Recombinant Burkholderia vietnamiensis ATP synthase subunit alpha (atpA), partial

What is Recombinant Burkholderia vietnamiensis ATP synthase subunit alpha (atpA)?

Recombinant Burkholderia vietnamiensis ATP synthase subunit alpha (atpA) is a critical component of the ATP synthase complex from the bacterium Burkholderia vietnamiensis, specifically strain G4/LMG 22486 (formerly classified as Burkholderia cepacia strain R1808). It functions as part of the F1 sector of ATP synthase (EC 3.6.3.14) and plays an essential role in the energy metabolism of the organism. The recombinant form is produced in yeast expression systems with a purity of >85% as determined by SDS-PAGE analysis . This protein is significant for both fundamental research on bacterial energy metabolism and applied research exploring potential therapeutic targets.

What are the optimal storage conditions for maintaining atpA stability and activity?

For optimal stability, store recombinant B. vietnamiensis atpA at -20°C for regular use, or at -80°C for extended storage periods. Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be strictly avoided as they significantly compromise protein integrity. For reconstitution, briefly centrifuge the vial before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To enhance stability, add glycerol to a final concentration between 5-50% (standard recommendation is 50%) before storing in aliquots . The shelf life varies based on storage conditions: approximately 6 months for liquid preparations and 12 months for lyophilized formulations when stored at -20°C/-80°C.

How does B. vietnamiensis atpA differ from ATP synthase subunits in other bacterial species?

B. vietnamiensis atpA shares structural and functional similarities with ATP synthase subunits from other bacteria but possesses species-specific sequence variations that reflect its adaptation to the unique metabolic requirements of B. vietnamiensis. Unlike the ATP synthase α subunit in Mycobacterium tuberculosis, which contains a distinctive 36-amino acid C-terminal domain that suppresses ATPase activity , B. vietnamiensis atpA lacks this suppressive domain. This allows it to function bidirectionally as both an ATP synthase and an ATPase. B. vietnamiensis belongs to the Burkholderia cepacia complex, which consists of multiple genomovars with significant genetic diversity despite their phenotypic similarities . This genetic diversity extends to their metabolic enzymes, including the ATP synthase components.

What molecular techniques are effective for identifying and characterizing B. vietnamiensis atpA in research samples?

Several molecular biology techniques are particularly effective for identifying and characterizing B. vietnamiensis atpA:

• PCR-based identification: While general Burkholderia-specific primers like BuRa-16-1 and BuRa-16-2 can identify the genus, specific primer sets must be used for B. vietnamiensis detection. The primer pair GB-F and GBN2-R, which works with most diazotrophic Burkholderia species, notably does not amplify B. vietnamiensis sequences .

• Restriction Fragment Length Polymorphism (RFLP) analysis: 16S rDNA RFLP analysis can help identify B. vietnamiensis from other Burkholderia species. For more definitive identification, recA gene RFLP analysis provides sufficient nucleotide sequence variation to separate all five B. cepacia complex genomovars .

• Amplified Ribosomal DNA Restriction Analysis (ARDRA): This technique has been successfully used to distinguish different Burkholderia species, with B. vietnamiensis showing characteristic ARDRA profiles .

• Sequence analysis: Complete recA nucleotide sequencing provides the most definitive identification, with B. vietnamiensis forming a distinct cluster in phylogenetic analyses based on recA sequences .

How can researchers differentiate B. vietnamiensis from other members of the Burkholderia cepacia complex at the genetic level?

Differentiating B. vietnamiensis from other members of the Burkholderia cepacia complex requires targeted genetic analysis:

What is the genomic organization of the atpA gene in B. vietnamiensis, and how does it compare to other Burkholderia species?

The atpA gene in B. vietnamiensis is located on the largest chromosome of its multi-replicon genome. Unlike earlier reports suggesting gene duplication in some Burkholderia species, comprehensive genetic analysis reveals that B. cepacia complex strains, including B. vietnamiensis, typically contain only a single copy of the recA gene on the largest chromosome . This contrasts with previous suggestions that B. cepacia might have a diploid recA gene distributed across two large chromosomes.

The atpA gene is part of the ATP synthase operon, which encodes the components of the F-type ATP synthase. The genomic context and organization of this operon in B. vietnamiensis is largely conserved among Burkholderia species, despite significant genomic diversity in other regions. The single-copy nature of atpA makes it a more reliable target for molecular diagnostics compared to the ribosomal RNA genes, which exist in multiple copies and are distributed across the multiple replicons of the B. cepacia complex genome .

What methods are most effective for assessing the enzymatic activity of recombinant B. vietnamiensis atpA?

Several complementary approaches can be used to effectively assess the enzymatic activity of recombinant B. vietnamiensis atpA:

• ATP Synthesis Assay: Measuring ATP production using luciferin-luciferase bioluminescence assays in reconstituted liposomes or inverted membrane vesicles containing the complete ATP synthase complex. This approach requires integration of recombinant atpA into a functional complex.

• ATP Hydrolysis Assay: Quantifying inorganic phosphate release using colorimetric methods (e.g., malachite green assay) to measure the reverse ATPase activity. Unlike Mycobacterium tuberculosis ATP synthase, which lacks reverse activity due to a 36-amino acid C-terminal extension in subunit α , B. vietnamiensis ATP synthase is expected to demonstrate both synthetic and hydrolytic activities.

• Proton Pumping Assays: Using pH-sensitive fluorescent dyes to measure proton movement across membranes during ATP hydrolysis in reconstituted proteoliposomes.

• Binding Affinity Studies: Isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) to measure nucleotide binding affinities and kinetics, which provide insights into the functional properties of the isolated α subunit.

• Conformational Change Analysis: Fluorescence resonance energy transfer (FRET) or limited proteolysis approaches to detect conformational changes associated with nucleotide binding and hydrolysis.

How does the partial recombinant form of B. vietnamiensis atpA affect its functionality in experimental systems?

The partial recombinant form of B. vietnamiensis atpA presents specific considerations for functional studies:

• Structural Implications: The partial nature of the recombinant protein may affect its folding and stability, potentially limiting certain structure-function analyses. Researchers should verify which domains are present in the partial construct (CSB-YP002344BPV) to determine suitability for specific experiments .

• Complex Assembly: Complete ATP synthase functionality requires assembly of multiple subunits. The partial atpA may have limited ability to form stable complexes with partner subunits, requiring careful experimental design when studying higher-order interactions.

• Nucleotide Binding: If the nucleotide-binding domains are intact in the partial construct, binding studies remain feasible, though binding kinetics may differ from the full-length protein.

• Functional Reconstitution: For studies requiring complete functional reconstitution, supplementation with the missing regions or co-expression with other subunits may be necessary to achieve physiologically relevant activities.

• Experimental Controls: Comparisons with full-length atpA or with the corresponding regions from related species can help interpret results obtained with the partial construct.

What are the key structural features of B. vietnamiensis atpA that determine its catalytic properties?

The catalytic properties of B. vietnamiensis atpA are determined by several key structural features:

• Nucleotide Binding Domain: Contains the Walker A and B motifs responsible for ATP binding and hydrolysis. The precise amino acid composition of these motifs influences nucleotide affinity and catalytic rate.

• DELSEED Region: This conserved motif (or its equivalent in bacterial systems) participates in energy coupling between catalytic sites and the rotating central stalk during catalysis.

• Catalytic Interface: The interface between α and β subunits forms the catalytic sites, with both subunits contributing residues that participate in ATP binding and hydrolysis.

• C-terminal Domain: Unlike M. tuberculosis, B. vietnamiensis atpA lacks the 36-amino acid C-terminal extension that suppresses ATPase activity , allowing this enzyme to function bidirectionally.

• N-terminal Domain: Involved in subunit interactions that stabilize the F1 sector of ATP synthase and influence the cooperativity between catalytic sites.

The specific amino acid sequence and structure of these regions in B. vietnamiensis atpA have evolved to optimize ATP synthesis under the organism's native environmental conditions, including pH, temperature optima, and metabolic requirements of this soil and rhizosphere bacterium.

What are the recommended protocols for reconstituting recombinant B. vietnamiensis atpA for functional studies?

For optimal reconstitution of recombinant B. vietnamiensis atpA (CSB-YP002344BPV), follow these methodological steps:

• Initial Preparation:

  • Briefly centrifuge the vial before opening to ensure all contents are at the bottom

  • Allow protein to warm to room temperature (if stored frozen)

• Reconstitution Procedure:

  • Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Gently mix by pipetting or slow vortexing; avoid generating foam

  • For long-term storage preparations, add glycerol to a final concentration of 5-50% (50% is recommended as the standard)

• For Functional Complex Assembly:

  • If studying the complete ATP synthase complex, combine with other purified subunits in a molar ratio matching the native complex stoichiometry

  • Incubate under controlled conditions (typically 4-25°C) to allow complex formation

  • Verify assembly by native PAGE, size exclusion chromatography, or electron microscopy

• Membrane Reconstitution:

  • For proton-pumping studies, reconstitute into liposomes using established protocols

  • Prepare liposomes from E. coli lipids or synthetic phospholipids (POPC/POPE mixtures)

  • Add detergent-solubilized protein to liposomes and remove detergent using Bio-Beads or dialysis

  • Verify orientation by protease protection assays

• Quality Control:

  • Verify protein integrity after reconstitution by SDS-PAGE

  • Assess functionality through ATP binding or hydrolysis assays

  • Monitor stability at working temperature over time

How can researchers troubleshoot common issues when working with recombinant B. vietnamiensis atpA?

What experimental systems are best suited for studying the interaction of B. vietnamiensis atpA with other ATP synthase subunits?

Several experimental systems are well-suited for studying the interactions between B. vietnamiensis atpA and other ATP synthase subunits:

• Yeast Two-Hybrid (Y2H) System:

  • Allows detection of binary protein-protein interactions

  • Useful for mapping interaction domains between atpA and other specific subunits

  • Limitations include potential false positives and requirement for nuclear localization

• Co-immunoprecipitation (Co-IP):

  • Enables detection of native protein complexes

  • Can be performed with tagged recombinant proteins or using subunit-specific antibodies

  • Allows identification of interaction partners under near-physiological conditions

• Surface Plasmon Resonance (SPR):

  • Provides quantitative binding kinetics and affinities

  • Allows real-time monitoring of association and dissociation

  • Requires immobilization of one interaction partner

• Fluorescence Resonance Energy Transfer (FRET):

  • Enables visualization of protein interactions in live cells or reconstituted systems

  • Provides spatial information about interacting components

  • Requires fluorescent labeling of interaction partners

• Cryo-Electron Microscopy:

  • Provides structural details of assembled complexes

  • Can reveal conformational changes associated with different functional states

  • Requires specialized equipment and sample preparation

• Bacterial Two-Hybrid System:

  • Similar to Y2H but functions in bacterial hosts, potentially providing a more native environment

  • Useful for membrane protein interactions

  • Less prone to certain artifacts compared to Y2H

• Proteoliposome Reconstitution:

  • Enables functional studies of assembled complexes

  • Allows assessment of how interactions affect enzymatic activity

  • Provides a membrane environment that better mimics native conditions

Selecting the appropriate system depends on the specific research question, with complementary approaches often providing the most comprehensive understanding of subunit interactions.

How can B. vietnamiensis atpA be utilized in research addressing bacterial pathogenicity and host interactions?

B. vietnamiensis atpA can serve as a valuable tool in pathogenicity and host interaction research:

• Cystic Fibrosis Research: As B. vietnamiensis belongs to the B. cepacia complex, which has significant implications for people with cystic fibrosis , its ATP synthase components can be studied to understand metabolic adaptations during infection. The atpA subunit may play a role in bacterial persistence under the unique conditions of CF lungs.

• Vaccine Development: As a conserved bacterial protein, atpA could be explored as a potential vaccine target. Studies could evaluate its immunogenicity, surface accessibility, and the protective efficacy of anti-atpA antibodies against B. vietnamiensis infections.

• Host-Pathogen Metabolic Interactions: Research can explore how ATP synthase activity modulates bacterial energy metabolism during host colonization, potentially revealing how B. vietnamiensis adapts its bioenergetic processes in different host microenvironments.

• Drug Target Identification: Comparative structural analysis between bacterial and human ATP synthase α subunits can identify unique features of B. vietnamiensis atpA that could be targeted by novel antimicrobials with minimal host toxicity.

• Biofilm Formation Studies: Investigating how atpA activity correlates with biofilm development could provide insights into persistence mechanisms, as energy metabolism is crucial for biofilm formation and maintenance.

• Genetic Manipulation Studies: Creating conditional atpA mutants could help determine its essentiality under different growth conditions relevant to pathogenesis.

What are the current challenges and emerging approaches in studying the structure-function relationship of B. vietnamiensis atpA?

Current challenges and emerging approaches in B. vietnamiensis atpA research include:

• Structural Determination Challenges:

  • Obtaining high-resolution structures of membrane protein complexes remains difficult

  • Emerging approaches: Leveraging advances in cryo-electron microscopy and integrative structural biology to determine complex structures without crystallization

• Functional Analysis in Native Environment:

  • Studying function within the native membrane context is challenging

  • Emerging approaches: Nanodiscs and native mass spectrometry to study membrane proteins in near-native lipid environments

• Dynamic Conformational Changes:

  • Capturing transient conformational states during catalysis

  • Emerging approaches: Time-resolved structural methods, single-molecule FRET, and molecular dynamics simulations to model conformational transitions

• Species-Specific Functions:

  • Understanding the unique aspects of B. vietnamiensis atpA compared to other species

  • Emerging approaches: Comparative genomics and molecular evolution analyses to identify selective pressures on atpA structure

• Integration with Metabolic Networks:

  • Connecting atpA function to broader metabolic adaptations

  • Emerging approaches: Systems biology approaches integrating proteomics, metabolomics, and fluxomics data

• Recombinant Expression Limitations:

  • Producing full-length, properly folded protein in sufficient quantities

  • Emerging approaches: Cell-free protein synthesis systems and novel fusion tags to enhance solubility

How does B. vietnamiensis atpA contribute to the organism's adaptation to different environmental conditions, particularly in nitrogen fixation contexts?

B. vietnamiensis atpA plays crucial roles in environmental adaptation, particularly in nitrogen fixation contexts:

• Energetic Support for Nitrogen Fixation: As a diazotrophic (nitrogen-fixing) Burkholderia species , B. vietnamiensis requires substantial energy for nitrogen fixation through nitrogenase. The ATP synthase complex, including atpA, provides the necessary ATP to power this energetically demanding process.

• Adaptation to Microoxic Conditions: Nitrogen fixation typically occurs under microoxic conditions, which affects respiratory ATP generation. The ATP synthase complex likely has adaptations to function optimally under these conditions, with atpA playing a key role in maintaining energy homeostasis.

• pH Adaptation: B. vietnamiensis colonizes diverse environments with varying pH levels, from acidic soils to the more neutral rhizosphere. AtpA likely contains structural adaptations that allow ATP synthase to function across these pH ranges, maintaining proton gradient conversion to ATP.

• Rhizosphere Colonization: In the plant rhizosphere, B. vietnamiensis engages in beneficial interactions with plants, particularly sugarcane and maize . AtpA contributes to the energy metabolism required for competitive colonization of these ecological niches.

• Metabolic Flexibility: Unlike some specialized bacteria, B. vietnamiensis can utilize diverse carbon sources. The ATP synthase complex, with atpA as a key component, supports this metabolic versatility by efficiently coupling proton gradients generated from different respiratory substrates to ATP synthesis.

• Stress Response Integration: Under environmental stresses like nutrient limitation, pH extremes, or oxidative stress, the regulation and activity of ATP synthase is modulated. The atpA subunit likely contains regulatory sites that respond to these stress conditions, allowing the bacterium to adjust its energy metabolism accordingly.

Understanding these adaptations provides insights into how B. vietnamiensis thrives in its ecological niches and contributes to its potential applications in sustainable agriculture as a plant growth-promoting bacterium.