Recombinant Burkholderia phymatum ATP synthase subunit b (atpF)

What is the role of ATP synthase subunit b (atpF) in Burkholderia phymatum metabolism?

ATP synthase subunit b serves as a critical structural component of the F₁F₀-ATP synthase complex in B. phymatum, functioning as part of the peripheral stalk that connects the membrane-embedded F₀ sector to the catalytic F₁ sector. This connection is essential for maintaining the structural integrity of the complex during rotation-driven ATP synthesis.

In B. phymatum, as in other bacteria, the ATP synthase complex plays a crucial role in energy conservation by utilizing the proton gradient across the cell membrane to generate ATP. The complex functionality is particularly important in this bacterium's adaptation to diverse environments, including both pathogenic and beneficial plant interactions. The ATP generated supports various cellular processes including nitrogen fixation in nodules, which requires substantial energy input.

Methodologically, researchers can assess atpF function through:

• Complementation assays with specific atpF mutants

• ATP production measurement in atpF knockdown strains

• Protein-protein interaction studies to determine stalk assembly

How does the atpF gene sequence in B. phymatum compare to homologs in other Burkholderia species?

The atpF gene in B. phymatum (now reclassified in some literature as Paraburkholderia phymatum) shares significant homology with other members of the Burkholderia genus, with sequence identity typically ranging from 75-90% depending on the species. Comparative genomic analyses have revealed conserved functional domains essential for ATP synthase operation while highlighting species-specific variations that may reflect adaptation to particular ecological niches.

For researchers interested in sequence comparisons, the following approach is recommended:

• Perform multiple sequence alignment of atpF sequences from various Burkholderia species

• Identify conserved domains using tools like PFAM or PROSITE

• Construct phylogenetic trees to visualize evolutionary relationships

• Map sequence variations to known functional regions of the protein

Notable sequence variations often occur in regions involved in species-specific interactions or environmental adaptations, which may correlate with B. phymatum's capacity to form symbiotic relationships with leguminous plants.

What methods are most effective for studying atpF expression regulation in B. phymatum?

Investigating the regulation of atpF expression in B. phymatum requires a multi-faceted approach:

• Transcriptional analysis:

  • RT-qPCR for quantitative measurement of atpF mRNA levels under different conditions

  • RNA-seq to identify co-regulated genes and transcriptional patterns

  • Promoter fusion reporters (e.g., β-galactosidase or GFP) to visualize expression in different growth conditions

• Protein-level analysis:

  • Western blotting with anti-atpF antibodies

  • Mass spectrometry-based proteomics

  • Pulse-chase experiments to determine protein turnover rates

• Regulatory element identification:

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding sites

  • DNase footprinting to map protein-DNA interactions

  • EMSA (electrophoretic mobility shift assay) to confirm specific binding interactions

Research indicates that atpF expression in Burkholderia species responds to environmental signals including oxygen availability, pH changes, and nutrient status. B. phymatum likely employs similar regulatory mechanisms, potentially with additional symbiosis-specific regulation factors that coordinate energy production with nitrogen fixation during plant interaction .

What expression systems yield optimal production of functional recombinant B. phymatum atpF protein?

The expression of functional recombinant B. phymatum atpF presents several challenges that researchers must address through careful system selection and optimization:

Recommended expression systems:

For optimal results, researchers should:

• Clone the atpF gene with appropriate affinity tags (His6 or Strep-tag II) for purification

• Test multiple expression conditions (temperature, induction time, media composition)

• Verify protein folding using circular dichroism spectroscopy

• Assess functionality through ATP hydrolysis assays if expressing as part of a complex

When expressing atpF alone, it's critical to consider its hydrophobic nature and potential toxicity to host cells. The use of fusion partners (such as MBP or SUMO) can enhance solubility and reduce host toxicity, though these must subsequently be removed for functional studies .

How can site-directed mutagenesis be applied to study functional domains in B. phymatum atpF?

Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationships of B. phymatum atpF. The following methodology is recommended:

• Target selection:

  • Conserved residues identified through multiple sequence alignments

  • Charged residues likely involved in subunit interactions

  • Residues corresponding to known functional sites in homologous proteins

  • Positions with natural variants in related Burkholderia species

• Mutagenesis techniques:

  • QuikChange site-directed mutagenesis for single mutations

  • Gibson Assembly or overlap extension PCR for multiple mutations

  • Alanine scanning for systematic functional mapping

  • Conservative vs. non-conservative substitutions to assess amino acid properties

• Functional assessment:

  • Complementation assays in atpF-deficient strains

  • In vitro reconstitution with purified components

  • ATP synthesis/hydrolysis measurements

  • Protein-protein interaction analyses using pull-down assays or crosslinking

Key residues to target include those involved in:

• Interaction with the δ subunit of F₁

• Dimerization of the b subunits

• Membrane association

• Potential regulatory sites specific to B. phymatum

Researchers should consider combining mutational analysis with structural studies (e.g., cryo-EM) to correlate functional effects with structural perturbations. Recent studies in related bacteria suggest that the C-terminal domain of subunit b is particularly important for assembly and function of the ATP synthase complex .

What are the challenges in purifying functional recombinant B. phymatum ATP synthase complexes?

Purification of functional ATP synthase complexes from B. phymatum presents several technical challenges that researchers must address:

Major challenges and solutions:

• Membrane protein nature:

  • Challenge: Hydrophobic components difficult to maintain in solution

  • Solution: Use appropriate detergents (DDM, LMNG) or amphipols; detergent screening is essential

• Complex stability:

  • Challenge: Multi-subunit complex may dissociate during purification

  • Solution: Employ gentle purification methods; crosslinking approaches; purification in the presence of nucleotides

• Functional verification:

  • Challenge: Ensuring the purified complex retains catalytic activity

  • Solution: Develop robust ATP synthesis/hydrolysis assays; liposome reconstitution to verify proton pumping

• Heterologous expression:

  • Challenge: Low expression yields and improper assembly

  • Solution: Consider native host expression; optimize codon usage; co-express all operon components

Recommended purification protocol:

• Membrane preparation using differential centrifugation

• Solubilization using optimized detergent conditions (typically 1% DDM)

• Affinity chromatography using tagged subunits (His-tagged atpF or other accessible subunits)

• Size exclusion chromatography to isolate intact complexes

• Functional verification through ATP synthesis assays using artificial proton gradients

Recent advances in cryo-EM have reduced the quantity of purified protein required for structural studies, making it feasible to determine the structure of the B. phymatum ATP synthase complex even with limited purification yields .

How does ATP synthase activity correlate with the symbiotic properties of B. phymatum?

B. phymatum forms symbiotic relationships with leguminous plants, particularly Mimosa species, where energy metabolism plays a critical role in establishing and maintaining the symbiosis. ATP synthase activity has several important connections to symbiotic function:

• Energy requirements for nitrogen fixation:

  • Nitrogen fixation by B. phymatum within root nodules is an energy-intensive process requiring significant ATP

  • ATP synthase activity must be tightly regulated to meet these demands

  • Research approach: Compare ATP synthase activity in free-living versus symbiotic states using membrane vesicle preparations

• Adaptation to microaerobic nodule environment:

  • Within nodules, oxygen levels are low to protect nitrogenase

  • ATP synthase regulation must adapt to microaerobic respiration

  • Research approach: Measure ATP synthase expression and activity under varying oxygen tensions

• pH and ion homeostasis during infection:

  • ATP synthase contributes to proton gradient maintenance, affecting pH homeostasis

  • Research approach: Use pH-sensitive fluorescent proteins to monitor internal pH during symbiotic stages

• Coordination with Type VI Secretion System:

  • B. phymatum contains two T6SS clusters that contribute to its competitive ability in plant infection

  • ATP supply may influence T6SS activity

  • Research approach: Create conditional atpF mutants and measure effects on T6SS function and competitive nodulation

Studies suggest that ATP synthesis is upregulated during early stages of plant infection, corresponding with the energy requirements of T6SS function and biofilm formation. Paraburkholderia phymatum T6SS mutants show reduced competitiveness in plant infection, potentially due to altered energy availability for secretion system operation .

What structural biology techniques are most effective for analyzing B. phymatum ATP synthase subunit b?

Multiple structural biology approaches can be employed to investigate the structure of B. phymatum atpF, each with distinct advantages:

For B. phymatum atpF, a hybrid approach is recommended:

Recent advances in membrane protein structural biology, particularly in cryo-EM, have revolutionized the study of ATP synthases. These techniques would be particularly valuable for understanding how B. phymatum ATP synthase may differ structurally from those of non-symbiotic bacteria .

What approaches can be used to study the interaction between atpF and other ATP synthase subunits in B. phymatum?

Investigating protein-protein interactions within the ATP synthase complex requires multiple complementary approaches:

• In vitro binding assays:

  • Pull-down assays with recombinant tagged proteins

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Microscale thermophoresis for interaction studies in solution

• Crosslinking strategies:

  • Chemical crosslinking coupled with mass spectrometry (XL-MS)

  • Photo-activatable crosslinkers for capturing transient interactions

  • In vivo crosslinking to capture physiologically relevant interactions

• Genetic approaches:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Suppressor mutation analysis to identify compensatory mutations

  • Synthetic lethality screens to identify functional interactions

• Structural visualization:

  • FRET (Förster resonance energy transfer) between labeled subunits

  • Co-immunoprecipitation followed by structural analysis

  • Negative stain EM of isolated subcomplexes

Key interactions to investigate include:

• atpF dimerization interface

• atpF-δ subunit interface (connecting to F₁)

• atpF-a and atpF-c subunit interactions (connecting to F₀)

These interactions may differ in B. phymatum compared to model organisms due to adaptations for symbiotic lifestyle and environmental flexibility .

How can comparative genomics be used to understand the evolution of atpF in B. phymatum and related species?

Comparative genomics offers powerful insights into the evolutionary trajectory of atpF in B. phymatum and its relatives:

Recommended methodological approach:

• Sequence collection and alignment:

  • Retrieve atpF sequences from diverse Burkholderia/Paraburkholderia species

  • Include representatives from both pathogenic and symbiotic lineages

  • Perform multiple sequence alignment using MUSCLE or MAFFT

  • Visualize conservation patterns with tools like Jalview

• Phylogenetic analysis:

  • Construct maximum likelihood or Bayesian phylogenetic trees

  • Test multiple evolutionary models to find best fit

  • Perform bootstrap analysis to assess branch support

  • Compare atpF tree to species tree to identify horizontal gene transfer events

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify sites under positive or purifying selection

  • Apply branch-site models to identify lineage-specific selection

  • Correlate selection patterns with ecological transitions (free-living to symbiotic)

• Structural mapping:

  • Map conserved and variable regions onto structural models

  • Identify co-evolving residues using methods like mutual information analysis

  • Correlate structural features with functional adaptation

Based on available information on Paraburkholderia phymatum, we would expect atpF to show evidence of adaptation related to the bacterium's transition to a symbiotic lifestyle. The analysis might reveal convergent evolution with other symbiotic bacteria or distinctive adaptations related to B. phymatum's unique ecological role .

What are the best approaches for studying ATP synthase activity in B. phymatum during different growth conditions?

To effectively measure ATP synthase activity across different physiological states:

• In vivo approaches:

  • Luciferase-based ATP sensing for real-time measurement

  • Membrane potential measurements using fluorescent dyes (DiSC3)

  • Oxygen consumption rate determination using respirometry

  • pH monitoring using fluorescent probes to track proton translocation

• In vitro approaches:

  • Inverted membrane vesicle preparation for ATP synthesis assays

  • ATP hydrolysis measurement using coupled enzyme assays

  • Reconstitution into proteoliposomes for controlled environment studies

  • Patch-clamp electrophysiology for direct measurement of proton currents

• Experimental conditions to test:

  • Aerobic vs. microaerobic growth (relevant to nodule environment)

  • Different carbon sources (glucose, succinate, etc.)

  • Plant-derived compounds present during symbiosis

  • pH variation and acid stress conditions

  • Nutrient limitation (N, P) mimicking host environments

• Data analysis considerations:

  • Normalize activity to enzyme concentration

  • Account for proton motive force variations

  • Consider ATP synthase expression level changes

  • Integrate with metabolomic data for comprehensive interpretation

Key findings from related research suggest ATP synthase activity in Burkholderia species shows regulatory adaptation to changing environmental conditions. For example, the ATP synthase of B. cenocepacia exhibits altered regulation under oxygen limitation, a feature that may be shared with B. phymatum and relevant to its adaptation to the nodule environment .

How does ATP synthase function relate to the hydroxyapatite solubilization abilities of Burkholderia sp.?

The ability of Burkholderia species to solubilize phosphate (particularly hydroxyapatite) represents an important ecological function that likely intersects with energy metabolism and ATP synthase activity:

• Mechanism of phosphate solubilization:

  • Burkholderia sp. Ha185 produces 2-ketogluconate as the predominant organic anion for phosphate solubilization

  • This process requires energy for both substrate uptake and metabolic transformation

  • ATP synthase function is crucial for providing the energy required for these processes

• Research approaches to study this relationship:

  • Create conditional atpF mutants and assess phosphate solubilization ability

  • Measure ATP levels during phosphate solubilization

  • Track expression of atpF and other ATP synthase genes during phosphate limitation

  • Analyze correlation between ATP synthesis rates and organic acid production

• Experimental design considerations:

  • Use hydroxyapatite-containing media to assess solubilization

  • Implement radioisotope labeling to track phosphate uptake and metabolism

  • Monitor pH changes as indicator of organic acid production

  • Establish gene expression profiles under phosphate-rich vs. phosphate-limited conditions

The HemX protein has been identified as essential for 2-ketogluconate production in Burkholderia sp. Ha185, likely through its role in providing redox cofactors. The relationship between this pathway and ATP synthase activity represents an important area for investigation, as energy availability likely influences the efficiency of phosphate solubilization .

What methods can be used to study the role of ATP synthase in B. phymatum's stress response?

Investigating how ATP synthase contributes to stress adaptation in B. phymatum requires integrating multiple experimental approaches:

• Stress exposure protocols:

  • Oxidative stress (H₂O₂, paraquat)

  • Acid/alkaline stress (pH range 4.5-9.0)

  • Osmotic stress (NaCl, sucrose)

  • Antibiotic stress (sub-inhibitory concentrations)

  • Host-derived antimicrobial compounds

• ATP synthase activity measurements:

  • ATP synthesis/hydrolysis assays under stress conditions

  • Proton gradient measurements using pH-sensitive fluorophores

  • Expression analysis of ATP synthase genes (RT-qPCR, RNA-seq)

  • Protein level assessment via western blotting

• Genetic manipulation approaches:

  • Construction of conditional atpF mutants

  • Complementation with wild-type or mutant variants

  • Reporter fusions to monitor expression changes

  • Site-directed mutagenesis of regulatory elements

• Physiological assessments:

  • Growth curves under various stress conditions

  • Cell viability/death assessment

  • Membrane integrity analysis

  • Biofilm formation capacity

Particularly relevant is the connection between ATP synthase function and biofilm formation, which represents an important stress response in Burkholderia species. Research on Paraburkholderia phymatum has shown that Type VI Secretion System (T6SS) mutants are defective in biofilm formation, suggesting a potential link between energy metabolism, secretion systems, and stress adaptation .

What are the most promising approaches for targeting B. phymatum ATP synthase for agricultural applications?

As a symbiotic bacterium, B. phymatum offers potential agricultural applications through enhancement of its beneficial properties:

• Engineering strategies:

  • Optimization of ATP synthase efficiency through targeted mutations

  • Regulatory modifications to enhance ATP production under symbiotic conditions

  • Creation of strains with improved stress tolerance through ATP synthase modifications

• Research approaches:

  • Structure-guided engineering of atpF for enhanced stability

  • Transcriptional fusion of ATP synthase genes to symbiosis-induced promoters

  • Metabolic modeling to predict effects of ATP synthase modifications

• Application scenarios:

  • Development of B. phymatum inoculants with enhanced nitrogen fixation capacity

  • Creation of strains with improved drought or salinity tolerance

  • Biocontrol applications utilizing the competitive advantage of B. phymatum

• Experimental validation:

  • Greenhouse trials with engineered strains

  • Metabolomic analysis to confirm enhanced energy metabolism

  • Competition assays against indigenous soil microbiota

  • Plant growth and yield measurements

The Type VI Secretion System (T6SS) of B. phymatum has been shown to contribute to its competitive ability for nodule formation, making it a potential target for enhancement in conjunction with ATP synthase optimization. By improving energy efficiency through ATP synthase modifications, the competitive advantage conferred by T6SS might be further enhanced .

How might systems biology approaches advance our understanding of ATP synthase function in B. phymatum?

Systems biology offers integrative frameworks to understand ATP synthase within the broader cellular context:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Track ATP synthase components across different growth conditions

  • Identify regulatory networks controlling ATP synthase expression

  • Map metabolic fluxes dependent on ATP availability

• Computational modeling approaches:

  • Constraint-based metabolic modeling (FBA)

  • Kinetic modeling of ATP synthesis and consumption

  • Agent-based modeling of B. phymatum in symbiotic contexts

  • Network analysis to identify key regulatory nodes

• Experimental validation strategies:

  • Targeted metabolomics focusing on energy carriers

  • 13C metabolic flux analysis

  • ChIP-seq to identify transcription factor binding to ATP synthase promoters

  • Ribosome profiling to assess translational regulation

• Integration with host interaction data:

  • Dual RNA-seq of plant-bacteria interfaces

  • Metabolite exchange modeling between symbiotic partners

  • Signaling network reconstruction across organismal boundaries

The BDSF (cis-2-dodecenoic acid) quorum sensing system in Burkholderia influences biofilm formation and virulence factors through the second messenger c-di-GMP. Systems biology approaches could reveal potential connections between this signaling system, ATP synthase regulation, and symbiotic functions, providing a more comprehensive understanding of B. phymatum's energy metabolism in its ecological context .