Recombinant Burkholderia multivorans ATP synthase subunit beta (atpD)

What is the biological significance of atpD in Burkholderia multivorans?

The atpD gene encodes the beta subunit of ATP synthase, an essential enzyme complex that produces ATP through oxidative phosphorylation. In B. multivorans, this protein plays a critical role in energy metabolism. Beyond its metabolic function, atpD is one of seven conserved housekeeping genes used in the MLST scheme for Burkholderia species identification . The conservation pattern of this gene makes it valuable for taxonomic studies while still containing sufficient variation to distinguish between closely related strains.

How is atpD utilized in Burkholderia species classification?

The atpD gene is part of the standardized seven-locus MLST scheme that includes atpD, gltB, gyrB, lepA, phaC, recA, and trpB . This MLST approach has provided important insights into population dynamics, diversity, and recombination events in the B. cepacia complex. The scheme has been expanded with redesigned primers to more reliably amplify these loci from all 17 B. cepacia complex species and additional Burkholderia species, including clinically relevant species like B. gladioli, B. mallei, and B. pseudomallei .

What are the optimal PCR strategies for amplifying the atpD gene from Burkholderia multivorans?

Researchers have redesigned PCR primers targeting the atpD gene to address limitations in the original MLST methodology. The improved primers aim to:

• More reliably amplify atpD from all Bcc species

• Extend amplification capability to additional Burkholderia species

• Enable the use of a single primer set for both amplification and DNA sequencing

This approach resolves previous challenges where approximately 10% of B. cepacia complex strains analyzed included at least one locus that could not be amplified using the original MLST primers . When amplifying atpD from B. multivorans specifically, researchers should consider the high GC content of Burkholderia genomes and optimize annealing temperatures and extension times accordingly.

What expression systems are most effective for producing recombinant B. multivorans atpD protein?

While optimizing recombinant expression of B. multivorans atpD, researchers should consider:

• Host selection: E. coli BL21(DE3) derivatives are commonly used for expressing Burkholderia proteins, but codon optimization may be necessary due to differences in codon usage between E. coli and Burkholderia species.

• Expression vectors: pET-based vectors with T7 promoters often yield good expression levels for bacterial proteins when fused with affinity tags (His6, GST, or MBP) to facilitate purification.

• Solubility considerations: As a component of the membrane-associated ATP synthase complex, atpD may present solubility challenges. Expression at lower temperatures (16-20°C) and inclusion of solubility-enhancing tags may improve yield of correctly folded protein.

• Functional validation: Activity assays measuring ATP hydrolysis can confirm proper folding of the recombinant protein.

How has homologous recombination influenced the evolution of atpD in the Burkholderia cepacia complex?

Research indicates that homologous recombination contributes significant genetic variation to many genes in the Burkholderia cepacia complex. Approximately 5.8% of core orthologous genes in the Bcc showed strong evidence of recombination . This high level of recombination between Bcc species blurs taxonomic boundaries, making species difficult to distinguish phenotypically .

As a core housekeeping gene, atpD is part of the set of genes that help define species boundaries despite recombination events. The selective pressure to maintain ATP synthase function limits the extent of allowed variation, making atpD a relatively stable marker for phylogenetic analysis despite recombination in other regions of the genome.

What evidence exists for positive selection on atpD compared to other core genes?

Genome-wide analysis of the Burkholderia cepacia complex has shown that approximately 1.1% of core orthologous genes showed evidence of positive selection . Genes involved in protein synthesis, material transport, and metabolism are particularly favored by selection pressure. While the atpD gene hasn't been specifically highlighted, its role in energy metabolism suggests it may experience purifying selection to maintain its critical function.

The relative contributions of recombination versus positive selection in shaping atpD evolution provide important insights into how B. multivorans adapts to different environments while maintaining essential cellular functions.

What are the critical functional domains and residues in B. multivorans atpD?

ATP synthase subunit beta contains several highly conserved functional domains:

• Nucleotide-binding domains: These regions contain Walker A and Walker B motifs essential for ATP binding and hydrolysis.

• Catalytic sites: Located primarily in the beta subunit, these sites are formed at the interface between alpha and beta subunits in the F1 portion of ATP synthase.

• DELSEED region: This conserved motif is involved in energy coupling between proton transport and ATP synthesis.

Experimental approaches to identify critical residues include site-directed mutagenesis followed by functional assays measuring ATP synthesis/hydrolysis activities. Comparative analysis with ATP synthase structures from model organisms can guide the identification of key residues in the B. multivorans protein.

How do structural variations in atpD correlate with functional differences between Burkholderia species?

Structural analysis of atpD across Burkholderia species would require:

• Homology modeling: Using resolved ATP synthase structures as templates to predict B. multivorans atpD structure.

• Molecular dynamics simulations: To assess potential functional implications of amino acid substitutions.

• Experimental validation: Using recombinant proteins to measure kinetic parameters and compare activities between species.

The correlation between structural variations and adaptation to different ecological niches (clinical vs. environmental isolates) may provide insights into the evolution of Burkholderia species.

How does atpD expression change under different environmental conditions relevant to pathogenesis?

During infection, B. multivorans encounters various stressors including oxygen limitation, nutrient restriction, and host immune responses. While specific data on atpD regulation in B. multivorans is limited, research on related Burkholderia species provides relevant insights.

In B. pseudomallei, the RegAB two-component signal transduction system functions as the master regulator of anaerobic physiology and significantly affects virulence . This system controls adaptation to oxygen-restricted environments often encountered during infection. As ATP synthase is crucial for energy production under both aerobic and anaerobic conditions, atpD expression is likely regulated in response to oxygen availability, potentially through mechanisms similar to those identified in B. pseudomallei.

Research approaches to study atpD expression during infection include:

• qRT-PCR analysis of bacteria recovered from infection models

• Transcriptomic analysis comparing expression under different oxygen concentrations

• Reporter gene fusions to monitor atpD promoter activity during host cell interaction

Can recombinant atpD be used for developing diagnostic tools or vaccines for Burkholderia infections?

Recombinant atpD protein has potential applications in both diagnostics and vaccine development:

Diagnostic applications:

• Generation of specific antibodies for immunodiagnostic assays

• Development of protein-based identification methods to complement DNA-based MLST

• Creation of diagnostic panels including multiple Burkholderia antigens to increase specificity

Vaccine development considerations:

• ATP synthase components are highly conserved across bacterial species, requiring careful epitope selection to avoid cross-reactivity

• Combining atpD with other Burkholderia antigens may enhance protective immunity

• Evaluation of recombinant atpD as a carrier protein for conjugate vaccines targeting Burkholderia surface polysaccharides

Given the challenges in distinguishing between closely related Burkholderia species that cause similar clinical presentations, molecular approaches incorporating atpD could improve diagnostic accuracy .

What are the challenges and solutions for resolving the structure of recombinant B. multivorans atpD?

Structural studies of recombinant atpD face several challenges:

• Protein stability: As part of a multi-subunit complex, isolated atpD may have stability issues. Solutions include co-expression with interaction partners or use of stabilizing mutations.

• Crystallization difficulties: ATP synthase components can be challenging to crystallize. Alternative approaches include cryo-electron microscopy or NMR for structural determination.

• Functional state capture: ATP synthase undergoes conformational changes during catalysis. Capturing different functional states requires careful selection of nucleotide analogs and experimental conditions.

• Membrane association: Though primarily part of the F1 soluble portion, interactions with membrane components may affect structure. Detergent selection or nanodiscs can help maintain native-like conformations.

How can synthetic biology approaches utilize recombinant atpD to engineer Burkholderia for biotechnological applications?

Engineered atpD variants could enable:

• Enhanced bioenergy production: Optimized ATP synthase could improve energy efficiency in biofuel-producing strains.

• Biosensors: atpD-reporter fusions could create cellular sensors for environmental monitoring.

• Attenuated strains: Engineered atpD variants with reduced efficiency could create attenuated strains for vaccine development.

• Protein engineering platforms: Understanding the structure-function relationship of atpD could inform the design of novel enzymes with altered nucleotide specificity.

Given the metabolic versatility of Burkholderia species and their ability to degrade complex compounds, engineered strains with modified energy metabolism could have applications in bioremediation and industrial biotechnology.

How does atpD compare to other core genes in terms of genetic diversity across Burkholderia species?

Comparative genomic analysis of 116 Burkholderia cepacia complex strains identified 1005 orthogroups consisting entirely of single-copy genes . These core orthologous genes showed significant differences in evolutionary properties across different functional categories.

A comprehensive analysis of atpD would include:

• Calculating nucleotide diversity (π) and dN/dS ratios

• Comparing sequence conservation with other MLST loci

• Analyzing codon usage bias and GC content

• Examining phylogenetic congruence between atpD and whole-genome trees

This comparative approach would position atpD within the broader context of Burkholderia genome evolution and help assess its reliability as a phylogenetic marker.

What computational methods are most appropriate for analyzing recombination events affecting atpD in Burkholderia?

To detect and characterize recombination events affecting atpD, researchers should consider:

• Detection methods:

  • Phylogenetic approaches (RDP4 software suite)

  • Sequence-based methods (GARD, MaxChi)

  • Compatibility-based methods (PhiTest)

• Validation strategies:

  • Applying multiple detection methods to increase confidence

  • Simulating sequence evolution with known recombination rates

  • Bayesian approaches to estimate recombination parameters

• Visualization techniques:

  • Recombination networks using SplitsTree

  • Heatmaps of sequence similarity

  • Sliding window analysis of phylogenetic signals

The high level of recombination between Bcc species requires robust analytical approaches to accurately characterize genetic exchange events affecting genes like atpD .

How does atpD interact with other components of energy metabolism networks in B. multivorans?

ATP synthase functions within a broader energy metabolism network. Systems biology approaches to study these interactions include:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with recombinant tagged atpD

  • Bacterial two-hybrid systems

  • Cross-linking mass spectrometry

• Metabolic flux analysis:

  • 13C-labeling to track carbon flow

  • Flux balance analysis incorporating ATP synthase activity

  • Metabolomic profiling under different energy states

• Regulatory network mapping:

  • ChIP-seq to identify transcription factors regulating atpD

  • RNA-seq under various conditions to identify co-regulated genes

  • Network analysis to position atpD within the broader metabolic network

This integrative approach would reveal how B. multivorans coordinates energy production with other cellular processes, particularly under the variable conditions encountered during infection.

What insights can multi-omics analysis provide about atpD function during host-pathogen interactions?

Integration of multiple omics approaches can reveal atpD's role during infection:

• Transcriptomics: RNA-seq analysis comparing gene expression during different infection stages

• Proteomics: Quantifying ATP synthase components during host adaptation

• Metabolomics: Measuring changes in ATP/ADP ratios and energy charge

• Interactomics: Identifying host factors interacting with bacterial ATP synthase

Such analysis could reveal whether atpD and ATP synthase function is modulated during infection, potentially contributing to virulence through optimized energy production in challenging host environments.