory influences present in the holoenzyme

  • Native complexes maintain quaternary structure constraints potentially absent in isolated subunits

  • Consider the impact of post-translational modifications potentially missing in recombinant systems

• Data reconciliation strategies:

  • Perform assays under increasingly native-like conditions (e.g., adding other F1 subunits to recombinant atpH)

  • Examine the effects of potential regulators like NADH in both systems

  • Validate findings using orthogonal techniques (e.g., SPR vs. ITC for binding studies)

• Statistical analysis approach:

  • Apply appropriate statistical tests to determine if differences are significant

  • Use regression analysis to identify factors influencing variability

  • Consider Bayesian approaches to incorporate prior knowledge about ATP synthase behavior

What experimental controls are essential when studying NADH-dependent regulation of ATP synthase involving atpH?

Based on the discovery of NADH-controlled gatekeeper mechanisms for ATP synthase , critical controls include:

• Specificity controls:

  • Test other nucleotides (NAD+, NADPH, NADP+) to confirm NADH specificity

  • Employ varying NADH concentrations to establish dose-dependency

  • Use NADH analogs to identify structural requirements for the regulatory effect

• Mechanistic controls:

  • Mutate potential NADH-binding residues in atpH to confirm direct interaction

  • Examine the effect of NADH in the presence/absence of other subunits

  • Test whether the effect persists under different pH conditions relevant to A. capsulatum

• System validation controls:

  • Compare results from multiple expression and purification batches

  • Verify protein integrity before and after NADH exposure

  • Include positive and negative controls for activity assays

• Physiological relevance controls:

  • Correlate in vitro findings with whole-cell measurements under varying metabolic conditions

  • Develop A. capsulatum mutants with altered NADH metabolism to test effects in vivo

  • Compare findings with data from other acidophilic bacteria

How can researchers effectively compare atpH sequence and functional conservation across different Acidobacteria species?

To conduct comparative analyses across Acidobacteria:

• Sequence analysis approach:

  • Perform multiple sequence alignment of atpH from diverse Acidobacteria

  • Calculate conservation scores for each residue

  • Identify domains with higher/lower conservation

  • Map conservation onto structural models (if available)

• Structure-function correlation:

  • Use homology modeling to predict structural features of atpH from different species

  • Identify species-specific insertions/deletions

  • Correlate structural differences with habitat-specific adaptations (e.g., pH optima)

• Experimental validation:

  • Express recombinant atpH from multiple Acidobacteria species

  • Compare biochemical properties under standardized conditions

  • Perform complementation studies in model organisms

• Phylogenetic analysis:

  • Construct phylogenetic trees based on atpH sequences

  • Compare with species trees based on 16S rRNA or whole genomes

  • Identify instances of possible horizontal gene transfer or convergent evolution

These comparative approaches can reveal how the atpH gene has evolved in response to different environmental pressures, particularly in acidophilic niches where Acidobacterium capsulatum is found .

What are common challenges in recombinant atpH expression and how can they be addressed?

Researchers frequently encounter these challenges when working with recombinant ATP synthase subunits:

• Poor solubility and inclusion body formation:

  • Solution: Lower induction temperature (16-20°C), reduce inducer concentration, use solubility-enhancing tags (SUMO, MBP)

  • Alternative: Develop refolding protocols from inclusion bodies using gradual dialysis

• Proteolytic degradation:

  • Solution: Use protease-deficient host strains, include multiple protease inhibitors, optimize purification speed

  • Identification: N-terminal sequencing or mass spectrometry to identify cleavage sites

• Low activity of purified protein:

  • Solution: Test multiple buffer conditions, include stabilizing agents (glycerol, reducing agents)

  • Validation: Compare with native ATP synthase activity, ensure proper folding using spectroscopic methods

• Inconsistent yields between batches:

  • Solution: Standardize growth conditions, harvest at consistent cell density, monitor expression using reporter systems

  • Analysis: Implement statistical process control to identify sources of variability

Drawing from approaches used for other ATP synthase components , consider:

• Co-expression of multiple subunits may enhance stability

• Expression in specialized hosts adapted to membrane protein production

How can researchers optimize crystallization conditions for structural studies of A. capsulatum atpH?

For successful crystallization:

• Pre-crystallization optimization:

  • Verify protein homogeneity using dynamic light scattering

  • Perform thermal shift assays to identify stabilizing buffer conditions

  • Consider limited proteolysis to remove flexible regions that might impede crystallization

• Initial screening strategy:

  • Employ sparse matrix screens at multiple protein concentrations (5-20 mg/ml)

  • Test both vapor diffusion and batch crystallization methods

  • Include additive screens with known ATP synthase ligands

• Optimization approaches:

  • Fine-tune promising conditions through grid screens varying pH, precipitant concentration, and salt

  • Implement seeding techniques to improve crystal quality

  • Consider crystallization with binding partners or antibody fragments

• Special considerations for atpH:

  • Test crystallization at pH values relevant to A. capsulatum's natural environment (pH 3.0-6.0)

  • Include NADH in crystallization trials based on potential regulatory interactions

  • Try co-crystallization with other ATP synthase subunits or fragments

What emerging technologies could advance our understanding of A. capsulatum atpH function and regulation?

Several cutting-edge approaches show promise for ATP synthase research:

• Cryo-electron tomography:

  • Visualize ATP synthase in its native membrane environment

  • Reveal supramolecular organization and potential interactions with other complexes

  • Capture different conformational states during the catalytic cycle

• Single-molecule techniques:

  • FRET-based studies to monitor real-time conformational changes

  • Optical tweezers to measure force generation during ATP synthesis

  • High-speed atomic force microscopy to observe structural dynamics

• Integrative structural biology:

  • Combine X-ray crystallography, NMR, and cryo-EM data

  • Integrate computational modeling with experimental restraints

  • Develop time-resolved structural methods to capture transient states

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Flux balance analysis to understand ATP synthase contribution to cellular energetics

  • Network modeling of energy metabolism in acidophilic conditions

Building on the discovered NADH-dependent regulation mechanisms , future research could explore:

• Redox-sensitive structural elements within atpH

• Integration of ATP synthase function with cellular metabolic state

• Adaptation mechanisms in extremophiles like A. capsulatum

How might understanding A. capsulatum atpH contribute to broader research on acidophilic adaptation mechanisms?

Studying atpH from A. capsulatum can provide insights into:

• Molecular basis of acid tolerance:

  • Identification of acid-resistant protein structural features

  • Understanding how energy conservation occurs efficiently at low pH

  • Revealing adaptations in proton-utilizing enzymes

• Evolutionary perspectives:

  • Comparison with ATP synthases from neutrophilic bacteria

  • Identification of convergent adaptations across different acidophilic lineages

  • Understanding the evolutionary history of Acidobacteria

• Ecological implications:

  • Role of efficient energy conservation in the dominance of Acidobacteria in acidic environments

  • Contribution to biogeochemical cycling in low pH ecosystems

  • Interactions with other microorganisms in acidic environments

• Biotechnological applications:

  • Development of acid-stable enzymes for industrial processes

  • Understanding energy metabolism in acidic conditions for bioremediation applications

  • Engineering acid-tolerant organisms for various biotechnological purposes