Recombinant Pasteurella multocida Na (+)-translocating NADH-quinone reductase subunit C (nqrC)

How does the structure of P. multocida NqrC compare to NqrC from other bacterial species?

While specific structural information for P. multocida NqrC is limited in the provided search results, we can infer from related research that NqrC is likely conserved across Na+-NQR-containing bacteria. The Na+-NQR complex is found in several important pathogens including Vibrio cholerae, Vibrio parahaemolyticus, Haemophilus influenzae, Neisseria gonorrhoeae, Pasteurella multocida, Porphyromonas gingivalis, Enterobacter aerogenes, and Yersinia pestis . NqrC typically contains conserved cysteine residues that are involved in covalent FMN binding, which is essential for electron transport within the complex. Sequence alignment analysis would likely reveal high conservation in functional domains while showing species-specific variations in other regions.

What are the standard expression systems used for recombinant production of P. multocida NqrC?

For recombinant expression of Na+-NQR components including NqrC from P. multocida, E. coli expression systems are commonly employed. Based on methodologies used for similar proteins, the process typically involves:

• Gene amplification using PCR with high-fidelity polymerases (such as PrimeSTAR Mix or Encyclo polymerase)

• Cloning into expression vectors (like pET series vectors or pBAD-TOPO TA)

• Expression in E. coli strains such as BL21

• Induction with appropriate inducers (like L-arabinose at 0.2% for pBAD-based systems)

• Purification via affinity chromatography using N-terminal or C-terminal His-tags

When expressing NqrC, it's crucial to co-express the maturation factors ApbE (for flavin attachment) and NqrM for proper assembly, as these are required for producing functional Na+-NQR subunits .

What is the optimal protocol for purifying active recombinant P. multocida NqrC?

The optimal protocol for purifying active recombinant P. multocida NqrC would follow these methodological steps:

• Express the His-tagged NqrC in E. coli BL21 or similar strains, co-expressing maturation factors ApbE and NqrM

• Grow cells at 32°C to mid-exponential phase (A600 ~0.3-0.4)

• Induce expression with 0.2% L-arabinose and continue growth for 3 hours

• Harvest cells by centrifugation (10,000 × g, 10 min)

• Wash cells twice with a buffer containing 300 mM NaCl, 5 mM MgSO4, and 10 mM Tris-HCl (pH 8.0)

• Lyse cells using French press or sonication

• Separate the soluble fraction by centrifugation

• Load the supernatant onto a Ni-NTA column equilibrated with 5 mM imidazole-HCl (pH 8.0)

• Wash with 10 mM imidazole-HCl

• Elute the His-tagged protein with 100 mM imidazole-HCl in buffer containing 300 mM NaCl and 10 mM Tris-HCl (pH 8.0)

• Concentrate the purified protein and store frozen in liquid N2

This methodology is adapted from protocols used for other Na+-NQR components and would need to be optimized specifically for NqrC .

How can researchers verify the proper folding and flavin incorporation in recombinant NqrC?

To verify proper folding and flavin incorporation in recombinant NqrC, researchers should employ multiple analytical techniques:

• Spectroscopic analysis: UV-visible absorption spectroscopy can confirm the presence of covalently bound FMN, which typically shows characteristic absorption peaks around 380 nm and 450 nm.

• Fluorescence spectroscopy: FMN fluorescence can be measured to confirm flavin incorporation.

• SDS-PAGE analysis: When properly flavinated, NqrC should show fluorescence under UV light prior to staining, and the band may show abnormal migration compared to predicted molecular weight due to covalent flavin binding.

• Western blot analysis: Using anti-His antibodies (1:1000 dilution) followed by fluorescent secondary antibodies (1:10,000 dilution) as described for other recombinant proteins .

• Activity assays: Measuring NADH:quinone oxidoreductase activity and its sodium dependency can confirm functional incorporation of the subunit into the complex.

Proper flavination of NqrC depends on co-expression with ApbE, which is responsible for the covalent attachment of FMN to specific threonine residues .

What are the critical considerations for designing mutagenesis studies of P. multocida NqrC?

When designing mutagenesis studies of P. multocida NqrC, researchers should consider the following critical factors:

• Target residue selection: Focus on conserved residues likely involved in:

  • Flavin binding (typically conserved threonine residues)

  • Subunit interactions with other Nqr components

  • Electron transfer pathways

  • Sodium binding sites

• Mutagenesis method: Site-directed mutagenesis using systems like the QuikChange II kit with carefully designed mutagenic primers that maintain proper annealing temperatures (typically around 60°C for 30s in PCR cycles) .

• Co-expression requirements: Ensure mutant NqrC is co-expressed with necessary maturation factors (ApbE and NqrM) to achieve proper assembly.

• Functional impact assessment: Design assays to measure:

  • Flavin incorporation efficiency

  • Electron transfer rates

  • Na+ translocation activity

  • Complex stability

• Control mutations: Include conservative substitutions as controls to distinguish between structural and functional effects of mutations.

The mutagenesis approach should follow similar protocols to those described for NqrM mutations, where specific conserved residues (such as cysteines) were systematically replaced to analyze their functional significance .

How can researchers effectively reconstitute the complete Na+-NQR complex containing recombinant NqrC?

Effective reconstitution of the complete Na+-NQR complex containing recombinant NqrC requires precise methodological approaches:

• Co-expression strategy: The most effective approach is to co-express all six Nqr subunits (NqrA-F) along with the maturation factors ApbE and NqrM in E. coli. This can be achieved using:

  • A polycistronic expression system containing the entire nqr operon

  • Compatible plasmids with different origins of replication (like p15A ori) for co-expression

  • Balanced expression levels to ensure proper stoichiometry

• Expression conditions optimization:

  • Growth at 32°C rather than 37°C to improve protein folding

  • Induction at mid-exponential phase (A600 ~0.3-0.4)

  • Use of 0.2% L-arabinose for pBAD-based systems

  • Extended expression time (3+ hours) for complex assembly

• Membrane isolation and complex purification:

  • Membrane fraction isolation via ultracentrifugation

  • Solubilization using appropriate detergents

  • Affinity purification via His-tagged subunits

  • Size exclusion chromatography to isolate the intact complex

• Functional verification:

  • NADH/dNADH oxidase activity measurements

  • Na+-stimulated quinone reductase activity assays

  • Inhibition studies with specific Na+-NQR inhibitors like HQNO

The integrity of the complex can be verified by observing Na+-stimulated, HQNO-inhibited dNADH oxidase activity, which is only present when the complete functional complex is assembled .

What are the latest techniques for studying the electron transfer mechanism within NqrC and the Na+-NQR complex?

Advanced techniques for studying electron transfer mechanisms within NqrC and the Na+-NQR complex include:

• Time-resolved spectroscopy: Ultrafast spectroscopic methods can track electron transfer between redox centers in real-time, allowing determination of transfer rates and pathways.

• EPR spectroscopy: For identifying paramagnetic intermediates during electron transfer and characterizing the electronic structure of redox cofactors.

• FRET-based approaches: Using strategically placed fluorophores to monitor conformational changes associated with electron transfer events.

• Electrochemical techniques:

  • Protein film voltammetry to determine redox potentials

  • Chronoamperometry to measure electron transfer kinetics

• Crystallographic and cryo-EM approaches: For high-resolution structural analysis of the complex in different redox states.

• Computational methods:

  • Molecular dynamics simulations

  • Quantum mechanical calculations of electron transfer pathways

  • Prediction of reorganization energies and transfer rates

• Specialized activity assays: Using artificial electron acceptors with different redox potentials to map the electron transfer pathway.

These techniques can help elucidate the unique electron transfer mechanism that involves both covalently bound FMN in NqrC and the iron-sulfur center formed between NqrD and NqrE subunits .

How can structural information about NqrC be leveraged for inhibitor design targeting Pasteurella multocida infections?

Leveraging structural information about NqrC for inhibitor design involves several sophisticated approaches:

• Structure-based drug design workflow:

  • Homology modeling of P. multocida NqrC if crystal structure is unavailable

  • Identification of druggable pockets near functional sites

  • Virtual screening of compound libraries against these sites

  • Molecular docking studies to predict binding modes

  • Molecular dynamics simulations to assess binding stability

• Target site selection strategies:

  • The FMN binding site in NqrC provides a unique target

  • Interfaces between NqrC and other subunits

  • Regions involved in electron transfer

  • Sodium channel pathways

• Rational design considerations:

  • Focus on inhibitors that disrupt electron transfer without affecting host proteins

  • Design molecules that block NqrC assembly into the complex

  • Target species-specific features of P. multocida NqrC

• Validation methodologies:

  • In vitro activity assays against isolated NqrC and reconstituted complexes

  • Binding affinity measurements using isothermal titration calorimetry

  • Bacterial growth inhibition assays

  • Cytotoxicity testing against mammalian cells

• Specialized approaches:

  • Fragment-based drug discovery targeting multiple sites

  • Covalent inhibitors targeting critical residues

  • Allosteric inhibitors affecting complex assembly

Since Na+-NQR is absent in mammals but essential for numerous bacterial pathogens, including P. multocida, inhibitors targeting this complex represent promising narrow-spectrum antimicrobial candidates with potentially minimal side effects in host animals .

What immunological considerations are important when using recombinant NqrC for vaccine development?

When developing vaccines based on recombinant NqrC from P. multocida, researchers should consider several critical immunological factors:

• Antigen design optimization:

  • Removal of the signal peptide as performed for other P. multocida recombinant proteins

  • Selection of antigenic epitopes that are surface-exposed in the native protein

  • Consideration of full-length versus subdomain constructs for optimal immunogenicity

• Expression and purification quality:

  • High purity preparations (>95%) are essential for vaccine applications

  • Endotoxin removal is critical for preventing non-specific immune responses

  • Protein folding verification to ensure epitope presentation matches native conformation

• Adjuvant selection:

  • Match adjuvants to the target species (bovine, avian, etc.)

  • Consider adjuvants that promote appropriate Th1/Th2 balance

  • Evaluate multiple adjuvant formulations for optimal response

• Immune response assessment:

  • Analyze both humoral and cell-mediated responses

  • Evaluate antibody isotype distribution

  • Assess cytokine profiles

  • Measure opsonization and bacterial killing activities

• Delivery system considerations:

  • Evaluate various delivery routes (subcutaneous, intramuscular, mucosal)

  • Consider prime-boost strategies

  • Test combination with other P. multocida antigens

Based on similar studies with other P. multocida recombinant proteins, standard vaccine preparation methods could include formaldehyde inactivation of whole bacteria combined with recombinant antigens for enhanced protection .

How can systems biology approaches enhance our understanding of NqrC function in the context of bacterial metabolism?

Systems biology approaches can provide comprehensive insights into NqrC function within P. multocida metabolism through:

• Multi-omics integration strategies:

  • Comparative proteomics between wild-type and nqrC mutants

  • Metabolomics to identify altered metabolic pathways in mutants

  • Transcriptomics to reveal regulatory networks connected to Na+-NQR function

  • Fluxomics to quantify changes in metabolic flux distributions

• Network analysis methodologies:

  • Construction of protein-protein interaction networks centered on NqrC

  • Metabolic control analysis to quantify NqrC's influence on metabolic fluxes

  • Identification of regulatory hubs connected to bioenergetic functions

• Computational modeling approaches:

  • Constraint-based models incorporating Na+-NQR function

  • Kinetic models of electron transport chain components

  • Integration of bioenergetic parameters into genome-scale metabolic models

• Experimental validation techniques:

  • Gene expression studies under various environmental conditions

  • Growth phenotyping under different nutrient and stress conditions

  • Real-time monitoring of membrane potential and pH in living cells

• Comparative analysis framework:

  • Cross-species comparison of Na+-NQR function in different pathogens

  • Evolutionary analysis of nqr genes to identify conserved functional networks

  • Host-pathogen interaction models incorporating bioenergetic components

This systems-level understanding would reveal how NqrC and the Na+-NQR complex integrate with broader metabolic networks and regulatory systems in P. multocida, potentially identifying new therapeutic targets or metabolic vulnerabilities .

What are common issues in recombinant NqrC expression and how can they be resolved?

Researchers commonly encounter several challenges when expressing recombinant P. multocida NqrC:

For optimal results, researchers should employ the co-expression strategy with all necessary maturation factors as demonstrated for other Na+-NQR components, using compatible plasmids with different origins of replication (e.g., p15A ori for maturation factors) .

How can researchers accurately measure the activity of recombinant NqrC in isolation and within the complex?

Accurate measurement of recombinant NqrC activity requires different approaches depending on whether the protein is isolated or part of the complete complex:

For isolated NqrC:

• Spectroscopic assays:

  • Monitor absorbance changes at 340 nm to track NADH oxidation

  • Follow flavin reduction/oxidation at specific wavelengths (450 nm)

  • Use artificial electron acceptors like menadione or ferricyanide

• Electrochemical methods:

  • Protein film voltammetry to determine redox potential

  • Chronoamperometry to measure electron transfer rates

For NqrC within the complete Na+-NQR complex:

• NADH/dNADH oxidation assays:

  • Measure oxidation at 340 nm spectrophotometrically

  • Compare rates with and without sodium to determine Na+ dependency

  • Use specific inhibitors like HQNO to confirm Na+-NQR specificity

• Quinone reductase activity:

  • Follow sodium-stimulated quinone reduction

  • Use ubiquinone-1 or other quinone analogs as substrates

  • Calculate activity as μmol of NADH oxidized per min per mg protein

• Ion translocation measurements:

  • Monitor Na+ movements using fluorescent indicators

  • Measure H+/Na+ antiport activity

  • Use reconstituted proteoliposomes for direct transport measurements

Standard assay conditions typically include:

• Temperature: 30°C

• Buffer: containing appropriate Na+ concentrations (100-300 mM)

• pH: typically 7.5-8.0

• Substrate concentrations: 100-200 μM NADH or dNADH

• Quinone concentration: 50-100 μM

The presence of Na+-stimulated, HQNO-inhibited dNADH oxidase activity is the hallmark of functional Na+-NQR complex assembly .

What precautions should be taken when handling recombinant proteins from pathogenic organisms like P. multocida?

When working with recombinant proteins derived from pathogenic organisms like P. multocida, researchers should implement the following precautions:

• Biosafety considerations:

  • Work at appropriate biosafety level (typically BSL-2 for recombinant P. multocida proteins)

  • Use certified biosafety cabinets for aerosol-generating procedures

  • Follow institutional biosafety guidelines and obtain proper approvals

• Laboratory practices:

  • Wear appropriate PPE (lab coat, gloves, eye protection)

  • Implement proper hand hygiene and avoid touching face

  • Use sealed centrifuge rotors and cups for centrifugation

  • Decontaminate all surfaces after work completion

• Sample handling protocols:

  • Clearly label all containers with biohazard symbols

  • Use sealed, leak-proof containers for storage and transport

  • Avoid creating aerosols during pipetting or mixing

  • Implement proper waste disposal procedures

• Personnel considerations:

  • Ensure proper training for all personnel

  • Consider vaccination where appropriate

  • Implement health monitoring if working extensively with pathogens

  • Restrict laboratory access to authorized personnel

• Specific precautions for P. multocida work:

  • Be aware of zoonotic potential (P. multocida can infect humans)

  • Take extra care with sharps handling to prevent inoculation

  • Implement immediate reporting procedures for potential exposures

Although recombinant proteins themselves are generally non-infectious, prudent biosafety practices should always be followed when working with components derived from pathogenic organisms .

What emerging technologies could advance our understanding of P. multocida NqrC structure and function?

Several cutting-edge technologies show promise for advancing our understanding of P. multocida NqrC:

• Advanced structural biology approaches:

  • Cryo-electron microscopy for high-resolution structure determination of the entire Na+-NQR complex

  • Integrative structural biology combining multiple techniques (X-ray, NMR, cryo-EM, crosslinking)

  • Time-resolved X-ray crystallography to capture conformational changes during catalysis

• Single-molecule techniques:

  • Single-molecule FRET to monitor conformational dynamics

  • Optical tweezers to study force-dependent structural changes

  • Single-particle tracking in live bacteria to understand complex assembly

• New spectroscopic methods:

  • Ultrafast multidimensional spectroscopy to track electron transfer events

  • Advanced EPR techniques (DEER, ENDOR) to map distances between redox centers

  • Raman microscopy for label-free detection of structural changes

• Genome engineering approaches:

  • CRISPR-Cas9 engineering of P. multocida to create precise mutations

  • In vivo crosslinking combined with mass spectrometry to map protein interactions

  • Proximity labeling methods to identify transient interaction partners

• Computational advancements:

  • Quantum mechanical/molecular mechanical (QM/MM) simulations of electron transfer

  • Machine learning approaches to predict functional residues and interactions

  • Advanced molecular dynamics simulations with polarizable force fields

These technologies will help address fundamental questions about the electron transfer mechanism, Na+ translocation pathway, and the unique structural features of the P. multocida Na+-NQR complex compared to other bacterial species .

How might comparative studies between NqrC and other respiratory complex subunits inform antimicrobial development?

Comparative studies between NqrC and other respiratory complex subunits can significantly inform antimicrobial development through several approaches:

• Structural comparison frameworks:

  • Identify unique structural features of NqrC absent in mammalian respiratory complexes

  • Compare binding pockets across bacterial species to design broad-spectrum inhibitors

  • Analyze structural differences between Na+-NQR and NDH-1 (Complex I) to ensure specificity

• Evolutionary analysis applications:

  • Identify highly conserved regions across pathogens that could serve as universal targets

  • Detect species-specific variations that could enable narrow-spectrum antibiotics

  • Track the co-evolution of resistance mechanisms to inform inhibitor design

• Functional comparison strategies:

  • Analyze differences in cofactor binding and electron transfer mechanisms

  • Compare the kinetics and thermodynamics of different bacterial respiratory complexes

  • Identify unique dependencies (like ApbE and NqrM for Na+-NQR) that could be exploited

• Translational research directions:

  • Develop high-throughput screening assays based on unique NqrC functions

  • Create animal infection models to test the efficacy of Na+-NQR inhibitors in vivo

  • Investigate combination therapies targeting multiple respiratory complexes

• Resistance mechanism predictions:

  • Anticipate potential resistance mechanisms by analyzing natural variations

  • Design inhibitors targeting multiple conserved regions to reduce resistance development

  • Develop assays to monitor emergence of resistance

Since Na+-NQR is present in multiple pathogens but absent in mammals, comparative studies can identify common features that could be targeted while ensuring minimal host toxicity. This approach is particularly valuable for developing treatments against multi-drug resistant pathogens where traditional antibiotics are failing .

What are the potential applications of NqrC in synthetic biology and bioengineering?

The unique properties of NqrC and the Na+-NQR complex offer several innovative applications in synthetic biology and bioengineering:

• Bioenergetic engineering applications:

  • Creation of synthetic sodium-motive force generators in engineered cells

  • Development of hybrid energy systems combining proton and sodium gradients

  • Engineering bacteria with enhanced bioenergetic efficiency for biotechnology applications

• Biosensor development:

  • NqrC-based biosensors for detecting sodium concentration changes

  • Redox-sensitive cellular reporters based on NqrC electron transfer

  • Whole-cell biosensors for screening Na+-NQR inhibitors

• Protein engineering opportunities:

  • Creation of chimeric proteins combining NqrC electron transfer capabilities with other functions

  • Engineering proteins with novel covalent flavin binding sites based on NqrC structure

  • Development of artificial electron transport chains with tailored properties

• Biotechnological production systems:

  • Engineering NqrC and Na+-NQR for improved biofuel production

  • Using Na+-based bioenergetics to drive product synthesis in industrial microorganisms

  • Creation of selective growth advantages in engineered probiotics

• Bioelectronic interfaces:

  • Development of NqrC-based bioelectronic devices that convert biological signals to electronic outputs

  • Creation of biofuel cells utilizing the electron transfer properties of modified NqrC

  • Integration of NqrC into hybrid biological-electronic systems

• Therapeutic enzyme design:

  • Engineering NqrC-based enzymes for targeted drug activation

  • Development of "Trojan horse" antimicrobials that hijack bacterial bioenergetics

  • Creation of enzyme systems for controlled electron transfer in therapeutic applications