Recombinant Shewanella oneidensis NADH-quinone oxidoreductase subunit A (nuoA)

What is the role of NADH-quinone oxidoreductase subunit A (nuoA) in Shewanella oneidensis?

NADH-quinone oxidoreductase subunit A (nuoA) in Shewanella oneidensis MR-1 functions as a critical component of the respiratory chain complex I (NADH dehydrogenase I). This membrane-bound protein (134 amino acids) participates in electron transfer from NADH to quinones, contributing to the organism's remarkable respiratory versatility. The protein is encoded by the nuoA gene (also designated as SO_1021) and forms part of the proton-translocating module of the complex that generates the proton motive force necessary for ATP synthesis. S. oneidensis is known for its diverse respiratory capabilities, including the reduction of various oxyanions and metals, making its electron transport components particularly significant in biogeochemical cycling and bioremediation applications .

What expression systems are recommended for recombinant nuoA protein production?

E. coli expression systems have been successfully employed for the production of recombinant Shewanella oneidensis nuoA protein as demonstrated in commercial applications . For optimal expression, consider the following methodology:

When expressing membrane proteins like nuoA, lower post-induction temperatures help reduce inclusion body formation and improve proper membrane integration. The recently developed electroporation method for S. oneidensis with efficiency of ~4.0 x 10^6 transformants/μg DNA provides an alternative approach for homologous expression, potentially yielding more naturally folded protein .

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

To maintain stability and functionality of recombinant Shewanella oneidensis nuoA protein, implement the following storage and handling protocols:

• Store lyophilized protein at -20°C/-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% (50% is recommended) for long-term storage

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• For working solutions, store aliquots at 4°C for up to one week

• Use Tris/PBS-based buffer at pH 8.0 with 6% trehalose for optimal stability

For experimental protocols, minimize exposure to room temperature and centrifuge vials briefly before opening to bring contents to the bottom. This helps preserve the structural integrity of this membrane protein, which is particularly important for functional studies.

How can I verify the identity and purity of recombinant nuoA protein?

Multiple complementary techniques should be employed to confirm the identity and purity of recombinant Shewanella oneidensis nuoA protein:

The complete amino acid sequence (MFADISAQHWAFAIYVIGAIAICLTMIGLAALLGGRAQGRTKNKPFESGVDSVGTARLRFSAKFYLVAMFFVIFDVEALYLFAWSVSVRESGWVGFIEATIFIGLLLIGLVYLWRIGALEWSPRKPQLNNKNTD) can be used as a reference for protein identification techniques . For membrane proteins like nuoA, consider using specialized detergent-based buffers during analysis to maintain protein solubility.

How does nuoA contribute to the diverse respiratory capabilities of Shewanella oneidensis?

Shewanella oneidensis MR-1 is renowned for its remarkable respiratory diversity, capable of utilizing numerous terminal electron acceptors including metal oxides, nitrate, fumarate, and various oxyanions . The nuoA protein, as part of Complex I, plays a significant role in this respiratory flexibility through:

• Primary electron input: Complex I (containing nuoA) represents a major entry point for electrons into the respiratory chain from NADH oxidation

• Proton translocation: Contributes to proton motive force generation necessary for energy conservation

• Respiratory adaptation: Participates in modulating electron flow depending on available terminal electron acceptors

While specific bromate reduction is mediated by dimethylsulfoxide reductase (encoded by dmsA) rather than nuoA directly , the nuoA-containing Complex I likely supplies electrons to this pathway under microaerobic conditions. This is supported by the observation that S. oneidensis MR-1 can reduce bromate effectively when exposed to concentrations between 0.15-1 mM .

The relationship between nuoA expression and alternative respiratory pathways in S. oneidensis presents a promising area for investigating the molecular basis of respiratory flexibility in environmental bacteria.

What approaches are most effective for studying nuoA structure-function relationships?

Investigating structure-function relationships in Shewanella oneidensis nuoA requires a multifaceted approach combining genetic, biochemical, and biophysical techniques:

A. Genetic Engineering Approaches:
The recently developed prophage-mediated genome engineering (recombineering) system for S. oneidensis represents a significant advancement for structure-function studies . This system achieves:

• An efficiency of ~5% recombinants among total cells

• Markerless mutations without antibiotic selection markers

• Precise editing using single-strand DNA oligonucleotides

B. Recommended Structure-Function Analysis Workflow:

The λ Red Beta homolog from Shewanella sp. W3-18-1 enables precise targeting of chromosomal alleles, facilitating systematic mutagenesis of nuoA to map functional domains . This approach can be particularly valuable for understanding how nuoA contributes to the respiratory versatility that makes S. oneidensis valuable for bioremediation applications.

How does the electron transfer mechanism involving nuoA compare between aerobic and anaerobic conditions?

The electron transfer mechanisms involving nuoA in Shewanella oneidensis differ significantly between aerobic and anaerobic respiratory conditions:

Aerobic Conditions:
Under aerobic conditions, nuoA-containing Complex I primarily couples NADH oxidation to quinone reduction, with oxygen serving as the terminal electron acceptor via cytochrome oxidases. This represents the classical aerobic respiratory chain configuration.

Anaerobic Conditions:
S. oneidensis demonstrates remarkable respiratory flexibility under anaerobic conditions. While nuoA still functions in electron input to the quinone pool, the downstream electron flow changes dramatically:

• Under microaerobic conditions with bromate present, electrons likely flow from Complex I (containing nuoA) through CymA (a membrane-anchored cytochrome) to terminal reductases like DmsA, which has been shown to reduce bromate effectively

• The extracellular electron transfer pathway, crucial for metal reduction, involves:

  • Initial electron input via nuoA-containing Complex I

  • Transfer to CymA (menaquinol:cytochrome c oxidoreductase)

  • Further transfer through the Mtr pathway (MtrB, MtrC)

  • Final reduction of extracellular substrates

This pathway adaptation is evidenced by experiments showing that mutants lacking key components such as MtrB, MtrC, CymA, GspD, and DmsA exhibit significantly reduced bromate reduction capacity . Specifically, when S. oneidensis MR-1 was exposed to bromate concentrations ranging from 0.15 to 1 mM, reduction efficiencies between 23-100% were observed within 12 hours, with the efficiency decreasing as bromate concentration increased .

What methodologies are most effective for studying protein-protein interactions involving nuoA?

Investigating protein-protein interactions (PPIs) involving the membrane-bound nuoA protein requires specialized techniques that accommodate its hydrophobic nature:

A. In vivo Crosslinking Mass Spectrometry (XL-MS):

• Treat intact S. oneidensis cells with membrane-permeable crosslinkers (e.g., DSS, formaldehyde)

• Isolate membrane fractions and purify crosslinked complexes

• Perform tryptic digestion followed by LC-MS/MS analysis

• Identify crosslinked peptides using specialized software (e.g., pLink, XlinkX)

This approach preserves native membrane environments and captures transient interactions within the respiratory complex.

B. Genetic Fusion Reporters for Membrane Proteins:

The recent development of efficient transformation methods in S. oneidensis (achieving ~4.0 x 10^6 transformants/μg DNA ) facilitates the genetic manipulation required for these techniques, allowing for more effective study of nuoA interactions within its native cellular context.

C. Co-purification Approaches:
When expressing recombinant nuoA with a His-tag , mild solubilization conditions using digitonin or amphipol can preserve protein-protein interactions during purification. Subsequent analysis by Blue Native PAGE or co-immunoprecipitation can identify interacting partners within the respiratory complex.

How can nuoA be incorporated into synthetic biology applications for enhanced extracellular electron transfer?

Shewanella oneidensis MR-1's remarkable ability to perform extracellular electron transfer makes it valuable for synthetic biology applications in bioremediation, bioelectrochemical systems, and microbial fuel cells. The nuoA protein, as part of Complex I, can be strategically manipulated to enhance these capabilities:

A. Metabolic Engineering Strategies:

• Overexpression approach:

  • Utilize the newly developed electroporation method (efficiency ~4.0 x 10^6 transformants/μg DNA)

  • Design expression constructs with tunable promoters controlling nuoA expression

  • Systematically vary Complex I component ratios to optimize electron flux

• Heterologous expression system:

  • Express S. oneidensis nuoA in other chassis organisms

  • Introduce additional components of the Shewanella electron transport chain

  • Create hybrid systems with enhanced electron transfer capabilities

B. Implementation workflow:

C. Current performance benchmarks:
S. oneidensis strains have demonstrated variable capacities for reducing different compounds. For example, with bromate, reduction efficiencies range from 23% (at 1 mM bromate) to 100% (at 0.15 mM bromate) within 12 hours . By engineering nuoA and related components, these capabilities could potentially be enhanced for specific applications.

The precise genome editing capabilities provided by the λ Red Beta homolog-based recombineering system enable the fine-tuning necessary to optimize nuoA function within synthetic biology constructs, opening new possibilities for environmental biotechnology applications.

What protocols are recommended for assessing nuoA involvement in different respiratory pathways?

To systematically evaluate nuoA's role in Shewanella oneidensis respiratory pathways, the following comprehensive protocol framework is recommended:

Genetic Approach: Gene Deletion and Complementation

• Generate nuoA deletion mutant using the λ Red Beta homolog-based recombineering system (achieving ~5% recombinants)

• Create complementation strains with wild-type and modified nuoA variants

• Verify genetic modifications by PCR and sequencing

B. Respiratory Phenotype Analysis:

C. Molecular Analysis:

• RNA-seq to determine transcriptional changes in respiratory pathways

• Proteomics to quantify changes in respiratory proteins

• Activity assays for NADH dehydrogenase using recombinant nuoA protein

This approach follows the experimental design utilized in bromate reduction studies, where gene deletion mutants and complemented strains were systematically evaluated to identify proteins involved in the reduction pathway . For nuoA specifically, deletion would be expected to impact multiple respiratory pathways if it indeed serves as a primary electron input component.

How can isotope labeling be used to track electron flow through nuoA-dependent pathways?

Isotope labeling provides powerful insights into electron flow through respiratory pathways involving nuoA in Shewanella oneidensis. The following methodological approaches are particularly effective:

A. 13C-Metabolic Flux Analysis:

• Culture S. oneidensis with 13C-labeled substrates (e.g., [1-13C]lactate, [U-13C]glucose)

• Extract metabolites at defined time points

• Analyze isotope distribution using LC-MS/MS or NMR

• Compare wild-type and nuoA mutant strains to identify pathway alterations

B. 2H (Deuterium) Labeling to Track Hydride Transfer:

• Incubate cells with deuterated substrates (e.g., [4-2H]NADH)

• Monitor deuterium incorporation into quinone pool and downstream electron carriers

• Quantify isotope distribution using mass spectrometry

• Map electron transfer routes through nuoA-containing Complex I

C. Experimental Design Framework:

This approach builds upon the knowledge that S. oneidensis can reduce various substrates through different electron transfer pathways . By tracking isotope labels through these pathways in wild-type versus genetically modified strains, researchers can quantitatively assess the contribution of nuoA to different respiratory modes.

What considerations are important when designing nuoA mutants for structure-function studies?

When designing nuoA mutants for structure-function studies in Shewanella oneidensis, several critical factors must be considered to ensure meaningful results:

A. Transmembrane Domain Preservation:
The nuoA protein (134 amino acids) contains multiple transmembrane helices that are essential for proper integration into the membrane and complex assembly. When designing mutations:

• Use topology prediction tools (TMHMM, TOPCONS) to identify transmembrane segments

• Avoid disrupting helix-helix interfaces unless specifically targeting interaction sites

• Consider conservative substitutions within transmembrane regions to maintain hydrophobicity

B. Strategic Mutation Planning:

C. Implementation Using Recombineering:
The recently developed λ Red Beta homolog-based recombineering system for S. oneidensis provides an ideal tool for creating precise mutations :

• Design single-stranded DNA oligonucleotides containing desired mutations

• Target specific chromosomal alleles for modification

• Achieve ~5% recombinants among total cells without antibiotic markers

• Screen for successful mutants using appropriate phenotypic assays

What techniques can resolve contradictory data when studying nuoA function in different respiratory conditions?

When faced with contradictory data regarding nuoA function in different respiratory conditions, a systematic troubleshooting and validation approach is essential:

A. Experimental Validation Matrix:

B. Source of Contradiction Resolution Strategies:

• Technical vs. Biological Variability:

  • Implement statistical approaches (e.g., ANOVA with post-hoc tests)

  • Increase biological replicates (n≥5) to distinguish patterns

  • Use multiple technical approaches to measure the same parameter

• Strain Background Effects:

  • Compare nuoA function in different S. oneidensis isolates

  • Sequence verify the entire nuoA operon to identify potential suppressors

  • Consider genomic context using the developed recombineering system

• Environmental Condition Standardization:

  • Precisely control oxygen levels (especially for microaerobic conditions)

  • Standardize media composition and growth phase

  • Monitor real-time environmental parameters during experiments

This approach draws inspiration from studies of bromate reduction in S. oneidensis, where initial contradictory results under anaerobic versus microaerobic conditions were resolved through systematic investigation . Under anaerobic conditions, S. oneidensis MR-1 did not exhibit bromate-respiring characteristics, yet under microaerobic conditions, it effectively reduced bromate to bromide. Such apparent contradictions can often be resolved through careful control of experimental conditions and comprehensive measurement approaches.

How can nuoA function be integrated into whole-cell models of Shewanella oneidensis metabolism?

Integrating nuoA function into comprehensive whole-cell models of Shewanella oneidensis metabolism requires a multi-scale modeling approach that connects molecular mechanisms to cellular physiology:

A. Model Integration Framework:

• Molecular Level: Incorporate nuoA structure and electron transfer kinetics

  • Include amino acid sequence data (134 residues)

  • Model transmembrane topology and protein interactions

  • Define electron transfer rates and proton translocation stoichiometry

• Metabolic Network Level: Position nuoA in genome-scale metabolic models

  • Connect to NADH-producing pathways (TCA cycle, glycolysis)

  • Link to various terminal electron acceptor pathways

  • Establish regulatory constraints based on experimental data

• Cellular Physiology Level: Model impact on growth and respiration

  • Predict growth rates under different respiratory conditions

  • Estimate ATP generation capacity

  • Simulate adaptation to changing electron acceptor availability

B. Implementation Methodology:

This integrative approach would build upon existing knowledge of S. oneidensis respiratory diversity, including its ability to reduce various compounds like bromate under microaerobic conditions , while incorporating the molecular details of nuoA function within Complex I. The resulting model would provide predictive power for understanding how nuoA contributes to the remarkable respiratory flexibility that makes S. oneidensis valuable for bioremediation applications.

What emerging technologies will advance our understanding of nuoA function in the next decade?

Several emerging technologies are poised to revolutionize our understanding of nuoA function in Shewanella oneidensis over the next decade:

A. Single-Molecule Technologies:

• Cryo-electron tomography: Will enable visualization of nuoA within intact membrane complexes in near-native states

• Single-molecule FRET: Can track conformational changes during electron transfer in real-time

• Nanopore-based single-molecule protein sequencing: Will provide direct readout of post-translational modifications

B. Advanced Genetic Technologies:

The recently developed recombineering system for S. oneidensis provides a foundation for these advanced genetic approaches, enabling precise genome editing that will be essential for next-generation studies.

C. Computational and Systems Biology Approaches:

• Quantum mechanics/molecular mechanics (QM/MM): Will provide atomic-level insights into electron transfer through nuoA

• Artificial intelligence-driven structure prediction: Will generate increasingly accurate models of nuoA and its complexes

• Multi-scale modeling: Will connect molecular events to cellular phenotypes