Recombinant Pseudomonas putida Ubiquinol oxidase subunit 2 (cyoA)

What is Pseudomonas putida Ubiquinol oxidase subunit 2 (cyoA) and what is its biological function?

Pseudomonas putida Ubiquinol oxidase subunit 2 (cyoA) is a critical component of the cytochrome o ubiquinol oxidase complex, which functions as the main terminal oxidase in the electron transport chain under highly aerobic conditions in Pseudomonas putida . This protein plays an essential role in cellular respiration by facilitating electron transfer from ubiquinol to oxygen, thereby contributing to energy generation through oxidative phosphorylation. The cyoA subunit (UniProt ID: Q9WWR1) comprises amino acids 24-314 of the mature protein and is part of the larger cytochrome bo3 complex .

The biological significance of cyoA extends beyond simple respiratory function. Research has demonstrated that cytochrome o ubiquinol oxidase is involved in regulatory processes, particularly in catabolite repression control. Inactivation of this terminal oxidase has been shown to decrease catabolic repression in both rich media and defined media containing alternative carbon sources like lactate or succinate . This suggests that the protein may function as a metabolic sensor, potentially monitoring the redox state of the cell and influencing gene expression accordingly.

How does recombinant cyoA compare to native cyoA in terms of structure and function?

Recombinant Pseudomonas putida cyoA proteins, particularly those expressed with affinity tags like the His-tag, generally maintain the core structural and functional properties of the native protein while providing advantages for purification and experimental manipulation. The recombinant version available commercially is expressed in E. coli with an N-terminal His-tag, allowing for efficient purification using metal affinity chromatography .

The primary differences between recombinant and native cyoA include:

Researchers should note that while the recombinant protein maintains core functionality, the presence of the His-tag and expression in a heterologous system may subtly affect certain interactions or enzymatic parameters, which should be considered when designing experiments.

How does inactivation of cytochrome o ubiquinol oxidase affect metabolic regulation in Pseudomonas putida?

Inactivation of cytochrome o ubiquinol oxidase has significant effects on metabolic regulation in Pseudomonas putida, particularly regarding catabolic repression control. Research has shown that mutations in genes encoding this terminal oxidase lead to decreased catabolic repression of the alkane degradation pathway encoded by the OCT plasmid of P. putida GPo1 . This is observed both in rich media and in defined media containing preferred carbon sources like lactate or succinate.

The mechanism appears distinct from other catabolic repression pathways, such as those involving the Crc protein. When mutations in both cyo genes and the crc gene were combined, an additive effect in relieving catabolic repression was observed, suggesting they operate through different regulatory pathways . This indicates that cytochrome o ubiquinol oxidase may function as part of a sensing system that monitors the cellular energy status or redox state, providing information that feeds into global regulatory networks controlling carbon source utilization.

The metabolic consequences of cyoA inactivation include:

• Altered expression of catabolic pathways

• Changed preference for carbon source utilization

• Modified electron flow through the respiratory chain

• Potential redox imbalance affecting gene regulation

• Shifts in energy generation efficiency

These findings suggest that terminal oxidases like cytochrome o ubiquinol oxidase serve as metabolic checkpoints, integrating respiratory activity with broader cellular regulatory processes.

What expression systems are most effective for producing recombinant cyoA protein?

While the search results don't specifically address cyoA expression systems, insights from recombinant protein expression research can be applied. For membrane proteins like cyoA, several expression systems have shown promise, with considerations needed for proper folding and membrane insertion.

Based on general principles of recombinant membrane protein expression and the specific information about the commercially available recombinant cyoA protein, the following expression systems are potentially effective:

• E. coli-based expression systems: The commercial recombinant cyoA is successfully expressed in E. coli with an N-terminal His-tag . This suggests that E. coli can properly process this protein, likely using vectors with strong promoters like T7 or tac.

• Vector design considerations: Research on recombinant antibody expression indicates that vector design significantly impacts yield. Three main vector types have different effectiveness:

  Vector Type	Characteristics	Applicability to cyoA
  Bicistronic vectors	Single promoter driving two genes	May be useful if expressing multiple subunits
  Monocistronic vectors	Separate promoters for each gene	Good for single subunit expression
  Dual promoter vectors	Two promoters in one vector	Shown to give highest yields in some systems

• Production process optimization: Low-temperature cultivation (typically 25-30°C instead of 37°C) can improve the yield of properly folded membrane proteins like cyoA by slowing protein synthesis and allowing more time for proper folding .

• Fed-batch culture: This approach can further improve protein yields by maintaining optimal nutrient levels throughout the cultivation period, preventing nutrient limitation while avoiding substrate inhibition .

For membrane proteins specifically, E. coli strains engineered for membrane protein expression (such as C41(DE3) or C43(DE3)) may provide superior results by accommodating the potential toxicity associated with membrane protein overexpression.

What methodologies are most effective for studying cyoA-protein interactions and complex formation?

Studying cyoA-protein interactions requires specialized techniques that account for its membrane-bound nature. Based on current research practices for similar proteins, the following methodologies are recommended:

• Co-immunoprecipitation (Co-IP): Using antibodies against cyoA or its interaction partners (other oxidase subunits) to pull down complexes from solubilized membranes. The His-tag on recombinant cyoA can be leveraged for pulldown assays using Ni-NTA or similar matrices.

• Blue Native PAGE (BN-PAGE): This technique preserves native protein complexes during electrophoresis, allowing visualization of intact cytochrome o ubiquinol oxidase complexes and assessment of proper assembly.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry can identify proteins in close proximity to cyoA in the membrane and provide insights into the spatial arrangement of complex components.

• Surface Plasmon Resonance (SPR): For studying direct binding kinetics between purified cyoA and potential interaction partners, particularly when using detergent-solubilized or reconstituted proteins.

• Bacterial two-hybrid systems: Modified for membrane proteins, these genetic systems can detect interactions in vivo.

• Fluorescence techniques:

  • Förster Resonance Energy Transfer (FRET) for detecting close associations between fluorescently labeled proteins

  • Fluorescence Recovery After Photobleaching (FRAP) for studying dynamics of protein complexes in membranes

Each method has specific advantages and limitations when applied to membrane proteins like cyoA, and combinations of complementary techniques typically provide the most robust results.

What are the optimal conditions for storing and handling recombinant cyoA protein?

Proper storage and handling of recombinant Pseudomonas putida cyoA protein is critical for maintaining its structural integrity and functional activity. Based on the manufacturer's recommendations for the commercially available His-tagged recombinant cyoA, the following conditions should be observed :

• Storage temperature:

  • Long-term storage: -20°C to -80°C

  • Working aliquots: 4°C for up to one week

• Physical form:

  • The protein is supplied as a lyophilized powder

  • Prior to opening, vials should be briefly centrifuged to bring contents to the bottom

• Reconstitution:

  • Use deionized sterile water to reconstitute to 0.1-1.0 mg/mL

  • Addition of glycerol (5-50% final concentration) is recommended for aliquoting and long-term storage

  • The manufacturer's default is 50% glycerol for optimal stability

• Buffer conditions:

  • The protein is supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0

  • This formulation helps maintain protein stability during freeze-thaw cycles

• Handling precautions:

  • Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

  • Prepare appropriate working aliquots during initial reconstitution

These storage and handling recommendations are designed to minimize protein denaturation, aggregation, and loss of activity that can occur with membrane proteins like cyoA, which are typically more sensitive to storage conditions than soluble proteins.

How can researchers validate the functional activity of recombinant cyoA protein?

Validating the functional activity of recombinant cyoA protein is essential for ensuring experimental reliability. Several complementary approaches can be employed:

• Ubiquinol oxidase activity assays:

  • Measure oxygen consumption rates using Clark-type oxygen electrodes

  • Monitor ubiquinol oxidation spectrophotometrically by following absorbance changes at specific wavelengths

  • These assays require reconstitution of cyoA with other subunits of the complex or use of membrane fractions containing the assembled complex

• Electron transfer capability:

  • Assess electron transfer using artificial electron donors and acceptors

  • Cytochrome c reduction assays can provide indirect evidence of electron transfer functionality

• Structural validation:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Limited proteolysis to verify correct folding based on protease accessibility patterns

  • Thermal shift assays to assess protein stability

• Complex formation assessment:

  • Blue Native PAGE to verify assembly into higher-order complexes

  • Size exclusion chromatography to analyze complex formation

  • Co-immunoprecipitation to confirm interactions with known partners

• Functional complementation:

  • Expressing recombinant cyoA in cyoA-deficient bacterial strains to assess restoration of respiratory function

  • Measuring growth rates under conditions requiring terminal oxidase activity

A combination of these methods provides robust validation of recombinant cyoA functionality, ensuring that experimental findings accurately reflect the protein's natural behavior.

What are common challenges in working with recombinant cyoA and how can they be addressed?

Working with recombinant membrane proteins like cyoA presents several challenges that require specific strategies to overcome:

Additionally, researchers should be aware that the presence of affinity tags like the His-tag on recombinant cyoA may occasionally interfere with certain functions or interactions. If activity issues are encountered, constructing versions with removable tags or with tags in alternative positions may be beneficial.

How can recombinant cyoA be used to study bacterial respiratory adaptation and regulation?

Recombinant cyoA provides a valuable tool for investigating how bacteria adapt their respiratory systems in response to environmental changes and metabolic demands. Several research applications include:

• Investigation of regulatory mechanisms: The inactivation of cytochrome o ubiquinol oxidase has been shown to relieve catabolic repression in P. putida . Recombinant cyoA can be used in complementation studies to dissect the molecular mechanisms underlying this regulation and identify the specific domains or residues involved.

• Study of respiratory chain assembly: Properly tagged recombinant cyoA enables tracking of respiratory complex formation, helping to elucidate the assembly pathway of the cytochrome o ubiquinol oxidase complex and the factors influencing this process.

• Analysis of electron transport chain flexibility: By manipulating cyoA expression or structure, researchers can examine how bacteria reroute electron flow through alternative branches of the respiratory chain under different conditions.

• Investigation of redox sensing mechanisms: Given the connection between cyoA function and metabolic regulation, recombinant variants can be used to probe how respiratory activity might serve as a cellular redox sensor that influences broader metabolic networks.

• Comparative studies across species: Recombinant cyoA proteins from different bacterial species can be analyzed to understand evolutionary adaptations in respiratory systems and how these relate to ecological niches.

These applications contribute to our fundamental understanding of bacterial bioenergetics while potentially informing biotechnological applications, including metabolic engineering for optimized bioproduction and development of novel antimicrobial strategies targeting respiratory systems.

What experimental designs are most effective for studying the role of cyoA in catabolic repression?

Based on the finding that cytochrome o ubiquinol oxidase inactivation relieves catabolic repression in Pseudomonas putida , several experimental approaches can be employed to further investigate this phenomenon:

These experimental approaches should be performed under carefully controlled growth conditions, with appropriate carbon sources (e.g., alkanes plus preferred carbon sources like lactate or succinate) to properly observe catabolic repression phenomena.

What are the future research directions for understanding cyoA function and applications?

Future research on Pseudomonas putida cyoA holds promise for advancing both fundamental understanding of bacterial metabolism and applied biotechnological capabilities. Several promising research directions include:

• Structural biology approaches: Obtaining high-resolution structures of the entire cytochrome o ubiquinol oxidase complex, including cyoA, would provide critical insights into the molecular mechanism of its function. Cryo-electron microscopy and X-ray crystallography of reconstituted complexes represent important future goals.

• Systems biology integration: Investigating how cyoA function integrates with global cellular networks through multi-omics approaches (proteomics, metabolomics, transcriptomics) would help elucidate its role in broader metabolic regulation beyond catabolic repression.

• Synthetic biology applications: Engineered variants of cyoA could potentially be used to create strains with modified regulatory properties, such as relief from catabolic repression, which might improve biotechnological processes requiring simultaneous utilization of multiple carbon sources.

• Comparative analysis across species: Expanding studies of terminal oxidases across diverse bacterial species could reveal evolutionary adaptations and species-specific regulatory mechanisms.

• Application in bioremediation: Given P. putida's importance in bioremediation and the role of cyoA in regulating alkane degradation pathways, engineered cyoA variants could potentially enhance biodegradation of environmental pollutants.

• Development as antimicrobial targets: As terminal oxidases are essential for bacterial respiration, understanding the structure-function relationship of cyoA could inform the development of novel antimicrobials targeting respiratory chains of pathogenic bacteria.