Recombinant Burkholderia phymatum NADH-quinone oxidoreductase subunit A (nuoA)

What is NADH-quinone oxidoreductase subunit A (nuoA) and what is its functional role in Burkholderia phymatum?

NADH-quinone oxidoreductase subunit A (nuoA) is a component of the NADH dehydrogenase complex (Complex I) in the respiratory chain of Burkholderia phymatum. This protein plays a crucial role in electron transport and energy metabolism. Specifically, the enzyme catalyzes the transfer of electrons from NADH to quinones with an EC number of 1.6.99.5. In Burkholderia species, nuoA is also known as NADH dehydrogenase I subunit A, NDH-1 subunit A, or NUO1, and is encoded by the nuoA gene .

The protein functions within the membrane-bound respiratory complex, contributing to proton translocation across the membrane and subsequent ATP synthesis. In the context of Burkholderia phymatum's symbiotic relationship with legumes, energy metabolism is critical for supporting nitrogen fixation processes that make this bacterium valuable as a plant symbiont .

How should researchers store and handle Recombinant Burkholderia phymatum NADH-quinone oxidoreductase subunit A?

For optimal research outcomes, Recombinant Burkholderia phymatum NADH-quinone oxidoreductase subunit A should be stored at -20°C for routine use, while extended storage is recommended at -20°C to -80°C. The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized to maintain protein stability .

Methodologically important handling notes:

• Avoid repeated freeze-thaw cycles as these can degrade protein structure and function

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

• For experimental protocols requiring longer incubation periods, maintain the protein in its optimized buffer

• When designing experiments, consider preparing multiple small aliquots during initial thawing to minimize future freeze-thaw events

What is the relationship between nuoA expression and nitrogen fixation in Burkholderia phymatum's symbiotic relationships?

The relationship between nuoA expression and nitrogen fixation capabilities involves complex metabolic interactions. Burkholderia phymatum is highly effective at fixing nitrogen in symbiosis with Mimosa species, demonstrating significant nitrogenase activity measured by acetylene reduction assay (ARA). In experimental studies with Mimosa pudica, Burkholderia phymatum STM815 demonstrated nitrogenase activity of 560.5 ± 32.1 nmol C₂H₄ per plant per hour, significantly outperforming other nitrogen-fixing bacteria such as Cupriavidus taiwanensis (223.9 ± 90.7 nmol C₂H₄ per plant per hour) .

The nuoA protein, as part of the respiratory chain, likely supplies the substantial energy required for the nitrogen fixation process. Research suggests that the expression of energy metabolism genes, including those in the respiratory chain, is upregulated during active nitrogen fixation in root nodules .

How does environmental temperature affect nuoA function and expression in Burkholderia phymatum?

Environmental temperature significantly impacts the expression patterns of various systems in Burkholderia phymatum, which likely extends to nuoA expression. Research on the Type VI Secretion Systems (T6SS) in P. phymatum has shown that T6SS-b expression is higher at 20/28°C compared to 37°C, while T6SS-3 expression is more prominent at 37°C .

By extension, NADH-quinone oxidoreductase complex components like nuoA may exhibit similar temperature-dependent expression patterns, optimized for the soil environments where symbiotic interactions occur (typically 20-28°C). This temperature range aligns with optimal conditions for symbiosis with legume partners like Mimosa pudica and Phaseolus vulgaris .

For experimental design, researchers should consider:

• Including temperature as a controlled variable in expression studies

• Monitoring nuoA expression at both free-living relevant temperatures (20-28°C) and potential stress temperatures (37°C)

• Correlating nuoA expression with nitrogen fixation efficiency at various temperatures

What approaches can be used to study the role of nuoA in Burkholderia phymatum's symbiotic competitiveness?

To investigate nuoA's role in Burkholderia phymatum's exceptional symbiotic competitiveness, researchers can employ several methodological approaches:

• Gene knockout studies: Creating nuoA deletion mutants can help determine its importance in competitive nodulation of legume hosts. This approach has been successfully used for studying T6SS components in P. phymatum .

• Comparative expression analysis: Quantifying nuoA expression levels during different stages of symbiosis (free-living, root colonization, nodule development, mature nodule) can reveal temporal patterns of importance.

• Co-inoculation experiments: Testing wild-type versus nuoA mutants in competition with other rhizobial strains (like Cupriavidus taiwanensis) can determine if energy metabolism through nuoA contributes to the competitive advantage observed in P. phymatum .

• Environmental condition variations: Studying nuoA expression under different carbon sources (citrate vs. succinate), temperatures, and pH conditions can provide insights into adaptation mechanisms .

• Host-specific response monitoring: Comparing nuoA expression patterns across different host plants (e.g., Mimosa pudica, Phaseolus vulgaris) can reveal whether energy metabolism is tailored to specific symbiotic relationships .

What protein expression systems are most effective for producing Recombinant Burkholderia phymatum NADH-quinone oxidoreductase subunit A?

For optimal expression of Recombinant Burkholderia phymatum NADH-quinone oxidoreductase subunit A, researchers should consider several expression system options:

• E. coli-based expression systems: BL21(DE3) or similar strains with T7 promoter systems typically yield good expression of bacterial proteins. For membrane proteins like nuoA, specialized strains such as C41(DE3) or C43(DE3) may provide better results by accommodating membrane protein overexpression.

• Expression tags and fusion proteins: While the specific tag types for nuoA may vary depending on the production process , common approaches include:

  • N-terminal His₆-tag for purification

  • Fusion with MBP (maltose-binding protein) to enhance solubility

  • SUMO fusion systems to improve folding

• Induction conditions: For membrane proteins like nuoA:

  • Lower temperatures (16-20°C) during induction

  • Reduced IPTG concentrations (0.1-0.5 mM)

  • Extended induction times (overnight)

• Extraction considerations: Given nuoA's membrane-associated nature, extraction protocols should incorporate:

  • Appropriate detergents (DDM, LDAO, or similar)

  • Gentle solubilization steps

  • Buffer optimization to maintain protein integrity

How can researchers effectively measure the enzymatic activity of purified nuoA?

Measuring enzymatic activity of NADH-quinone oxidoreductase subunit A requires specific approaches due to its role in the electron transport chain:

• Spectrophotometric NADH oxidation assay:

  • Monitor decrease in NADH absorbance at 340 nm

  • Reaction buffer typically contains:

    • 50 mM phosphate buffer (pH 7.5)

    • 100 μM NADH

    • Appropriate quinone electron acceptor (ubiquinone-1 or decylubiquinone)

  • Calculate activity using extinction coefficient of NADH (ε₃₄₀ = 6,220 M⁻¹cm⁻¹)

• Oxygen consumption measurements:

  • Use oxygen electrode (Clark-type) to monitor oxygen reduction

  • System should contain NADH, appropriate quinones, and purified enzyme

• Reconstitution into liposomes:

  • Incorporate purified nuoA into phospholipid vesicles

  • Measure proton translocation using pH-sensitive dyes or electrodes

  • This approach can help determine the protein's native functionality

• Inhibitor studies:

  • Use known Complex I inhibitors (rotenone, piericidin A)

  • Compare activity in presence/absence of inhibitors to confirm specificity

What techniques are recommended for studying nuoA expression during different growth phases and symbiotic stages?

To effectively study nuoA expression throughout Burkholderia phymatum's growth phases and symbiotic interactions, researchers should consider these methodological approaches:

• Quantitative RT-PCR (RT-qPCR):

  • Design primers specific to nuoA sequence

  • Extract RNA from bacteria at different:

    • Growth phases (log, stationary)

    • Environmental conditions (temperature, pH, carbon source)

    • Symbiotic stages (free-living, root colonization, nodule formation)

  • Normalize expression against stable reference genes

• Reporter gene fusions:

  • Create transcriptional or translational fusions of nuoA promoter with reporter genes (GFP, LacZ)

  • This approach has been successful for studying T6SS expression in P. phymatum under different conditions

  • Monitor expression in real-time during plant interaction

• Western blotting:

  • Develop specific antibodies against nuoA

  • Extract protein samples at different growth and symbiotic stages

  • Quantify relative protein abundance

• Proteomics approach:

  • Use LC-MS/MS to identify and quantify nuoA in different experimental conditions

  • Compare with other respiratory proteins to identify co-regulated systems

• In planta studies:

  • Use bacteroid isolation from nodules at different developmental stages

  • Combine with any of the above techniques to track nuoA expression during symbiosis

How does the structure of nuoA relate to its function in the NADH dehydrogenase complex?

The structure-function relationship of nuoA in the NADH dehydrogenase complex centers on its role as a membrane domain component. Based on the amino acid sequence analysis, nuoA contains multiple transmembrane segments that anchor it within the bacterial membrane .

Key structural aspects include:

• Transmembrane helices: The sequence "MNLAAYYPVLLFLLVGTGLGIALVSIGKLLGPNKPDVEKNAPYECGFEAFEDARMKFDVRYYLIAILFIIFDLETAFLFPWGVALRDIGWPGFIAMMIFLLEFLLGFAYIWKKGGLDWE" contains hydrophobic regions characteristic of membrane-spanning domains .

• Functional domains: Within the NADH dehydrogenase complex, nuoA likely contributes to:

  • Stabilization of the membrane domain structure

  • Formation of the proton translocation pathway

  • Maintenance of proper interaction with other complex subunits

• Evolutionary conservation: The nuoA sequence shows conservation patterns that highlight functionally important residues across bacterial species.

Methodologically, researchers can investigate structure-function relationships through:

• Site-directed mutagenesis of conserved residues

• Protein-protein interaction studies with other complex components

• Computational modeling based on homologous structures from related organisms

What techniques are most effective for studying the interaction of nuoA with other components of the respiratory chain?

To investigate interactions between nuoA and other respiratory chain components, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against nuoA or epitope tags

  • Identify interacting partners through mass spectrometry

  • Preserve native membrane protein interactions using appropriate detergents

• Blue Native PAGE:

  • Analyze intact respiratory complexes

  • Identify complex composition and assembly intermediates

  • Combine with Western blotting to confirm nuoA presence

• Crosslinking coupled with mass spectrometry:

  • Use membrane-permeable crosslinkers to capture transient interactions

  • Identify crosslinked peptides to map interaction interfaces

  • This approach is particularly valuable for membrane protein complexes

• Bacterial two-hybrid systems:

  • Adapted for membrane proteins to detect protein-protein interactions

  • Screen for interactions between nuoA and other respiratory components

• Cryo-electron microscopy:

  • Visualize the entire respiratory complex structure

  • Locate nuoA within the larger assembly

  • Determine structural changes under different conditions

How might nuoA function contribute to Burkholderia phymatum's exceptional nitrogen-fixing capabilities?

Burkholderia phymatum demonstrates exceptional nitrogen-fixing capabilities in symbiosis with legumes, particularly Mimosa species. When inoculated with B. phymatum STM815, Mimosa pudica plants showed significantly higher nitrogenase activity (560.5 ± 32.1 nmol C₂H₄ per plant per hour) compared to plants inoculated with Cupriavidus taiwanensis (223.9 ± 90.7 nmol C₂H₄ per plant per hour) .

The nuoA protein likely contributes to this exceptional capability through:

• Energy provision: As part of the respiratory chain, nuoA contributes to ATP generation needed for the energy-intensive nitrogen fixation process.

• Redox balance maintenance: The NADH dehydrogenase complex helps maintain appropriate redox conditions within bacteroids for optimal nitrogenase activity.

• Adaptation to nodule microenvironment: The respiratory chain components may be optimized for the low-oxygen environment of root nodules.

• Host-specific optimization: The superior nitrogen-fixing capabilities observed in Mimosa species may relate to specialized energy metabolism adaptations, potentially involving nuoA expression or activity patterns .

What role might nuoA play in Burkholderia phymatum's competitiveness during legume colonization?

Burkholderia phymatum demonstrates exceptional competitiveness in colonizing legume hosts, outcompeting other rhizobial strains. While the Type VI Secretion Systems (T6SS) have been identified as contributing to this competitive advantage , nuoA may also play significant roles:

• Energy supply for colonization: Efficient energy metabolism through properly functioning respiratory complexes containing nuoA likely supports the energy demands of competitive colonization processes.

• Adaptation to host environments: The ability to modulate energy metabolism under different environmental conditions (temperature, pH, carbon source availability) may contribute to B. phymatum's adaptability during host colonization .

• Support for secretion systems: The energy generated by respiratory complexes containing nuoA likely powers the T6SS and other secretion systems that directly contribute to competitive advantage .

• Metabolic flexibility: The respiratory chain components may enable B. phymatum to utilize diverse carbon sources available during different stages of plant colonization.

Researchers investigating this relationship should consider designing experiments that combine nuoA expression analysis with competitive nodulation assays across different host plants and environmental conditions.

How does nuoA in Burkholderia phymatum compare to homologous proteins in other bacterial species?

Comparative analysis of nuoA across bacterial species provides insights into evolutionary adaptations and functional conservation:

• Sequence conservation: While the search results don't provide direct comparison data, typical analysis would examine:

  • Conserved residues across alpha- and beta-proteobacteria

  • Lineage-specific adaptations in the Burkholderia genus

  • Variations correlated with different ecological niches (symbiotic vs. free-living bacteria)

• Structural variations: Comparing transmembrane domains and functional motifs across species can reveal adaptations specific to Burkholderia's lifestyle.

• Phylogenetic context: The reclassification of this organism from Burkholderia phymatum to Paraburkholderia phymatum reflects ongoing taxonomic refinement based on molecular phylogeny, which extends to the analysis of individual proteins like nuoA.

For researchers conducting comparative studies, recommended methodological approaches include:

• Multiple sequence alignments with homologs from diverse bacteria

• Construction of phylogenetic trees based on nuoA sequences

• Correlation of sequence variations with ecological niches and metabolic capabilities

• Structural prediction and comparison across species

What can genomic context analysis of the nuoA gene reveal about its regulation and evolution?

Genomic context analysis of the nuoA gene can provide valuable insights into its regulation, evolution, and functional relationships:

• Operon structure: The nuoA gene typically exists within the nuo operon containing multiple subunits of the NADH dehydrogenase complex. Analysis of this operon structure across Burkholderia species can reveal:

  • Conservation of gene order

  • Potential regulatory elements

  • Co-evolution of complex components

• Regulatory elements: Examining upstream regions may identify:

  • Promoter sequences

  • Transcription factor binding sites

  • Environmental response elements (oxygen, temperature, carbon source)

• Horizontal gene transfer assessment: Comparative genomic analysis can determine if:

  • nuoA shows evidence of horizontal transfer

  • The gene maintains synteny across related species

  • There are signs of selection pressure specific to symbiotic lifestyles

• Relationship to other metabolic genes: Examining genomic neighborhoods may reveal functional relationships with:

  • Other respiratory chain components

  • Nitrogen fixation genes

  • Type VI Secretion System components

Methodologically, researchers should combine bioinformatic approaches with experimental validation of predicted regulatory elements through reporter gene studies and transcriptomic analysis.

What emerging technologies could advance understanding of nuoA function in Burkholderia phymatum?

Several emerging technologies hold promise for advancing understanding of nuoA function in Burkholderia phymatum:

• CRISPR-Cas9 genome editing:

  • Precise modification of nuoA sequence without disrupting operon structure

  • Introduction of point mutations to test specific hypotheses about functionally important residues

  • Creation of conditional knockdowns to study essential functions

• Cryo-electron tomography:

  • Visualization of respiratory complexes in their native membrane environment

  • Study of supercomplexes involving NADH dehydrogenase in bacteroids within nodules

  • Structural comparison between free-living and symbiotic states

• Single-cell transcriptomics:

  • Analysis of nuoA expression heterogeneity within bacterial populations

  • Correlation with nitrogen fixation activity at single-cell level

  • Identification of regulatory networks controlling expression

• Metabolic flux analysis:

  • Tracking energy metabolism differences between wild-type and nuoA mutants

  • Quantifying contribution to ATP production during symbiosis

  • Measuring impact on nitrogen fixation efficiency

• Synthetic biology approaches:

  • Engineering optimized nuoA variants for enhanced energy efficiency

  • Creating biosensors to monitor respiratory activity during symbiosis

  • Developing minimal systems to study nuoA function independent of other variables

What are the potential applications of understanding nuoA function for improving agricultural sustainability?

Understanding nuoA function in Burkholderia phymatum has several potential applications for agricultural sustainability:

• Enhanced biological nitrogen fixation:

  • Optimizing energy metabolism could improve nitrogen fixation efficiency

  • Developing more efficient Burkholderia strains as biofertilizers

  • Reducing dependence on chemical nitrogen fertilizers

• Expanded host range engineering:

  • Understanding the energetic requirements for successful symbiosis could help engineer strains capable of nodulating additional crop species

  • B. phymatum already shows an exceptionally broad host range, nodulating over 40 different Mimosa species and important crops like Phaseolus vulgaris (common bean)

• Stress tolerance improvement:

  • Enhancing respiratory efficiency under environmental stresses (temperature, pH, drought)

  • Developing more resilient symbiotic relationships for changing climate conditions

• Competitive inoculant development:

  • Using knowledge of how energy metabolism contributes to competitive advantage to develop strains that can outcompete less efficient indigenous soil bacteria

  • B. phymatum has already demonstrated superior competitiveness compared to other rhizobial strains

• Sustainable agriculture systems:

  • Integrating optimized Burkholderia strains into farming practices

  • Reducing environmental impacts of nitrogen fertilization

  • Supporting organic and low-input agriculture systems

For researchers pursuing these applications, interdisciplinary approaches combining molecular biology, agricultural science, and ecology will be most effective in translating fundamental understanding of nuoA function into practical agricultural solutions.