Recombinant Pseudomonas putida NADH-quinone oxidoreductase subunit G (nuoG), partial

What is NADH-quinone oxidoreductase subunit G (nuoG) in Pseudomonas putida?

NADH-quinone oxidoreductase subunit G (nuoG) is a component of the NADH dehydrogenase I complex (NDH-1) in Pseudomonas putida. This protein functions as part of the electron transport chain, playing a crucial role in energy metabolism by catalyzing the transfer of electrons from NADH to quinones. In P. putida, nuoG is part of a larger complex that contributes to the organism's remarkable metabolic versatility . The protein exists as a partial recombinant form with a molecular weight of approximately 42.9 kDa when tagged, and typically comprises amino acids 2-399 of the full sequence .

To study this protein, researchers typically use recombinant expression systems, with E. coli being the most common host. Expression vectors usually incorporate features such as His-tags to facilitate purification, and the resulting protein is often used for structural studies, enzymatic assays, and investigation of metabolic pathways in P. putida .

How does P. putida nuoG function in bacterial energy metabolism?

NuoG functions as a critical component of P. putida's respiratory chain, contributing to the organism's ability to thrive in diverse environments. Methodologically, researchers investigate this function through:

The specific activity of purified recombinant P. putida nuoG has been measured at ≥35 μmol/min/μg, indicating its high catalytic efficiency in electron transfer reactions . This activity is crucial for P. putida's ability to metabolize diverse carbon sources and adapt to changing environmental conditions .

What expression systems are optimal for producing recombinant P. putida nuoG?

Several expression systems have been employed for the production of recombinant P. putida nuoG, each with distinct advantages depending on research objectives:

E. coli expression systems:
The most commonly used approach involves E. coli host strains with vectors containing strong promoters (T7, tac) and affinity tags. Typical expression conditions include:

• Host strains: BL21(DE3), Rosetta, or C41(DE3) for membrane proteins

• Induction: 0.1-1.0 mM IPTG at OD600 of 0.6-0.8

• Temperature: 16-30°C for 4-18 hours to balance yield and solubility

• Media: LB or auto-induction media supplemented with appropriate antibiotics

Yeast expression systems:
For studies requiring eukaryotic post-translational modifications:

• P. pastoris or S. cerevisiae hosts

• Methanol-inducible or constitutive promoters

• Secretory expression with α-factor signal sequence

• Glycerol batch/methanol fed-batch cultivation strategies

Baculovirus expression systems:
For complex protein folding requirements:

• Sf9 or Hi5 insect cells

• Recombinant bacmid generation via Tn7 transposition

• Infection at 1-2 MOI

• Harvest 48-72 hours post-infection

Protein purification typically employs a combination of affinity chromatography (Ni-NTA for His-tagged proteins), followed by size exclusion and/or ion exchange chromatography. Final preparations of P. putida nuoG typically achieve ≥74% purity in aqueous buffer containing 40 mM Tris-HCl, pH 8.0, 110 mM NaCl, 2.2 mM KCl, 200 mM imidazole, and 20% glycerol .

How does nuoG contribute to P. putida's environmental adaptability and metabolic versatility?

P. putida is renowned for its metabolic versatility and ability to thrive in diverse environments, from soil to clinical settings. The nuoG subunit plays a significant role in this adaptability through several mechanisms:

Methodological approaches to study these mechanisms include:

The metabolic versatility of P. putida, supported by nuoG function, has been harnessed for various biotechnological applications including bioremediation of contaminated areas, improvement of fossil fuels, biocatalytic production of fine chemicals, and production of bioplastics . This versatility is reflected in the organism's genome, which contains numerous pathways for degradation of aromatic compounds and other xenobiotics.

What is the relationship between nuoG function and pathogenicity in clinical P. putida isolates?

Although P. putida is generally considered non-pathogenic, clinical isolates have been associated with opportunistic infections in immunocompromised patients. Research into the role of nuoG in potential pathogenicity requires sophisticated approaches:

• Comparative genomics of clinical vs. environmental isolates: Analysis of nuoG sequence variations and surrounding genetic elements has revealed differences between clinical and environmental strains. For example, the clinical isolate HB3267 exhibits extensive antibiotic resistance and enhanced pathogenicity, potentially linked to altered respiratory chain function .

• Virulence models: Several experimental models have been developed to assess pathogenicity:

  • Human tissue culture systems show cytotoxicity and tissue damage

  • Mammalian (Wistar rat) skin models demonstrate structural tissue alterations

  • Insect larvae (Chrysoperla carnea) survival models assess systemic effects

• Transcriptional profiling during host interaction: RNA-seq of P. putida during infection reveals nuoG expression changes in response to host environments.

• Biofilm formation assays: Crystal violet staining and confocal microscopy to quantify biofilm formation capacity, which correlates with pathogenicity potential.

Data from these studies have revealed a significant variability in the pathogenic potential of clinical P. putida isolates. For example, the strain HB3267 showed extensive tissue damage in multiple models and high antibiotic resistance, while strain HB13667 exhibited no pathogenic traits in the same models . The table below summarizes these variations:

These findings suggest that respiratory chain components like nuoG may contribute to the organism's ability to colonize and survive in clinical environments, potentially through mechanisms involving energy production under host-associated conditions .

What methodologies are most effective for studying nuoG's role in electron transport chain assembly?

Investigating nuoG's contribution to electron transport chain assembly and function requires specialized techniques:

• Blue native polyacrylamide gel electrophoresis (BN-PAGE):

  • Allows visualization of intact respiratory complexes

  • Combined with activity staining using NADH and tetrazolium salts

  • Western blotting with anti-nuoG antibodies to confirm presence in complexes

  • Second-dimension SDS-PAGE to analyze subunit composition

• Cryo-electron microscopy (cryo-EM):

  • High-resolution structural analysis of fully assembled complexes

  • Sample preparation typically involves membrane solubilization with mild detergents

  • Data collection at 300 kV with direct electron detectors

  • Image processing using software like RELION or cryoSPARC

• Protein crosslinking coupled with mass spectrometry:

  • Identification of protein-protein interactions within the complex

  • MS/MS analysis to determine crosslinked residues

  • Molecular modeling to predict interface regions

• Fluorescence resonance energy transfer (FRET):

  • Real-time assembly monitoring using fluorescently tagged subunits

  • Expression of donor-acceptor pairs in P. putida

  • Live-cell imaging to track complex formation

• Genetic complementation assays:

  • Chromosomal integration of nuoG variants at specific loci

  • Evaluation of respiratory function restoration

  • Growth rate measurements under different carbon sources

When integrating heterologous or modified nuoG genes into the P. putida chromosome, researchers have identified optimal integration sites that do not affect growth rate but can significantly affect expression levels. Studies have shown that insertion location can cause expression variation of up to 27-fold, highlighting the importance of careful locus selection for genetic engineering .

How can structural analysis of nuoG inform inhibitor design for antimicrobial development?

With the emergence of multi-drug-resistant (MDR) P. putida strains in clinical settings, nuoG has emerged as a potential antimicrobial target. Structural analysis approaches include:

• X-ray crystallography:

  • Protein crystallization typically requires 10-15 mg/ml highly purified protein

  • Crystallization conditions: vapor diffusion method with PEG-based precipitants

  • Data collection at synchrotron radiation sources

  • Structure determination by molecular replacement using homologous structures

• Computational modeling and docking:

  • Homology modeling based on related bacterial nuoG structures

  • Virtual screening of compound libraries against predicted binding sites

  • Molecular dynamics simulations to assess protein flexibility

  • Binding energy calculations to prioritize candidate inhibitors

• Fragment-based drug design:

  • NMR or X-ray crystallography to identify fragment binding

  • Structure-activity relationship studies to optimize fragments

  • Fragment linking or growing strategies to develop lead compounds

• Structure-guided mutagenesis:

  • Site-directed mutagenesis of predicted catalytic or binding site residues

  • Enzymatic assays to validate functional importance

  • Thermal shift assays to assess structural stability

These approaches have revealed important structural features of nuoG that could be exploited for selective inhibition. For instance, analysis of antibiotic-resistant clinical isolates like HB3267 has identified specific mutations that may contribute to resistance mechanisms, providing targets for circumvention strategies .

What techniques are most effective for studying nuoG's role in P. putida's stress response mechanisms?

P. putida's remarkable adaptability to environmental stressors partially depends on its energy generation systems. To investigate nuoG's contribution to stress responses, researchers employ:

• Stress exposure experiments:

  • Growth in the presence of oxidative agents (H₂O₂, paraquat)

  • Nutrient limitation studies (carbon, nitrogen, phosphorus)

  • Heavy metal exposure (Cu²⁺, Cd²⁺, Zn²⁺)

  • Temperature and pH stress conditions

• Quantitative reverse transcription PCR (RT-qPCR):

  • Measurement of nuoG expression changes under stress conditions

  • Reference genes: rpoD, proC, and rpoB

  • Normalization using multiple reference genes

  • Statistical analysis with REST software

• Proteomics approaches:

  • SILAC or TMT labeling for quantitative comparison

  • Isolation of membrane fractions for enrichment

  • LC-MS/MS analysis for protein identification

  • Pathway analysis of co-regulated proteins

• Metabolomics:

  • Targeted analysis of redox-related metabolites (NAD⁺/NADH ratio)

  • Untargeted GC-MS or LC-MS metabolic profiling

  • Flux analysis using isotope-labeled substrates

  • Integration with transcriptomic data

• Reporter systems:

  • Transcriptional fusions of nuoG promoter with fluorescent proteins

  • Flow cytometry for single-cell analysis

  • Microfluidics for real-time monitoring

  • Chromosomal integration at neutral loci for stable expression

Studies utilizing these approaches have demonstrated that nuoG expression and activity are modulated in response to various stressors, contributing to the remarkable environmental adaptability of P. putida. For instance, genome-scale metabolic models have identified nuoG as part of the core metabolic network that enables P. putida to utilize diverse carbon sources and adapt to changing environmental conditions .

What are common challenges in recombinant P. putida nuoG expression and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant P. putida nuoG:

• Protein solubility issues:

  • Problem: Formation of inclusion bodies in E. coli expression systems

  • Solution: Lower induction temperature (16-20°C), reduce IPTG concentration (0.1-0.5 mM), co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Alternative: Use solubility-enhancing fusion partners (MBP, SUMO, Trx)

• Protein stability concerns:

  • Problem: Rapid degradation during expression or purification

  • Solution: Include protease inhibitors (PMSF, leupeptin, aprotinin), reduce purification temperature (4°C)

  • Optimization: Identify and mutate protease recognition sites without affecting function

• Low expression yield:

  • Problem: Insufficient protein production for downstream applications

  • Solution: Codon optimization for expression host, use stronger promoters, optimize media composition

  • Enhancement: High-density fermentation techniques with controlled feeding strategies

• Purification difficulties:

  • Problem: Co-purification of contaminants or degradation products

  • Solution: Multi-step purification combining affinity, ion exchange, and size exclusion chromatography

  • Refinement: Optimize binding and washing conditions (imidazole concentration, salt concentration, pH)

• Activity loss during purification:

  • Problem: Reduced enzymatic activity after purification

  • Solution: Include stabilizing agents (glycerol 20%, reducing agents), avoid freeze-thaw cycles

  • Preservation: Flash-freeze aliquots in liquid nitrogen and store at -80°C

Successful expression and purification protocols typically achieve ≥74% purity with specific activity ≥35 μmol/min/μg for recombinant P. putida nuoG . The optimal formulation for maintaining stability includes 40 mM Tris-HCl (pH 8.0), 110 mM NaCl, 2.2 mM KCl, 200 mM imidazole, and 20% glycerol .

How can researchers effectively integrate nuoG variants into P. putida for functional studies?

Genetic manipulation of P. putida to study nuoG function requires careful consideration of integration methods and locations:

• Selection of integration sites:

  • Evaluate multiple chromosomal loci to identify neutral integration sites

  • Avoid disrupting essential genes or operons

  • Consider the orientation relative to downstream genes

  • Analyze intergenic regions of varying lengths (12-211 bp)

• Integration methods:

  • Homologous recombination using suicide vectors like pK18mob-sacB

  • Counter-selection with sucrose for plasmid backbone excision

  • CRISPR-Cas9 systems for precise genomic modifications

  • Transposon-based random integration with subsequent mapping

• Verification strategies:

  • PCR screening for successful integration

  • Whole genome resequencing to confirm precise insertion

  • RT-qPCR to verify expression levels

  • Western blotting to confirm protein production

• Phenotypic validation:

  • Growth rate measurements under various conditions

  • Fluorescent reporter assays if using reporter fusions

  • Enzymatic activity assays for functional confirmation

  • Metabolic profiling to assess pathway integration

Research has shown that integration location significantly impacts heterologous protein expression and host phenotype, with expression levels varying by up to 27-fold depending on the integration site . For nuoG studies, integration sites that maintain normal growth rates while allowing sufficient expression are most desirable. Researchers have identified that insertion at certain loci, such as PP_5388, can result in integration into adjacent genes rather than the intended intergenic region, highlighting the importance of thorough verification .

What analytical methods provide the most comprehensive assessment of nuoG enzymatic activity?

Comprehensive evaluation of nuoG enzymatic activity requires multiple complementary approaches:

• Spectrophotometric assays:

  • NADH oxidation monitoring at 340 nm (ε = 6.22 mM⁻¹cm⁻¹)

  • Coupled enzyme assays with artificial electron acceptors (ferricyanide, DCPIP)

  • Continuous monitoring in 96-well format for high-throughput screening

  • Standard conditions: 50 mM phosphate buffer pH 7.4, 100 μM NADH, 25°C

• Oxygen consumption measurements:

  • Clark-type oxygen electrode or optical oxygen sensors

  • Comparison of activity with different quinone substrates

  • Inhibitor studies with rotenone, piericidin, or stigmatellin

  • Determination of kinetic parameters (KM and Vmax)

• Electron paramagnetic resonance (EPR) spectroscopy:

  • Detection of iron-sulfur cluster redox states

  • Sample preparation in anaerobic conditions

  • Low-temperature measurements (4-100K)

  • Identification of intermediate species during catalysis

• Protein film voltammetry:

  • Direct electrochemistry of immobilized enzyme

  • Determination of redox potentials

  • Analysis of electron transfer kinetics

  • Effect of pH and temperature on activity

• Isothermal titration calorimetry (ITC):

  • Thermodynamic analysis of substrate binding

  • Determination of binding constants and stoichiometry

  • Enthalpy and entropy contributions to binding energy

  • Temperature dependence studies

These methods have revealed that P. putida nuoG maintains specific activity ≥35 μmol/min/μg under optimal conditions . Kinetic analysis indicates that the enzyme follows typical Michaelis-Menten kinetics with substrate inhibition at high NADH concentrations. The pH optimum lies between 7.5-8.0, and the temperature optimum is typically around 30°C, reflecting P. putida's mesophilic nature .

How might systems biology approaches advance our understanding of nuoG in P. putida metabolism?

Systems biology offers powerful frameworks for understanding nuoG's role in the context of P. putida's whole-cell metabolism:

• Genome-scale metabolic modeling:

  • Integration of nuoG function into existing P. putida metabolic reconstructions

  • Flux balance analysis to predict phenotypic effects of nuoG modifications

  • In silico gene knockout studies to identify synthetic lethal interactions

  • Comparison with experimental data from growth phenotyping arrays

• Multi-omics integration:

  • Correlation of transcriptomic, proteomic, and metabolomic data

  • Network analysis to identify co-regulated genes and proteins

  • Regulatory network reconstruction focusing on nuoG regulation

  • Machine learning approaches to predict nuoG function under various conditions

• Synthetic biology applications:

  • Design of artificial electron transport chains with modified nuoG

  • Engineering electron flow for improved production of value-added compounds

  • Creation of biosensors based on nuoG activity

  • Development of nuoG variants with altered substrate specificity

• Comparative systems analysis:

  • Cross-species comparison of nuoG function in various Pseudomonas species

  • Evolutionary analysis of respiratory chain adaptations

  • Identification of conserved and divergent features across strains

  • Correlation with ecological niches and metabolic capabilities

P. putida's metabolic versatility has been extensively mapped through genome-scale metabolic reconstructions, which have identified 746 genes, 950 reactions, and 911 metabolites in the core metabolic network . These models have already shown that understanding electron flow through respiratory complexes containing nuoG is critical for predicting the organism's behavior in diverse environments and for optimizing biotechnological applications.

What potential applications exist for engineered P. putida nuoG variants in biotechnology?

Engineered nuoG variants offer several promising biotechnological applications:

• Bioremediation enhancement:

  • Engineering nuoG for improved electron transfer to toxic compounds

  • Creation of variants with altered substrate specificity for pollutant degradation

  • Integration with pathways for degradation of recalcitrant compounds

  • Development of whole-cell biosensors for environmental monitoring

• Biocatalysis optimization:

  • Coupling nuoG-driven NADH regeneration to oxidoreductase reactions

  • Engineering electron bifurcation for simultaneous catalysis of multiple reactions

  • Development of artificial enzyme cascades with improved electron efficiency

  • Creation of redox-balanced pathways for fine chemical production

• Bioelectrochemical systems:

  • Engineered nuoG variants for improved electron transfer to electrodes

  • Development of microbial fuel cells with enhanced power output

  • Creation of bioelectrosynthesis systems for electricity-driven production

  • Engineering of biofilm formation on electrodes for improved performance

• Antibiotic target exploration:

  • Structure-based design of nuoG inhibitors as novel antimicrobials

  • Development of narrow-spectrum antibiotics targeting pathogenic Pseudomonas species

  • Exploration of nuoG as an adjuvant target to potentiate existing antibiotics

  • Creation of screening platforms for nuoG inhibitor discovery

P. putida's remarkable metabolic capabilities have already been harnessed for various biotechnological applications including bioremediation, improvement of fossil fuels, production of fine chemicals, and bioplastic synthesis . Engineered nuoG variants have the potential to significantly enhance these applications by optimizing energy metabolism and redox balance.

How might nuoG contribute to P. putida's potential as a chassis organism for synthetic biology?

P. putida is increasingly recognized as a promising chassis for synthetic biology applications, with nuoG playing a key role in its suitability:

• Metabolic robustness contributions:

  • NuoG's role in maintaining redox balance during stress

  • Engineering of electron transport for improved carbon utilization efficiency

  • Development of strains with optimized NADH/NAD⁺ ratios for specific applications

  • Creation of cells with enhanced tolerance to toxic compounds

• Integration with heterologous pathways:

  • Optimizing chromosomal integration sites for nuoG variants

  • Engineering electron transport chain components for coupling with synthetic pathways

  • Balancing energy generation with production pathway demands

  • Development of orthogonal redox systems for pathway isolation

• Genetic stability enhancements:

  • Identification of genomic safe harbors for stable integration

  • Engineering of strain lineages with reduced mutation rates in respiratory complexes

  • Development of selection systems based on respiratory function

  • Creation of genetic firewalls to prevent horizontal gene transfer

• Chassis optimization strategies:

  • Genome reduction to create streamlined platforms

  • Fine-tuning of respiratory chain components for specific applications

  • Engineering of regulatory systems for controlled expression

  • Development of modular systems for plug-and-play metabolic engineering

Studies have demonstrated that careful selection of chromosomal integration sites is critical for maximizing heterologous gene expression in P. putida. Integration location can affect expression levels by up to 27-fold, highlighting the importance of this consideration in synthetic biology applications . Furthermore, the integration of heterologous genes like muconate importers has shown significant phenotypic differences depending on the integration locus, with expression varying approximately 3-fold between sites .

What standardized protocols enable consistent nuoG research across laboratories?

Establishing standardized methods is essential for collaborative research on P. putida nuoG:

• Strain documentation and distribution:

  • Complete genome sequencing of working strains

  • Deposition in public strain collections (ATCC, DSMZ)

  • Detailed growth and maintenance protocols

  • Verification of strain identity through specific markers

• Expression and purification standardization:

  • Defined expression vectors with standardized features

  • Detailed protocols for induction conditions

  • Step-by-step purification procedures with quality control metrics

  • Activity assays with defined conditions and controls

• Enzymatic activity measurements:

  • Spectrophotometric assays under defined conditions

  • Specific activity calculation methods

  • Standard substrate concentrations and buffer compositions

  • Inclusion of reference standards for inter-lab comparison

• Data reporting requirements:

  • Minimum information standards for experimental details

  • Raw data deposition in public repositories

  • Standardized metadata for experimental conditions

  • Statistical analysis methods and significance thresholds

• Genetic manipulation techniques:

  • Validated chromosomal integration sites with known effects

  • Standardized cloning strategies and verification methods

  • Well-characterized genetic parts (promoters, RBSs, terminators)

  • Protocols for genome editing with efficiency metrics

Researchers studying P. putida have established that nuoG protein preparations should meet minimum standards of ≥74% purity with specific activity ≥35 μmol/min/μg . Optimal storage conditions include buffer containing 40 mM Tris-HCl (pH 8.0), 110 mM NaCl, 2.2 mM KCl, 200 mM imidazole, and 20% glycerol at -80°C .

How can researchers effectively compare nuoG function across different P. putida strains?

Comparative analysis of nuoG across strains requires systematic approaches:

• Sequence-structure-function analysis:

  • Multiple sequence alignment of nuoG from different strains

  • Identification of conserved and variable regions

  • Homology modeling based on available structures

  • Prediction of functional differences based on sequence variations

• Standardized functional assessments:

  • Activity assays under identical conditions

  • Growth phenotyping in defined media with various carbon sources

  • Stress tolerance tests (oxidative stress, temperature, pH)

  • Host-interaction models for clinical isolates

• Cross-strain complementation studies:

  • Deletion of native nuoG and complementation with variants

  • Integration at identical chromosomal loci for fair comparison

  • Expression level normalization through promoter engineering

  • Phenotypic assessment under standardized conditions

• Evolutionary context analysis:

  • Phylogenetic tree construction for nuoG across strains

  • Correlation with ecological niches and metabolic capabilities

  • Analysis of selection pressure on different protein domains

  • Identification of horizontal gene transfer events

Research has revealed significant variability among P. putida strains, particularly between environmental and clinical isolates. Clinical isolates like HB3267 exhibit extensive antibiotic resistance (to 84% of tested antibiotics) and enhanced pathogenicity, while others like HB13667 show no pathogenic traits in the same models . These differences likely extend to nuoG function and regulation, highlighting the importance of strain-specific characterization.

What ethical considerations apply to research on potentially pathogenic P. putida strains expressing recombinant nuoG?

Research on clinical P. putida isolates and recombinant nuoG requires careful ethical considerations:

• Biosafety level assessment:

  • Proper classification based on pathogenic potential

  • Implementation of appropriate containment measures

  • Development of emergency response protocols

  • Regular risk assessment and mitigation planning

• Dual-use research concerns:

  • Evaluation of potential misuse of engineered strains

  • Implementation of security measures for strain storage

  • Careful consideration of information sharing and publication

  • Consultation with institutional biosafety committees

• Clinical sample acquisition and use:

  • Ensuring proper informed consent for patient-derived isolates

  • De-identification of patient data associated with samples

  • Compliance with institutional review board requirements

  • Adherence to international guidelines for human subject research

• Environmental and ecological considerations:

  • Assessment of environmental release risks

  • Development of biological containment strategies

  • Consideration of horizontal gene transfer potential

  • Implementation of kill switches for engineered strains

• Antibiotic resistance concerns:

  • Responsible use of antibiotic resistance markers

  • Development of marker-free systems where possible

  • Consideration of resistance spread in clinical environments

  • Proper disposal of antibiotic-resistant organisms

Studies have shown that some clinical P. putida isolates harbor plasmids containing multiple antibiotic resistance genes, such as the 80 kb megaplasmid pPC9 found in strain HB3267 . These plasmids can potentially be transferred to other pathogens, highlighting the importance of proper containment and responsible research practices. Additionally, research has indicated that certain P. putida strains can cause infections in immunocompromised individuals, necessitating appropriate biosafety measures .