Recombinant Gluconacetobacter diazotrophicus ATP synthase subunit alpha (atpA), partial
Description
Overview of ATP Synthase in Gluconacetobacter diazotrophicus
ATP synthase in G. diazotrophicus comprises multiple subunits, including:
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Subunit alpha (atpA): Part of the F1 sector, involved in ATP synthesis/hydrolysis.
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Subunit a (atpB): Essential for proton translocation in the F0 sector .
The genome of G. diazotrophicus (strain PAL5) includes a 3.9 Mb chromosome with genes encoding ATP synthase components, highlighting its metabolic versatility .
Recombinant Protein Production
Recombinant ATP synthase subunits from G. diazotrophicus are typically expressed in E. coli systems for structural and functional studies. Examples include:
| Subunit | Expression Host | Tag | Form | Source |
|---|---|---|---|---|
| Subunit c (atpE) | E. coli | N-terminal His | Lyophilized powder | |
| Subunit a (atpB) | E. coli | Not specified | Lyophilized powder |
Key features of recombinant production:
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Storage: Lyophilized proteins are stable for 12 months at -20°C/-80°C .
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Reconstitution: Requires sterile water and glycerol for solubility .
Nitrogen Fixation and Energy Metabolism
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G. diazotrophicus relies on ATP synthase for energy during nitrogen fixation under microaerobic conditions .
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Disruption of nitrogen-associated genes (e.g., amtB1, amtB2) impacts cellular fitness, indirectly linking ATP synthase activity to diazotrophic growth .
Membrane Complex Assembly
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Studies on the AldFGH complex (a related membrane-bound dehydrogenase) revealed that subunit interactions (e.g., AldF for membrane binding) are critical for enzymatic activity .
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Heterologous expression of G. diazotrophicus AldFGH in Acetobacter restored ubiquinone reduction (45 mU/mg protein) and oxygen consumption (56 mU/mg protein) , illustrating the importance of subunit cooperativity.
Genomic Context
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The G. diazotrophicus genome contains 3,938 coding sequences, with ATP synthase genes forming part of its core metabolic machinery .
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Comparative genomics identified 894 core genes shared with related bacteria, including those for energy transduction .
Protein Secretion Systems
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A type II secretory system (e.g., lsd gene cluster) facilitates extracellular enzyme transport in G. diazotrophicus, which could inform recombinant protein secretion strategies .
Research Gaps and Future Directions
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Direct Studies on atpA: Current literature focuses on subunits atpB and atpE; recombinant atpA characterization remains unexplored.
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Functional Assays: Enzyme activity assays (e.g., ATP hydrolysis rates) for recombinant subunits are needed.
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Structural Biology: Cryo-EM or X-ray crystallography could elucidate atpA’s role in the F1F0 complex.
Applications in Biotechnology
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Plant Growth Promotion: G. diazotrophicus enhances host nutrient uptake via nitrogen fixation and phytohormone synthesis . Engineered strains with modified ATP synthase subunits could improve metabolic efficiency.
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Industrial Enzymes: Recombinant ATP synthase subunits may serve as templates for bioenergy applications (e.g., synthetic ATP production systems) .
Enzyme Activity in Recombinant Systems
| Strain/Plasmid | Intracellular LsdA Activity (U mg⁻¹) | Extracellular LsdA Activity (U ml⁻¹) |
|---|---|---|
| G. diazotrophicus SRT4 | 20 ± 4 | 32 ± 4 |
| G. diazotrophicus M31 | 75 ± 6 | 5 ± 2 |
| M31(pALS117) | 19 ± 3 | 28 ± 4 |
Table adapted from studies on the type II secretion system .
Product Specs
Target Background
KEGG: gdi:GDI0694
STRING: 272568.GDI_0694
Q&A
What is Gluconacetobacter diazotrophicus and why is its ATP synthase important for research?
Gluconacetobacter diazotrophicus is a nonpathogenic, nitrogen-fixing endophyte primarily found in sugarcane and other sucrose-rich crops . The bacterium's ATP synthase complex is crucial for energy metabolism, particularly during nitrogen fixation, which requires substantial energy input. The alpha subunit (atpA) forms part of the F1 catalytic domain that synthesizes ATP. Research on this subunit helps understand the energy metabolism supporting nitrogen fixation capabilities, which makes G. diazotrophicus valuable for sustainable agriculture applications.
What are the typical expression patterns of ATP synthase genes in G. diazotrophicus?
ATP synthase genes in G. diazotrophicus are typically constitutively expressed, similar to other housekeeping genes required for basic cellular metabolism. The expression pattern may resemble that of the levansucrase (lsdA) gene, which is constitutively expressed to facilitate the utilization of plant sucrose . The gene likely follows the characteristic G+C content (64-74%) and codon usage patterns observed in other G. diazotrophicus genes, with a strong preference for G and C in the third position of codons .
What are the optimal conditions for expressing recombinant G. diazotrophicus atpA in E. coli expression systems?
For successful expression of recombinant G. diazotrophicus atpA in E. coli, researchers should consider the following optimization parameters:
| Parameter | Recommended Condition | Rationale |
|---|---|---|
| Host strain | BL21(DE3) or Rosetta | Rosetta strains compensate for rare codons found in G. diazotrophicus |
| Expression vector | pET system with T7 promoter | Provides tight regulation and high expression |
| Induction temperature | 18-22°C | Lower temperatures reduce inclusion body formation |
| IPTG concentration | 0.1-0.5 mM | Lower concentrations improve soluble protein yield |
| Growth medium | LB supplemented with 1% glucose | Glucose reduces basal expression before induction |
| Induction time | OD600 of 0.6-0.8 | Mid-log phase optimizes expression |
| Post-induction period | 16-18 hours | Extended expression at lower temperatures |
The high G+C content (64-74%) and unique codon usage of G. diazotrophicus genes necessitate codon optimization or use of strains supplemented with rare tRNAs for efficient heterologous expression.
What purification strategies are most effective for isolating recombinant G. diazotrophicus atpA?
A multi-step purification approach yields the highest purity recombinant atpA protein:
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Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins
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Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol
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Imidazole gradient: 20-250 mM for washing and elution
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Intermediate Purification: Ion exchange chromatography
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Q-Sepharose column at pH 8.0 (atpA theoretical pI ~5.5)
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Linear NaCl gradient (50-500 mM)
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Polishing Step: Size exclusion chromatography
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Superdex 200 column in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol
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Quality Control:
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SDS-PAGE analysis: >95% purity
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Western blot confirmation with anti-His or anti-atpA antibodies
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Mass spectrometry verification
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The purification strategy should account for potential protein-protein interactions, as atpA naturally forms complexes with other ATP synthase subunits.
How can the ATP synthase activity of recombinant G. diazotrophicus atpA be measured in vitro?
The ATP synthase activity can be measured using the following methodologies:
Coupled Enzyme Assay:
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Reaction mixture: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 50 mM KCl, 2.5 mM ATP
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Coupling enzymes: Pyruvate kinase and lactate dehydrogenase
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Substrates: Phosphoenolpyruvate and NADH
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Measurement: Spectrophotometric monitoring of NADH oxidation at 340 nm
ATP Hydrolysis Assay:
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Reaction mixture: 40 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 2 mM ATP
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Reaction time: 10-30 minutes at 37°C
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Detection: Released inorganic phosphate using malachite green reagent
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Quantification: Absorbance measurement at 620 nm against a phosphate standard curve
Reconstitution Studies:
Combine recombinant atpA with other ATP synthase subunits to reconstitute partial or complete F1 complex for more authentic activity measurements.
How does nitrogen fixation in G. diazotrophicus affect ATP synthase expression and activity?
The nitrogen fixation process is energetically demanding, requiring substantial ATP input. Research indicates that during nitrogen fixation:
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Expression Regulation: ATP synthase gene expression likely increases during active nitrogen fixation to meet elevated energy demands
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Metabolic Adaptation: The bacterium may adjust its ATP synthase activity to balance energy production with nitrogen fixation requirements
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Environmental Response: ATP synthase activity changes in response to oxygen levels, as nitrogenase is oxygen-sensitive
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Host Plant Interaction: When associated with host plants, G. diazotrophicus modulates ATP synthase expression based on carbon source availability and nitrogen status
The constitutive expression pattern observed in other G. diazotrophicus genes like levansucrase suggests that atpA may be similarly regulated, with potential fine-tuning mechanisms to accommodate varying energy demands during nitrogen fixation.
How can recombinant G. diazotrophicus atpA be utilized for structural biology studies?
For structural biology studies of G. diazotrophicus atpA, researchers should consider:
X-ray Crystallography Approach:
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Protein preparation: High concentration (>10 mg/mL) of ultra-pure protein
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Crystallization screening: Commercial sparse matrix screens at varying temperatures (4°C, 16°C, 20°C)
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Optimization: Fine-tuning of promising crystallization conditions
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Data collection: Synchrotron radiation facilities for high-resolution diffraction patterns
Cryo-EM Studies:
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Sample preparation: Purified atpA alone or reconstituted with other ATP synthase subunits
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Grid preparation: Appropriate hole size and ice thickness optimization
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Data collection parameters: Defocus range -1.0 to -3.0 μm, total electron dose 40-60 e-/Ų
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Processing: Motion correction, CTF estimation, particle picking, classification, and refinement
NMR Studies for Dynamics:
For specific domains or smaller constructs of atpA that are amenable to NMR analysis:
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Isotopic labeling: ¹⁵N, ¹³C, and ²H incorporation during expression
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Spectral assignment: Triple-resonance experiments
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Dynamics measurements: Relaxation experiments (T₁, T₂, NOE)
How does G. diazotrophicus atpA compare to homologous proteins in related bacterial species?
Comparative analysis reveals significant insights into the evolutionary adaptation of G. diazotrophicus atpA:
| Species | Sequence Identity (%) | Notable Differences | Functional Implications |
|---|---|---|---|
| Acetobacter aceti | 85-90 (estimated) | Conservative substitutions in catalytic regions | Maintained ATP synthesis function |
| Gluconobacter oxydans | 80-85 (estimated) | Variations in regulatory domains | Different regulatory responses |
| Komagataeibacter xylinus | 75-80 (estimated) | Unique surface-exposed residues | Adaptation to different cellular environments |
| Azospirillum brasilense | 65-70 (estimated) | Significant differences in non-catalytic domains | Adaptation to different ecological niches |
The high G+C content characteristic of G. diazotrophicus genes (64-74%) is likely reflected in the atpA gene as well, influencing its codon usage and potentially its expression efficiency in heterologous systems.
What can G. diazotrophicus atpA tell us about the evolution of energy metabolism in plant-associated bacteria?
The ATP synthase alpha subunit in G. diazotrophicus provides several evolutionary insights:
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Endophytic Adaptation: Comparison with free-living relatives reveals adaptations specific to the plant-associated lifestyle
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Metabolic Efficiency: Modifications that optimize energy production in the sucrose-rich plant environment, complementing the bacterium's dependence on plant-derived sucrose through secreted levansucrase
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Co-evolution Signatures: Sequence features that reflect long-term association with specific host plants
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Horizontal Gene Transfer: Assessment of whether any atpA features were acquired through horizontal gene transfer events
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Selection Pressure: Analysis of synonymous vs. non-synonymous substitutions to identify regions under positive selection
Similar to the conservation observed in the levansucrase-levanase (lsdA-lsdB) locus across different G. diazotrophicus strains , the ATP synthase genes likely show conservation patterns that reflect their essential metabolic function.
What are common issues when working with recombinant G. diazotrophicus atpA and how can they be addressed?
| Challenge | Possible Causes | Solutions |
|---|---|---|
| Low expression yield | Codon bias, toxicity | Optimize codon usage, use Rosetta strains, lower induction temperature |
| Inclusion body formation | Rapid overexpression, improper folding | Reduce IPTG concentration, express at 16-18°C, add solubility tags |
| Protein instability | Loss of native interactions | Co-express with other ATP synthase subunits, optimize buffer conditions |
| Limited solubility | Hydrophobic regions, aggregation | Add solubilizing agents (0.1% Triton X-100), optimize salt concentration |
| Proteolytic degradation | Exposed cleavage sites | Add protease inhibitors, reduce expression time, purify at 4°C |
| Loss of activity | Denaturation, cofactor loss | Include Mg²⁺ in all buffers, avoid freeze-thaw cycles, store with glycerol |
The high G+C content (64-74%) and unique codon usage patterns observed in G. diazotrophicus genes contribute significantly to expression challenges in heterologous systems.
How can researchers verify the authenticity and integrity of purified recombinant G. diazotrophicus atpA?
A comprehensive validation approach includes:
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Identity Confirmation:
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Mass spectrometry peptide fingerprinting
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N-terminal sequencing
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Western blot with specific antibodies
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Structural Integrity:
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Circular dichroism to assess secondary structure
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Thermal shift assay to evaluate stability
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Size exclusion chromatography to verify oligomeric state
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Functional Validation:
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ATP hydrolysis activity measurements
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Binding assays for nucleotides (ATP, ADP)
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Reconstitution with other ATP synthase subunits to assess complex formation
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Quality Control Metrics:
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Purity >95% by SDS-PAGE and size exclusion chromatography
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Endotoxin levels <0.1 EU/mg for cellular applications
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Absence of proteolytic fragments by Western blot
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What are promising research areas involving G. diazotrophicus atpA for agricultural applications?
Several innovative research directions hold promise:
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Engineered Energy Efficiency: Creating G. diazotrophicus strains with modified ATP synthase to enhance nitrogen fixation efficiency under agricultural conditions
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Stress Adaptation: Understanding how ATP synthase contributes to bacterial survival under drought, salinity, or temperature stresses in crop systems
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Host Range Expansion: Investigating whether ATP synthase modifications could help extend G. diazotrophicus colonization to non-traditional crop species
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Signaling Roles: Exploring potential secondary functions of ATP synthase components in plant-microbe signaling pathways
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Consortia Engineering: Designing bacterial consortia with complementary ATP synthesis capacities for enhanced plant growth promotion
These directions build upon the established importance of G. diazotrophicus as a beneficial endophyte in sugarcane and other crops, potentially expanding its agricultural applications .
How might systems biology approaches enhance our understanding of G. diazotrophicus atpA in the context of nitrogen fixation?
Systems biology offers powerful frameworks to understand the role of ATP synthase in nitrogen fixation:
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Multi-omics Integration:
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Transcriptomics: Correlation of atpA expression with nitrogen fixation genes
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Proteomics: ATP synthase complex stoichiometry under different conditions
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Metabolomics: ATP/ADP ratios and energy charge measurements
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Fluxomics: Carbon flux through central metabolism to support ATP production
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Computational Modeling:
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Genome-scale metabolic models incorporating ATP synthase constraints
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Kinetic models of nitrogen fixation energy requirements
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Ecological models of plant-microbe energy exchange
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Single-Cell Approaches:
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Heterogeneity in ATP synthesis capacity within bacterial populations
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Correlation between ATP synthase activity and nitrogen fixation at single-cell level
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In Planta Studies:
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ATP dynamics during colonization and establishment
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Energy contribution to the plant-microbe mutualism
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These approaches would complement traditional biochemical characterization to provide a comprehensive understanding of how energy metabolism supports the beneficial plant-microbe interaction involving G. diazotrophicus.
