Recombinant Burkholderia vietnamiensis ATP synthase subunit delta (atpH)

What is Burkholderia vietnamiensis ATP synthase subunit delta (atpH)?

Burkholderia vietnamiensis ATP synthase subunit delta (atpH) is a critical component of the F-type ATP synthase complex in this bacterial species. The protein functions as part of the central stalk of the F1 sector, connecting the catalytic F1 portion with the membrane-embedded Fo portion. The protein consists of 179 amino acids and plays a crucial role in energy transduction during ATP synthesis. This subunit is encoded by the atpH gene and has the UniProt accession number A4JA32 in B. vietnamiensis strain G4/LMG 22486 .

How does atpH function within the ATP synthase complex?

The delta subunit serves as a crucial structural component of the F1Fo ATP synthase complex, forming part of the central stalk that connects the F1 and Fo portions. During ATP synthesis, proton translocation through the Fo domain drives rotation of the central stalk, which includes the delta subunit. This rotation causes conformational changes in the catalytic beta subunits, facilitating ATP synthesis. The delta subunit therefore plays an essential role in the mechanical coupling between proton translocation and ATP synthesis, contributing to the fundamental process of energy conversion in bacterial cells.

What expression systems are suitable for producing recombinant B. vietnamiensis atpH?

The recombinant B. vietnamiensis ATP synthase subunit delta can be successfully expressed in yeast expression systems as evidenced by commercial preparations . Other potential expression systems include:

For optimal expression, codon optimization for the target expression system should be considered, particularly when expressing bacterial proteins in eukaryotic hosts.

What is the recommended protocol for storage and reconstitution of recombinant atpH protein?

For optimal preservation of recombinant B. vietnamiensis ATP synthase subunit delta:

Storage:

• Store at -20°C for short-term storage

• For extended storage, conserve at -20°C or -80°C

• Avoid repeated freeze-thaw cycles, which can significantly reduce protein activity

Reconstitution:

• Briefly centrifuge the vial before opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is recommended for optimal stability)

• Aliquot for long-term storage to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

How can the purity and activity of recombinant atpH be assessed?

The purity of recombinant B. vietnamiensis ATP synthase subunit delta can be assessed through:

• SDS-PAGE analysis: Commercial preparations typically show >85% purity by SDS-PAGE

• Western blotting: Using antibodies specific to atpH or to an added tag

• Size exclusion chromatography: To verify the homogeneity of the protein preparation

• Mass spectrometry: For precise molecular weight determination and sequence verification

Functional assessment can include:

• ATP synthase reconstitution assays: Incorporating the recombinant delta subunit into ATP synthase complexes

• Binding assays with other ATP synthase subunits (particularly gamma and alpha subunits)

• Structural integrity assessment through circular dichroism spectroscopy

• Thermal stability assays to evaluate proper folding

How can CRISPRi be applied to study atpH gene expression in B. vietnamiensis?

CRISPRi (CRISPR interference) provides a powerful tool for gene silencing in Burkholderia species. Based on research with similar Burkholderia species, a broad-host-range CRISPRi toolkit can be applied to B. vietnamiensis for silencing atpH expression:

• Design multiple guide RNAs (gRNAs) targeting either the promoter region or early coding sequence of atpH. For most effective repression, target the template strand shortly after the transcription start site .

• Express the gRNA along with catalytically inactive Cas9 (dCas9) under a rhamnose-inducible promoter.

• A typical approach involves:

  • Integration of codon-optimized dcas9 into the chromosome using the mini-CTX1 integration vector, which targets the serine tRNA attB site

  • Delivery of the gRNA on a separate plasmid

• Validate repression through:

  • RT-qPCR to measure atpH mRNA levels

  • Western blotting to assess protein expression

  • Phenotypic assays such as growth rate analysis and ATP synthesis assays

• The system can be tuned by varying rhamnose concentrations (0.005% to 0.2%) to achieve different levels of repression, creating an effective dose-dependent knockdown .

This approach allows for precise temporal control of atpH expression and avoids the challenges associated with essential gene deletion.

What approaches can be used to study protein-protein interactions involving atpH in ATP synthase complexes?

Several methodological approaches can be employed to investigate protein-protein interactions involving B. vietnamiensis atpH:

• Co-immunoprecipitation (Co-IP)

  • Using antibodies against atpH or epitope tags to pull down interacting partners

  • Mass spectrometry analysis of co-precipitated proteins

• Bacterial Two-Hybrid Systems

  • Modified for use in Burkholderia or heterologous hosts

  • Allows screening for direct protein-protein interactions

• Surface Plasmon Resonance (SPR)

  • Provides real-time, label-free detection of molecular interactions

  • Can determine binding kinetics and affinity constants

• Crosslinking Mass Spectrometry

  • Chemical crosslinking of protein complexes followed by MS analysis

  • Identifies specific interaction surfaces

• Förster Resonance Energy Transfer (FRET)

  • Requires fluorescent protein fusions

  • Allows visualization of interactions in living cells

• Reconstitution Studies

  • In vitro reconstitution of ATP synthase complexes with purified components

  • Functional studies to assess the impact of mutations or subunit alterations

These techniques can reveal the specific interfaces between atpH and other subunits of the ATP synthase complex, providing insights into the structural basis of complex assembly and function.

How can site-directed mutagenesis be applied to investigate key functional residues in atpH?

Site-directed mutagenesis is a valuable approach for identifying critical functional residues in B. vietnamiensis atpH:

This approach can reveal residues essential for structural integrity, subunit interactions, and the mechanical function of atpH in the ATP synthase complex.

How does B. vietnamiensis atpH compare to homologous proteins in other bacterial species?

Comparative analysis of ATP synthase delta subunits across bacterial species reveals important evolutionary relationships and functional conservation:

These comparisons can provide insights into the essential conserved features of ATP synthase delta subunits and highlight species-specific adaptations that might be relevant to bacterial physiology and energy metabolism.

What are the implications of atpH research for understanding bacterial energy metabolism?

Research on B. vietnamiensis atpH has several important implications for understanding bacterial energy metabolism:

• Bioenergetic Efficiency: Investigation of atpH structure and function can reveal adaptations that optimize ATP synthesis efficiency in different environmental conditions.

• Bacterial Adaptation: Comparative studies across Burkholderia species can illuminate how ATP synthase components have evolved to support survival in diverse ecological niches.

• Antimicrobial Targets: ATP synthase is an essential complex, making it a potential target for novel antimicrobials. Structural and functional differences between bacterial and human ATP synthases can be exploited for selective targeting.

• Biotechnological Applications: Understanding the molecular details of bacterial ATP synthases can inform the development of bioengineered systems for energy production or biotransformation processes.

• Environmental Adaptations: Burkholderia species occupy diverse ecological niches, and variations in their ATP synthase components may reflect adaptations to specific energy availability and environmental conditions.

What are common troubleshooting strategies when working with recombinant atpH?

Researchers working with recombinant B. vietnamiensis ATP synthase subunit delta may encounter several challenges. Here are evidence-based troubleshooting strategies:

• Poor Expression Yield:

  • Optimize codon usage for the expression host

  • Test different promoter strengths and induction conditions

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

  • Reduce expression temperature to 16-20°C to improve protein folding

• Protein Aggregation:

  • Include mild detergents or stabilizing agents in the buffer

  • Optimize salt concentration (typically 150-300 mM NaCl)

  • Add glycerol (5-10%) to stabilize the protein

  • Consider co-expression with chaperones

• Low Protein Activity:

  • Verify protein folding using circular dichroism

  • Ensure proper disulfide bond formation if applicable

  • Test different buffer conditions and pH ranges

  • Maintain reducing conditions if necessary (DTT or β-mercaptoethanol)

• Degradation During Storage:

  • Add protease inhibitors during purification

  • Store in small aliquots to avoid repeated freeze-thaw cycles

  • Add stabilizing agents like glycerol (up to 50%)

  • Consider flash-freezing in liquid nitrogen

These strategies address common issues while maintaining the protein's structural integrity and functional properties.

How can genetic manipulation tools be optimized for studying atpH in B. vietnamiensis?

Optimizing genetic manipulation tools for studying atpH in B. vietnamiensis requires consideration of species-specific factors:

• CRISPRi System Adaptation:

  • Use the codon-optimized dcas9 gene designed for GC-rich Burkholderia species

  • Integrate dcas9 into the chromosome using the mini-CTX1 integration vector targeting the serine tRNA attB site

  • Design gRNAs targeting the template strand of atpH shortly after the transcription start site for maximum repression efficiency

  • Implement a rhamnose-inducible promoter system for tunable expression control

• gRNA Design Considerations:

  • Target the template strand a short distance downstream of the transcription start site

  • Design multiple gRNAs as efficacy can vary significantly (repression levels can range from 3-fold to over 100-fold)

  • Validate gRNA efficiency using RT-qPCR to measure target gene repression

• Expression Control:

  • The rhamnose-inducible system allows for dose-dependent gene repression

  • Use rhamnose concentrations between 0.005% and 0.2% for variable levels of repression

  • Consider the potential for uneven distribution of sugar transporters after cell division, which might affect induction uniformity

• Phenotypic Analysis:

  • Develop robust assays for ATP synthesis activity

  • Monitor growth parameters and energy metabolism markers

  • Consider microscopy techniques to visualize ATP synthase complex formation and localization

These optimized approaches leverage recent advances in Burkholderia genetic manipulation tools to enable precise study of atpH function.

What emerging technologies could advance our understanding of B. vietnamiensis atpH?

Several cutting-edge technologies show promise for advancing research on ATP synthase subunit delta:

• Cryo-Electron Microscopy:

  • High-resolution structural determination of the entire ATP synthase complex

  • Visualization of conformational changes during the catalytic cycle

  • Potential to capture intermediate states during rotary catalysis

• Single-Molecule Biophysics:

  • Direct observation of rotary motion in reconstituted ATP synthase complexes

  • Measurement of torque generation and mechanical properties

  • Correlation of structural features with mechanical function

• Integrative Structural Biology:

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

  • Computational modeling of dynamic protein interactions

  • Molecular dynamics simulations to explore conformational dynamics

• Advanced Genetic Tools:

  • Expansion of CRISPRi technologies for precise temporal control of gene expression

  • Development of CRISPR-based imaging techniques to visualize ATP synthase assembly

  • Synthetic biology approaches to engineer novel functionalities

• Systems Biology Approaches:

  • Multi-omics integration to understand the broader metabolic context

  • Flux analysis to quantify the impact of atpH variants on cellular energetics

  • Network modeling to predict effects of perturbations on bacterial physiology

These technologies promise to provide unprecedented insights into the structure, function, and regulation of bacterial ATP synthases.

How might research on B. vietnamiensis atpH contribute to broader scientific understanding?

Research on B. vietnamiensis ATP synthase subunit delta has implications that extend beyond this specific protein:

• Fundamental Bioenergetics:

  • Deeper understanding of the molecular mechanisms of biological energy conversion

  • Insights into the evolutionary optimization of energy transduction systems

  • Clarification of structure-function relationships in rotary molecular motors

• Bacterial Physiology:

  • Understanding how energy metabolism is adapted to specific ecological niches

  • Elucidation of regulatory mechanisms controlling bacterial energetics

  • Insights into bacterial adaptations to energy-limited environments

• Antimicrobial Development:

  • Identification of unique structural features that could be targeted by novel antibiotics

  • Understanding of resistance mechanisms involving ATP synthase modifications

  • Development of species-specific inhibitors based on structural differences

• Synthetic Biology Applications:

  • Design principles for creating artificial molecular motors

  • Engineering of ATP synthase variants with altered properties for biotechnology

  • Development of minimal energy conversion systems for synthetic cells

• Evolutionary Biology:

  • Insights into the evolution of complex molecular machines

  • Understanding of co-evolution between ATP synthase subunits

  • Elucidation of adaptation mechanisms in energy metabolism

These broader impacts demonstrate the significance of focused research on specific ATP synthase components for advancing multiple scientific fields.