Recombinant Bacillus weihenstephanensis NADH-quinone oxidoreductase subunit A (nuoA)

What is the structural composition of Bacillus weihenstephanensis NADH-quinone oxidoreductase subunit A?

Bacillus weihenstephanensis NADH-quinone oxidoreductase subunit A (nuoA) is a 122-amino acid membrane protein component of the respiratory chain. Its primary sequence (MASVYENSYMIVLIFLLLGILLPVVALTLGKMLRPNKPSAAKATTYESGIEPFHDANIRFHARYYIFALLFVIFDVETLFLYPWAVAYDKLGLFALIEMLIFVVMLLVGLAYAWKKKVLQWL) contains predominantly hydrophobic residues that anchor it within the bacterial membrane . This protein functions as part of the NDH-1 complex, which is involved in the electron transport chain of many bacterial species, facilitating NADH oxidation and quinone reduction through the FAD cofactor .

How does B. weihenstephanensis nuoA differ from similar proteins in other Bacillus species?

While the nuoA protein maintains similar function across Bacillus species, B. weihenstephanensis nuoA exhibits adaptations that may reflect its psychrotolerant (cold-tolerant) nature. Comparative sequence analysis reveals subtle but significant differences in amino acid composition that potentially contribute to protein flexibility and function at lower temperatures. Unlike mesophilic Bacillus species, B. weihenstephanensis exhibits specific signature sequences in cold-adaptive genes and proteins, including those involved in respiratory functions . These adaptations are part of a broader metabolic adjustment that allows the organism to maintain energy production through the electron transport chain at temperatures as low as 7°C .

What are the challenges in expressing and purifying recombinant B. weihenstephanensis nuoA?

Recombinant expression of B. weihenstephanensis nuoA presents several methodological challenges due to its membrane-associated nature. Successful protocols typically involve:

• Expression system selection: E. coli is the preferred heterologous host for nuoA expression

• Solubilization strategies: Extraction requires careful membrane disruption with detergents

• Purification approach: His-tag affinity chromatography followed by size-exclusion chromatography

• Storage considerations: The purified protein requires stabilization with glycerol (typically 50%) and storage at -20°C/-80°C to preserve activity

Researchers should note that repeated freeze-thaw cycles significantly reduce protein integrity and function, necessitating aliquoting of purified protein .

What methodologies can be employed to study nuoA protein-quinone interactions?

To investigate nuoA interactions with quinone substrates, researchers can employ:

• In silico docking studies: Using high-resolution structures (≤2.15Å) to model quinone binding poses and predict molecular interactions

• Site-directed mutagenesis: Systematic modification of predicted quinone-binding residues to assess their contribution to function

• Enzyme kinetics: Measuring NADH oxidation rates in the presence of various quinone substrates to determine specificity

• Biophysical techniques: Isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) to quantify binding affinities

Recent in silico studies with related NDH-2 structures revealed that quinone binding occurs with remarkably few molecular interactions, primarily involving hydrophobic contacts with the quinone head group . The binding mode positions one carbonyl oxygen of the quinone to form a hydrogen bond with the N5 atom of the FAD cofactor .

How can researchers assess nuoA function within the context of B. weihenstephanensis psychrotolerance?

To investigate nuoA's role in cold adaptation, researchers should employ a multi-faceted approach:

• Temperature-dependent activity assays: Measure NADH oxidation rates across temperatures ranging from 4°C to 43°C

• Comparative expression analysis: Quantify nuoA expression levels under various temperature conditions using RT-PCR

• Mutant complementation studies: Express B. weihenstephanensis nuoA in mesophilic Bacillus strains to assess functional replacement

• Structural dynamics investigation: Use hydrogen-deuterium exchange mass spectrometry to compare protein flexibility at different temperatures

This methodology should consider that B. weihenstephanensis exhibits optimal growth and enzyme activity at temperatures between 7-30°C, with significant metabolic changes occurring below 7°C .

What protocols are recommended for studying nuoA involvement in biofilm formation?

To investigate potential nuoA roles in biofilm processes:

• Biofilm growth assays: Compare wild-type and nuoA-deficient strains under various conditions

• Nuclease activity assays: Since extracellular DNA is a key biofilm component, assess whether nuoA influences nuclease production using methods similar to those employed for NucB characterization

• Gene expression analysis: Use RT-PCR to analyze nuoA expression changes during biofilm development stages

• Microscopy techniques: Employ confocal microscopy with fluorescent probes to visualize respiratory activity within biofilm structures

When designing these experiments, consider that B. weihenstephanensis forms robust biofilms at lower temperatures (12-20°C), which may influence the expression and activity of respiratory chain components .

How might the taxonomic reclassification of B. weihenstephanensis affect interpretation of nuoA research data?

Recent genomic analyses have proposed B. weihenstephanensis as a later heterotypic synonym of Bacillus mycoides , necessitating careful consideration when interpreting nuoA research. Researchers should:

• Use multilocus sequence typing (MLST) to accurately classify strains used in nuoA studies

• Compare nuoA sequences between traditionally classified B. weihenstephanensis and B. mycoides strains

• Evaluate whether observed functional differences correlate with psychrotolerant phenotype rather than species designation

• Consider evolutionary perspectives: nuoA sequence conservation may reflect its essential respiratory function despite taxonomic reclassification

Research may need to be reinterpreted in light of evidence that psychrotolerance and specific signature sequences in 16S rRNA and cspA genes may not properly distinguish B. weihenstephanensis from some other B. cereus sensu lato members .

What insights can comparative genomics provide about the evolution of nuoA in psychrotolerant Bacillus species?

Comparative genomic approaches to nuoA evolution should:

• Construct phylogenetic trees using nuoA sequences from multiple Bacillus species

• Identify positive selection signatures in nuoA coding regions across temperature-diverse species

• Analyze regulatory elements controlling nuoA expression across psychrotolerant and mesophilic strains

• Examine horizontal gene transfer patterns in respiratory chain components

These approaches can reveal whether nuoA adaptations represent convergent evolution or vertical inheritance patterns. Current evidence suggests that psychrotolerant characteristics in B. weihenstephanensis represent specific niche adaptations rather than distant evolutionary divergence from related Bacillus species .

How can researchers integrate nuoA function with broader metabolic networks in B. weihenstephanensis?

To understand nuoA's role within broader cellular metabolism:

• Metabolic flux analysis: Use isotope-labeled substrates to track carbon flow through respiratory and fermentative pathways at different temperatures

• Systems biology modeling: Develop mathematical models integrating transcriptomic and proteomic data for respiratory chain components

• Comparative multi-omics: Analyze how nuoA expression correlates with global metabolic shifts during cold adaptation

• Protein-protein interaction studies: Identify nuoA interaction partners beyond the immediate NDH complex

This integrated approach should consider that B. weihenstephanensis exhibits unique metabolic adaptations, including melanin-like pigment production and cereulide production at temperatures as low as 8°C in some strains .

What are effective strategies to overcome expression and solubility issues with recombinant nuoA?

When encountering difficulties with nuoA expression:

• Expression system optimization:

  • Test multiple E. coli strains (BL21(DE3), C41(DE3), C43(DE3))

  • Vary induction conditions (temperature, IPTG concentration)

  • Consider alternative expression systems (B. subtilis, cell-free)

• Fusion tag strategies:

  • N-terminal His-tag appears most effective for nuoA purification

  • Test MBP or SUMO fusion to enhance solubility

  • Include TEV or other protease cleavage sites for tag removal

• Membrane protein-specific approaches:

  • Use mild detergents (DDM, LDAO) for extraction

  • Consider bicelles or nanodiscs for functional studies

  • Test different buffer compositions to maintain stability

• Reconstitution methods:

  • Carefully monitor protein concentration during reconstitution (0.1-1.0 mg/mL recommended)

  • Add glycerol (5-50%) to prevent aggregation

  • Avoid repeated freeze-thaw cycles

How can researchers validate the structural integrity of purified recombinant nuoA?

To ensure properly folded nuoA:

Researchers should note that properly folded nuoA shows >90% purity by SDS-PAGE and maintains activity when stored appropriately .

What methodological approaches can address potential data inconsistencies when comparing nuoA from different psychrotolerant Bacillus strains?

When encountering data inconsistencies:

• Strain verification protocols:

  • Perform 16S rRNA sequencing

  • Check for psychrotolerant growth phenotype (growth at 7°C but not 43°C)

  • Verify signature sequences in 16S rRNA and cspA genes

• Standardized expression conditions:

  • Establish consistent cultivation temperatures

  • Standardize growth media composition

  • Define harvest points based on growth phase

• Data normalization strategies:

  • Use internal reference proteins

  • Apply statistical methods appropriate for between-strain comparisons

  • Report experimental conditions comprehensively

• Consideration of genetic context:

  • Analyze horizontal gene transfer events affecting respiratory genes

  • Check for mobile genetic elements altering expression patterns

  • Examine taxonomic classification using multiple genetic markers

This approach acknowledges that B. weihenstephanensis strains show genetic diversity that might influence nuoA expression and function.

How might nuoA be exploited as a potential antimicrobial target considering its absence in mammalian systems?

As NDH-2 proteins are absent in mammals but essential in many bacteria, nuoA represents a promising antimicrobial target . Research approaches should include:

• High-throughput screening strategies:

  • In silico screening using the high-resolution NDH-2 structure

  • Biochemical assays measuring NADH oxidation inhibition

  • Whole-cell assays with B. weihenstephanensis

• Structure-based drug design:

  • Focus on the quinone-binding site, which has limited molecular interactions

  • Explore allosteric sites that may affect nuoA function

  • Consider species selectivity to target specific pathogens

• Resistance development analysis:

  • Assess mutation frequencies in target sites

  • Evaluate horizontal gene transfer potential

  • Monitor compensatory metabolic pathways

• Delivery approaches for membrane-targeted compounds:

  • Nanoparticle formulations

  • Prodrug strategies

  • Combination approaches with membrane permeabilizers

What are the implications of studying nuoA function for understanding bacterial adaptation to extreme environments?

B. weihenstephanensis nuoA research has broader implications for understanding bacterial adaptation:

• Cold adaptation mechanisms:

  • Investigate how nuoA structural modifications maintain function at low temperatures

  • Explore energy conservation strategies in psychrotolerant bacteria

  • Compare with other extremophile respiratory adaptations

• Environmental resilience connections:

  • Examine links between respiratory efficiency and spore formation

  • Study how nuoA function relates to germination at different temperatures

  • Investigate potential roles in biofilm formation and persistence

• Evolutionary perspectives:

  • Analyze horizontal vs. vertical acquisition of respiratory chain adaptations

  • Compare nuoA sequences across psychrotolerant species from diverse environments

  • Evaluate convergent evolution in cold-adapted respiratory components

This research could provide insights applicable to bioremediation, food safety, and biotechnology applications involving cold-adapted organisms.

How might systems biology approaches advance our understanding of nuoA's role in B. weihenstephanensis metabolism?

Integrative systems approaches offer powerful tools for nuoA research:

• Multi-omics integration strategies:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Model metabolic flux changes at different temperatures

  • Analyze regulatory networks controlling respiratory processes

• Computational modeling approaches:

  • Develop genome-scale metabolic models incorporating temperature effects

  • Simulate electron transport chain dynamics under varying conditions

  • Predict metabolic adaptations to respiratory chain perturbations

• Single-cell analysis technologies:

  • Investigate cell-to-cell variability in nuoA expression

  • Examine respiratory heterogeneity within bacterial populations

  • Study nuoA dynamics during lifecycle transitions

• Synthetic biology applications:

  • Engineer optimized nuoA variants for biotechnological processes

  • Design minimal respiratory systems incorporating modified nuoA

  • Develop cold-adapted biocatalysts based on psychrotolerant principles

These approaches could reveal how nuoA contributes to the remarkable adaptability of B. weihenstephanensis across temperature ranges and environmental conditions.