Recombinant Burkholderia cenocepacia ATP synthase subunit beta (atpD)

What is atpD and what is its functional significance in B. cenocepacia?

ATP synthase subunit beta (atpD) is a critical component of the F1 sector of ATP synthase (EC 3.6.3.14) in Burkholderia cenocepacia. This protein plays an essential role in cellular energy metabolism, participating in the synthesis of ATP from ADP and inorganic phosphate during oxidative phosphorylation. The beta subunit contains the catalytic sites responsible for ATP synthesis and is highly conserved across bacterial species due to its fundamental role in energy production.

In B. cenocepacia, atpD is particularly important as this opportunistic pathogen requires efficient energy metabolism to survive in diverse environments, from soil and plant rhizospheres to the respiratory tract of cystic fibrosis patients . The protein consists of 464 amino acids in B. cenocepacia strain AU 1054, as documented in the UniProt database (Q1BRB0) .

How is B. cenocepacia classified taxonomically based on recent genomic studies?

Recent genomic analyses have revealed that strains currently classified as Burkholderia cenocepacia actually represent at least two distinct species. Phylogenetic analyses and whole genome average nucleotide identity (ANI) studies suggest a significant taxonomic division within this group .

The two main clades identified are:

• A clade containing most clinical isolates, including the highly virulent ET12 lineage, which possesses multiple key virulence factors

• A clade enriched in environmental isolates that lacks several genetic traits involved in human virulence

Digital DNA-DNA hybridization (dDDH) analysis supports this species delimitation, with values ranging from 49.9% to 61% between the two clades, well below the 70% threshold for considering organisms as the same species . Additionally, some strains originally described as B. cenocepacia show closer genomic identity with other Burkholderia species:

These findings indicate that what has been called B. cenocepacia may actually be B. cenocepacia sensu stricto plus a separate species that shows different virulence characteristics .

How can recombinant atpD be used for phylogenetic studies of Burkholderia species?

Recombinant ATP synthase subunit beta (atpD) serves as an excellent phylogenetic marker for studying evolutionary relationships among Burkholderia species for several reasons:

• Optimal evolutionary rate: atpD contains sufficient sequence variation to discriminate between closely related species while maintaining enough conservation to establish deeper evolutionary relationships.

• Single-copy nature: Unlike 16S rRNA genes which may exist in multiple copies, atpD typically exists as a single copy in bacterial genomes, avoiding the complications of paralogous gene analysis.

• Essential function: As a component of a critical metabolic pathway, atpD is present in all Burkholderia species, making it universally applicable for comparative studies.

For phylogenetic studies, researchers can:

• Amplify and sequence the atpD gene from multiple Burkholderia isolates

• Express and analyze recombinant atpD proteins to examine structural and functional differences

• Combine atpD sequence data with other housekeeping genes in multilocus sequence typing approaches

In the context of the recent findings showing that B. cenocepacia comprises at least two distinct species, atpD sequence analysis can contribute significantly to clarifying taxonomic classifications and understanding the divergence between clinical and environmental isolates .

What role might atpD play in the virulence or host adaptation of B. cenocepacia?

While ATP synthase subunit beta (atpD) is primarily involved in energy metabolism rather than being a classical virulence factor, it may contribute to virulence and host adaptation in B. cenocepacia through several indirect mechanisms:

• Energy provision for virulence:

  • Efficient ATP production is crucial for powering energy-intensive virulence processes

  • Adaptation of energy metabolism to different host environments may involve atpD modifications

• Survival under stress conditions:

  • In host environments, pathogens face various stresses (nutrient limitation, immune response)

  • Optimized ATP synthase function could contribute to stress tolerance and persistence

• Potential moonlighting functions:

  • Some metabolic enzymes, including ATP synthase components, have been shown to have secondary functions

  • atpD could potentially play roles beyond energy metabolism, such as in adhesion or immune evasion

How can atpD be used as a serological marker for diagnostic applications?

Insights from the use of ATP synthase subunit beta (AtpD) as a serological marker in other bacteria, such as Mycoplasma pneumoniae, suggest potential applications for B. cenocepacia atpD in diagnostic contexts .

The development pathway for atpD-based diagnostics could include:

• Antigen identification and validation:

  • Express and purify recombinant B. cenocepacia atpD

  • Validate recognition by sera from patients with confirmed B. cenocepacia infections

• Assay development:

  • Develop enzyme-linked immunosorbent assays (ELISA) for detecting IgM, IgA, and IgG antibodies

  • Optimize assay conditions for maximum sensitivity and specificity

• Performance assessment:

  • Evaluate using well-characterized serum panels from infected patients and healthy controls

  • Compare performance with existing diagnostic methods

Based on studies with M. pneumoniae, combining atpD with other B. cenocepacia antigens might improve diagnostic performance . For example, researchers found that combining the recombinant AtpD and the C-terminal fragment of adhesin P1 provided maximum discrimination between M. pneumoniae-infected patients and healthy subjects for IgM detection .

Note: This table is an example based on M. pneumoniae studies; actual values for B. cenocepacia would require specific validation.

What are the optimal conditions for expressing and purifying recombinant B. cenocepacia atpD?

Based on available information and established protocols for similar proteins, the following approach is recommended for efficient expression and purification of recombinant B. cenocepacia atpD:

Expression System:

• Host: E. coli is the documented expression system for B. cenocepacia atpD

• Vector: pET-series vectors with T7 promoter for high-level expression

• Tags: Consider His6-tag for purification; fusion partners like MBP or SUMO may improve solubility

Expression Conditions:

• Temperature: 16-20°C for overnight expression often improves solubility

• Induction: 0.1-0.5 mM IPTG for controlled expression

• Media: LB or TB medium for standard expression; defined media for isotope labeling if NMR studies are planned

• Duration: 16-24 hours at lower temperatures

Purification Strategy:

• Cell lysis in appropriate buffer (typically Tris or phosphate buffer, pH 7.5-8.0)

• Initial purification by immobilized metal affinity chromatography (IMAC)

• Tag removal if necessary using site-specific proteases

• Further purification by ion exchange and size exclusion chromatography

• Quality control by SDS-PAGE (targeting >85% purity)

Storage Recommendations:

• For liquid formulations: Store at -20°C/-80°C for up to 6 months

• For lyophilized preparations: Store at -20°C/-80°C for up to 12 months

• Addition of glycerol (5-50% final concentration) is recommended before freezing

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

Reconstitution:

• For lyophilized protein, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Consider adding glycerol (5-50% final concentration) for long-term storage

How can the activity of recombinant atpD be assayed in vitro?

Assessing the activity of recombinant ATP synthase subunit beta (atpD) in vitro requires specialized approaches since this protein normally functions as part of the multisubunit ATP synthase complex:

1. ATP/ADP Binding Assays:

• Fluorescence-based methods:

  • Measure changes in intrinsic tryptophan fluorescence upon nucleotide binding

  • Use fluorescent ATP analogs (e.g., TNP-ATP) to monitor binding directly

• Isothermal titration calorimetry (ITC):

  • Quantitatively determine binding affinities and thermodynamic parameters

2. Partial Reactions of Catalysis:

• ATP hydrolysis activity:

  • Isolated beta subunits may exhibit some ATPase activity

  • Measure inorganic phosphate release using colorimetric assays (malachite green)

  • Monitor ADP formation using coupled enzyme assays

Experimental Setup for ATP Hydrolysis Assay:

• Incubate at 37°C for 15-60 minutes

• Measure inorganic phosphate release using malachite green assay

• Calculate phosphate release using a standard curve

3. Structural Integrity Assessment:

• Circular dichroism (CD) spectroscopy to verify proper folding

• Differential scanning fluorimetry to assess thermal stability and nucleotide effects

4. Reconstitution Experiments:

• Combine recombinant atpD with other purified ATP synthase subunits

• Measure ATP synthesis or hydrolysis activities of reconstituted complexes

These approaches allow for comprehensive evaluation of recombinant atpD functionality even in the absence of the complete ATP synthase complex.

What bioinformatic approaches are useful for analyzing atpD sequence conservation across B. cenocepacia strains?

Analyzing ATP synthase subunit beta (atpD) sequence conservation across different strains of B. cenocepacia requires sophisticated bioinformatic approaches:

1. Sequence Alignment and Conservation Analysis:

• Multiple sequence alignment using tools like MUSCLE, MAFFT, or Clustal Omega

• Conservation scoring using entropy-based methods or substitution matrix-based scoring

• Sliding window analysis to identify regions of high/low conservation

2. Phylogenetic Analysis:

• Maximum Likelihood methods (RAxML, IQ-TREE) for tree construction

• Evolutionary model testing to find best-fit models

• Comparison between clinical and environmental B. cenocepacia clades to identify clade-specific signatures

3. Selection Pressure Analysis:

• Calculate dN/dS ratios to assess selective pressures using PAML or HyPhy

• Site-specific selection detection to identify positively or negatively selected residues

• Branch-site tests for lineage-specific selection potentially related to host adaptation

4. Structural Bioinformatics:

• Homology modeling of atpD structures using tools like SWISS-MODEL or AlphaFold

• Mapping sequence variation to structure to identify if variable regions correspond to surface-exposed areas

• Assessing if conserved regions map to functional sites or protein-protein interfaces

Comparative Analysis Framework:

This comprehensive bioinformatic toolkit enables thorough investigation of atpD sequence conservation patterns across B. cenocepacia strains, providing insights into evolutionary dynamics and potential adaptation to different ecological niches, including the differences between clinical and environmental isolates identified in recent genomic studies .

What are the key challenges in expressing and purifying functional recombinant atpD from B. cenocepacia?

Expressing and purifying functional recombinant ATP synthase subunit beta (atpD) from B. cenocepacia presents several technical challenges:

• Protein solubility: atpD normally functions as part of a multi-subunit complex, and when expressed alone, it may form inclusion bodies due to exposed hydrophobic surfaces that would normally interact with other ATP synthase components.

• Proper folding: Ensuring correct folding is crucial for maintaining native structure and function, particularly challenging when expressing a protein from a gram-negative bacterium like B. cenocepacia in heterologous systems.

• Stability during purification: atpD may be susceptible to degradation during purification, necessitating the use of protease inhibitors and optimized buffer conditions.

• Functional assessment: Unlike enzymes with easily assayable activities, testing the functionality of isolated atpD is difficult since it normally operates as part of the ATP synthase complex.

• Endotoxin contamination: When expressed in E. coli, purified recombinant proteins may contain lipopolysaccharide (LPS) contamination, which can interfere with immunological studies.

Strategies to overcome these challenges include:

• Using solubility-enhancing tags like MBP or SUMO

• Optimizing expression conditions (lower temperature, reduced inducer concentration)

• Employing specialized E. coli strains designed for expressing difficult proteins

• Implementing rigorous purification protocols with appropriate quality control steps

• Considering co-expression with other ATP synthase subunits

How do the differences in atpD between clinical and environmental B. cenocepacia strains relate to host adaptation?

The genomic analyses indicating that B. cenocepacia likely comprises at least two distinct species—one clade dominated by clinical isolates and another by environmental isolates—raises important questions about the role of atpD in host adaptation .

While specific differences in atpD between these clades are not explicitly detailed in the available research, several hypotheses can be proposed:

• Sequence variations: Clinical isolates might exhibit specific amino acid substitutions that optimize ATP synthase function in host environments, while environmental isolates may have adaptations for variable external conditions.

• Regulatory adaptations: Expression levels and regulation of atpD might differ between clinical and environmental isolates to meet different energetic demands.

• Energy efficiency: Clinical isolates might have evolved more efficient ATP synthesis to thrive in the specific conditions of host environments, while environmental isolates might prioritize versatility over efficiency.

To definitively characterize these differences, comparative studies would need to:

• Sequence and analyze atpD from multiple strains of both clades

• Compare biochemical properties, stability, and enzymatic activities

• Investigate structural differences using techniques like X-ray crystallography

The finding that the clade enriched in environmental isolates lacks multiple key virulence factors, which are conserved in the clade where most clinical isolates fall , suggests that even conserved proteins like atpD might show subtle but functionally significant differences that contribute to adaptation to different ecological niches.

What potential exists for atpD as a target for new antimicrobial strategies against B. cenocepacia?

ATP synthase is an attractive target for antimicrobial development due to its essential role in bacterial energy metabolism. Several aspects make B. cenocepacia atpD a potential target for novel therapeutic approaches:

• Essential function: Disrupting ATP synthase activity would severely compromise bacterial energy production and survival.

• Surface accessibility: The beta subunit has regions that could potentially be accessed by inhibitors.

• Structural uniqueness: Differences between bacterial and human ATP synthases could be exploited for selective targeting.

Research approaches could include:

• High-throughput screening for small molecule inhibitors specific to B. cenocepacia atpD

• Structure-based drug design targeting unique features of the protein

• Inhibitory antibody development against surface-exposed regions

• Peptide inhibitors designed to disrupt atpD interactions with other ATP synthase subunits

This approach is particularly relevant for B. cenocepacia infections, which are notoriously difficult to treat due to intrinsic antibiotic resistance. Targeting essential metabolic enzymes represents a promising alternative strategy that might be less prone to resistance development.

How might comparative studies of atpD across the two B. cenocepacia clades inform our understanding of host adaptation mechanisms?

Comparative studies of atpD between the clinical-dominated and environmental-dominated clades of B. cenocepacia could provide valuable insights into bacterial adaptation to different ecological niches :

• Identification of adaptive signatures: Detailed sequence and structural comparisons could reveal specific substitutions that contribute to host adaptation.

• Functional consequences: Biochemical characterization of atpD variants from both clades might demonstrate differences in catalytic efficiency, regulatory properties, or stability under different conditions.

• Co-evolutionary patterns: Analysis of atpD evolution in concert with other ATP synthase subunits might reveal coordinated changes that optimize energy production in different environments.

• Integration with virulence studies: Correlating atpD variations with the documented differences in virulence factor distribution between clades could illuminate the relationship between energy metabolism and pathogenicity.

A comprehensive research program would include:

• Sequencing atpD from multiple strains representing both clades

• Recombinant expression and biochemical characterization

• Structural studies to identify meaningful differences

• In vivo studies to assess the impact of atpD variants on bacterial fitness in different environments

Such work would contribute to our fundamental understanding of bacterial adaptation mechanisms and potentially identify new targets for therapeutic intervention.