Recombinant Bacillus cereus subsp. cytotoxis NADH-quinone oxidoreductase subunit A (nuoA)

What expression systems are recommended for producing recombinant nuoA protein?

For research purposes, recombinant Bacillus cereus nuoA protein can be successfully expressed in E. coli expression systems, particularly when fused to an N-terminal His-tag to facilitate purification . This heterologous expression approach allows for the production of the full-length protein (amino acids 1-122) with high purity (>90% as determined by SDS-PAGE) . The recombinant protein is typically obtained as a lyophilized powder, which provides stability during storage.

The expression system should be carefully optimized considering that nuoA is a membrane protein, which can present challenges for proper folding and solubility. The use of E. coli as an expression host offers practical advantages for laboratory-scale protein production, including rapid growth, high yields, and established protocols for membrane protein expression .

What are the optimal storage and handling conditions for recombinant nuoA protein?

Proper storage and handling of recombinant nuoA protein is critical for maintaining its structural integrity and functional activity. Recommended storage protocols include:

• Store lyophilized protein at -20°C to -80°C upon receipt

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (typically 50%) for long-term storage

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• For short-term storage, working aliquots can be kept at 4°C for up to one week

Repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of activity. When working with the reconstituted protein, it is advisable to briefly centrifuge the vial before opening to bring contents to the bottom .

What experimental conditions are optimal for assaying NADH:quinone oxidoreductase activity?

Based on studies with B. cereus KCTC 3674, the optimal conditions for assaying NADH:quinone oxidoreductase activity vary depending on the electron acceptor used:

How do respiratory chain inhibitors affect NADH:quinone oxidoreductase activity, and what mechanistic insights do these provide?

NADH:quinone oxidoreductase activity in B. cereus exhibits differential sensitivity to respiratory chain inhibitors, providing valuable insights into its mechanism and structure:

The enzyme shows remarkable resistance to several common respiratory chain inhibitors:

• Rotenone (a classic Complex I inhibitor)

• Capsaicin

• Silver nitrate (AgNO₃)

• 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO)

This inhibitor profile distinguishes the B. cereus enzyme from many other bacterial and mitochondrial NADH:quinone oxidoreductases. The HQNO sensitivity indicates that the quinone-binding site is accessible to this inhibitor, suggesting a specific structural arrangement in this region of the enzyme complex. The resistance to rotenone is particularly noteworthy as it implies a structural divergence from mitochondrial Complex I.

For experimental approaches, researchers should consider using HQNO as a specific inhibitor when characterizing mutant forms of nuoA or studying the respiratory chain in B. cereus. The inhibitor profile also suggests that the B. cereus NADH:quinone oxidoreductase lacks a canonical energy coupling site despite containing FAD as a cofactor .

What methods can be used to investigate the membrane topology and functional domains of nuoA?

Investigating the membrane topology and functional domains of nuoA requires a multi-faceted approach:

• Computational prediction analysis:

  • Hydropathy plot analysis of the amino acid sequence reveals multiple hydrophobic regions consistent with transmembrane domains

  • The amino acid sequence shows characteristic features of a membrane-embedded subunit with transmembrane helices

• Experimental topology mapping:

  • Cysteine scanning mutagenesis: Systematically replace residues with cysteine and probe accessibility with sulfhydryl reagents

  • PhoA/LacZ fusion approach: Create fusion proteins at different positions to determine cytoplasmic versus periplasmic orientation

  • Protease protection assays on membrane preparations

• Structure-function studies:

  • Site-directed mutagenesis of conserved residues, particularly those in predicted functional domains

  • Cross-linking studies to identify interaction partners within the NDH-1 complex

  • Truncation analysis to identify minimal functional domains

When designing these experiments, researchers should keep in mind that nuoA is part of a multi-subunit complex, and its proper folding and function may depend on interactions with other respiratory chain components.

How does nuoA contribute to electron transfer pathways in the Bacillus cereus respiratory chain?

The electron transfer role of nuoA can be investigated using several methodological approaches:

• Spectroscopic analysis:

  • UV-visible spectroscopy to monitor NADH oxidation rates

  • Electron paramagnetic resonance (EPR) to detect transient radical species

  • Analysis of flavin reduction/oxidation states during catalysis

• Artificial electron acceptor studies:

  • Comparative analysis using different electron acceptors (ubiquinone-1 and menadione)

  • The differential activity with ubiquinone-1 (8-fold enhancement after Triton X-100 extraction) versus menadione (4-fold enhancement) suggests specific electron transfer pathways

• Membrane potential measurements:

  • Determine if electron transfer through nuoA contributes to proton translocation

  • The evidence suggests that B. cereus NADH:quinone oxidoreductase lacks an energy coupling site, making its role in energy conservation distinct from proton-pumping Complex I enzymes

Researchers should note that B. cereus preferentially oxidizes NADH over deamino-NADH, indicating substrate specificity that may be partly determined by nuoA structure . This substrate preference provides a useful experimental parameter when characterizing mutant forms of the enzyme.

What experimental approaches can assess potential interactions between nuoA and virulence factors in Bacillus cereus?

While nuoA itself has not been directly linked to virulence, its role in the respiratory chain may indirectly influence the expression or activity of established virulence factors. Methodological approaches to investigate such interactions include:

• Cell culture models:

  • Intestinal epithelial cell (IEC) models can be used to assess cytotoxicity

  • Polarized colon epithelial cells (Ptk6) provide a physiologically relevant system

  • Flow cytometry with propidium iodide staining can quantify cell death

• Genetic approaches:

  • Construction of single and double deletion mutants (as demonstrated with ΔnheBC and Δsph strains)

  • Complementation studies to confirm phenotypes

  • Transcriptomic analysis to identify coordinated gene regulation

• In vivo models:

  • Galleria mellonella larval model provides a useful system for assessing pathogenicity

  • Mortality rates can be monitored over time (e.g., 3-7 days)

  • Comparison between wild-type and mutant strains can reveal contributions to virulence

The B. cereus NVH 0075-95 strain has been well-characterized in terms of cytotoxicity and virulence factors, making it a useful reference strain for comparative studies involving respiratory chain components .

How do extraction methods affect the study of membrane-associated enzymes like NADH:quinone oxidoreductase?

The choice of membrane extraction methods critically impacts the observed properties of NADH:quinone oxidoreductase:

These considerations are essential when interpreting activity data and designing experiments to study structure-function relationships in nuoA and the NADH:quinone oxidoreductase complex.

What genomic approaches can be used to study nuoA variants across Bacillus cereus strains?

Understanding nuoA conservation and variation across B. cereus strains can provide insights into its evolutionary importance and functional constraints. Methodological approaches include:

• Comparative genomics:

  • Whole-genome sequencing of multiple B. cereus isolates

  • Alignment of nuoA sequences to identify conserved regions and polymorphisms

  • Analysis of selection pressure (dN/dS ratios) to identify functionally important residues

• Structure prediction:

  • Homology modeling based on related respiratory chain components

  • Integration of sequence conservation data with structural predictions

  • Molecular dynamics simulations to predict effects of variants

• Functional validation:

  • Expression of variant nuoA proteins in heterologous systems

  • Activity assays under standardized conditions

  • Assessment of susceptibility to HQNO and other inhibitors

This genomic perspective can help researchers contextualize experimental findings and identify strains with naturally occurring variations for further functional studies.

How can systems biology approaches integrate nuoA function within the broader metabolic network of Bacillus cereus?

The function of nuoA and NADH:quinone oxidoreductase exists within a broader metabolic context that can be explored through systems-level approaches:

• Metabolic flux analysis:

  • ¹³C labeling experiments to trace carbon flow through central metabolism

  • Quantification of NADH/NAD⁺ ratios under different growth conditions

  • Integration of respiratory chain activity with glycolysis, TCA cycle, and fermentation pathways

• Transcriptomic and proteomic correlation:

  • RNA-Seq to identify co-expressed genes under varying oxygen tensions

  • Proteomics to quantify respiratory chain components

  • Integration of gene expression data with metabolic models

• Growth phenotype analysis:

  • Phenotypic microarrays to assess growth across diverse nutrients and conditions

  • Comparison of wild-type and nuoA mutant strains

  • Identification of conditions where NADH:quinone oxidoreductase activity becomes critical

These approaches can help position nuoA function within the broader adaptive strategies of B. cereus and potentially reveal unexpected connections to virulence mechanisms or stress responses.