Recombinant Salmonella schwarzengrund Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is UbiB and what role does it play in Salmonella metabolism?

UbiB is a protein required for the first monooxygenase step in coenzyme Q (ubiquinone) biosynthesis. In Salmonella and other bacteria, UbiB functions in the early stages of the ubiquinone biosynthetic pathway, which is essential for respiratory electron transport. The protein is part of the UbiB family, a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member . UbiB's involvement in ubiquinone biosynthesis is critical for Salmonella's energy metabolism, particularly under aerobic conditions, as ubiquinone serves as an electron carrier in the respiratory chain .

How is UbiB structurally characterized in Salmonella schwarzengrund?

UbiB in Salmonella schwarzengrund belongs to the UbiB family, which contains an atypical kinase/ATPase domain similar to that found in Coq8, an essential protein for CoQ synthesis that resides on the matrix face of the inner mitochondrial membrane in eukaryotes . While complete structural characterization specifically for S. schwarzengrund UbiB is limited in the literature, comparative analysis with homologous proteins suggests it contains conserved catalytic motifs necessary for ATP binding and potential phosphoryl transfer activity . The protein is likely to contain specific residues that are essential for its functional activity, similar to those identified in other UbiB family members where core protein kinase-like (PKL) family residues are required for function .

What are the recommended expression systems for recombinant Salmonella UbiB production?

For recombinant expression of Salmonella UbiB, E. coli-based expression systems are commonly employed due to their genetic similarity and ease of manipulation. BL21(DE3) or its derivatives are preferred host strains due to their reduced protease activity and compatibility with T7 promoter-based expression vectors . For optimal expression, vectors containing strong inducible promoters (like T7 or tac) with appropriate affinity tags (His6, GST, or MBP) facilitate both expression and subsequent purification. Temperature modulation (lowering to 16-25°C after induction) often improves the solubility of recombinant UbiB, which like other membrane-associated proteins, may have tendency to form inclusion bodies at higher expression temperatures .

What purification strategies yield the highest activity for recombinant UbiB?

Purification of recombinant UbiB presents challenges due to its potential membrane association and limited solubility. A multi-step purification approach is recommended:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

• Ion exchange chromatography (typically anion exchange at pH 7.5-8.5)

• Size exclusion chromatography for final polishing

Throughout purification, maintaining a stabilizing buffer system is critical:

Note that UbiB is often recalcitrant to recombinant protein purification, as observed with the homologous protein Cqd1 in yeast . Testing multiple constructs with different tags and expression conditions may be necessary to obtain functional protein.

How can researchers verify the functional activity of purified recombinant UbiB?

Verification of recombinant UbiB activity can be approached through several complementary methods:

What is the specific biochemical function of UbiB in the ubiquinone biosynthetic pathway?

UbiB is required for the first monooxygenase step in ubiquinone biosynthesis, specifically the hydroxylation reaction that converts 2-octaprenylphenol to the next intermediate in the pathway . Strains with mutations in ubiB accumulate 2-octaprenylphenol, indicating a block at this specific step . While UbiB has been classified as part of a protein kinase family, its exact mechanistic role may involve:

• Acting as a direct hydroxylase (though this is less supported by current evidence)

• Functioning as a regulatory kinase that activates another enzyme through phosphorylation

• Serving as an ATPase that couples energy from ATP hydrolysis to drive conformational changes required for the hydroxylation reaction

• Participating in protein-protein interactions that stabilize a larger biosynthetic complex

Recent research suggests that UbiB family proteins like Coq8 and Cqd1 in yeast depend on core protein kinase-like family residues for their function, supporting a model where UbiB's activity relies on atypical kinase/ATPase activity .

How does UbiB function differ between aerobic and anaerobic conditions?

Ubiquinone biosynthesis in bacteria can proceed through both O₂-dependent and O₂-independent pathways, with UbiB participating primarily in the aerobic pathway . Under anaerobic conditions, bacteria utilize an alternative pathway involving UbiT, UbiU, and UbiV proteins that form a novel class of O₂-independent hydroxylases . These proteins allow for ubiquinone biosynthesis in the absence of molecular oxygen.

Key differences in UbiB function between conditions include:

This adaptability allows bacteria like Salmonella to synthesize ubiquinone across varying oxygen concentrations, which is particularly important for pathogens that encounter different oxygen levels during infection .

What protein complexes does UbiB form during ubiquinone biosynthesis?

While specific complexes involving UbiB in Salmonella schwarzengrund have not been directly characterized in the provided literature, insights can be drawn from studies of the ubiquinone biosynthetic pathway in related bacteria. UbiB likely functions within a larger metabolon (multi-enzyme complex) dedicated to ubiquinone biosynthesis .

Recent research has identified important protein-protein interactions in this pathway, particularly the UbiJ-UbiK complex that appears to serve as an interface between the membrane and other enzymes in the ubiquinone biosynthetic pathway . The UbiJ-UbiK₂ heterotrimer plays a key role in releasing newly synthesized ubiquinone into the membrane .

Although direct evidence for UbiB's participation in this complex is limited, its early role in the pathway suggests potential interactions with:

• Substrate-providing enzymes that generate 2-octaprenylphenol

• Subsequent pathway enzymes that process the hydroxylated intermediate

• Potential scaffolding or regulatory proteins that organize the biosynthetic machinery

• Membrane components that facilitate localization to sites of ubiquinone synthesis

How does UbiB from Salmonella schwarzengrund differ from UbiB in other Salmonella serovars?

Comparative genomic analysis of UbiB across Salmonella serovars reveals high conservation of the core enzymatic domains, reflecting the essential nature of ubiquinone biosynthesis. Salmonella schwarzengrund UbiB maintains the characteristic UbiB family domain architecture with specific residues required for activity that are conserved across serovars .

Minor variations in non-catalytic regions may influence:

• Protein stability under different environmental conditions

• Interaction specificity with other proteins in the biosynthetic pathway

• Regulatory features that control expression or activity

What genetic regulatory elements control ubiB expression in Salmonella schwarzengrund?

The expression of ubiB in Salmonella is regulated by several factors that respond to metabolic and environmental conditions. While specific details for S. schwarzengrund are not explicitly covered in the provided literature, regulatory patterns in related bacteria suggest:

• Oxygen-responsive regulation: Given ubiquinone's role in aerobic respiration, oxygen availability likely influences ubiB expression through transcription factors responsive to oxygen levels .

• Metabolic regulation: Expression may be coordinated with central carbon metabolism and energy status, potentially through global regulators such as CRP (cAMP receptor protein).

• Growth phase-dependent expression: Ubiquinone requirements vary with growth phase, suggesting potential regulation by stationary phase sigma factors.

• Genetic organization: In E. coli, the ubiB gene has been found to be closely linked with ubiD, which is involved in a subsequent reaction in the pathway, suggesting potential co-regulation .

• Stress responses: Oxidative stress may induce expression as part of cellular defense mechanisms, as ubiquinone also functions as an antioxidant.

How does environmental adaptation influence UbiB function in Salmonella schwarzengrund during infection?

During infection, Salmonella schwarzengrund encounters diverse host environments with varying oxygen availability, nutrient concentrations, and immune pressures. These environmental transitions significantly impact UbiB function and ubiquinone biosynthesis:

• Oxygen gradient adaptation: As Salmonella transitions from the oxygen-rich intestinal lumen to oxygen-limited intracellular environments, the bacterium likely shifts between the UbiB-dependent aerobic pathway and the UbiT-UbiU-UbiV-dependent anaerobic pathway for ubiquinone biosynthesis . This adaptability ensures continuous energy production across diverse niches.

• Nutrient availability response: Host-imposed nutrient restriction may alter the availability of ubiquinone precursors, potentially requiring regulatory adjustments to UbiB activity to optimize limited resources.

• Immune evasion considerations: During macrophage invasion, Salmonella faces oxidative bursts as part of host defense. Ubiquinone's antioxidant properties may become particularly important during this phase, potentially elevating the significance of the UbiB pathway.

• Growth rate modulation: Different infection stages require distinct metabolic states, from rapid replication to persistence. UbiB activity likely adjusts to support the appropriate energy generation capacity for each stage .

This environmental responsiveness of UbiB function contributes to Salmonella's remarkable adaptability as a pathogen, enabling it to thrive across diverse host environments while maintaining essential energy metabolism .

How can UbiB-engineered Salmonella strains be developed as vaccine vectors?

Developing UbiB-engineered Salmonella strains as vaccine vectors involves several strategic approaches:

• Attenuation strategy selection: Creating defined mutations in ubiB can generate strains with controlled attenuation. These strains maintain immunogenicity while ensuring safety. Partial ubiB mutations that allow limited growth but prevent full virulence are particularly valuable .

• Antigen expression optimization: The foreign antigens should be expressed under control of promoters that are active at appropriate times during infection. Options include:

  • Constitutive promoters (e.g., PtrcA)

  • In vivo-inducible promoters (e.g., PphoP, PpagC)

  • Regulatable systems like arabinose-inducible PBAD

• Plasmid stabilization: Implementing balanced-lethal systems that eliminate the need for antibiotic selection markers improves both stability and safety. Examples include:

  • asd-based balanced-lethal systems

  • thyA-based complementation strategies

• Controlled delayed attenuation: Engineering strains that display wild-type phenotypes upon initial administration but become attenuated after reaching target tissues. This approach combines safety with improved immunogenicity .

• Multiple antigen delivery: Designing constructs that express multiple protective antigens to provide broader protection against target pathogens .

What are the advantages and limitations of using UbiB-modified Salmonella as vaccine vectors?

What immunological responses are generated by recombinant Salmonella expressing UbiB?

Recombinant Salmonella vectors generate complex, multi-faceted immune responses that contribute to their efficacy as vaccine platforms. The specific immunological profile depends on the degree of UbiB modification and the expressed foreign antigens, but typically includes:

• Innate immune activation:

  • Pattern recognition receptor engagement (TLR4, TLR5, NOD receptors)

  • Dendritic cell activation and maturation

  • Pro-inflammatory cytokine production (IL-12, TNF-α, IL-6)

  • Neutrophil and macrophage recruitment

• Adaptive immunity:

  • Humoral responses:

    • Robust serum IgG production against expressed antigens

    • Secretory IgA at mucosal surfaces

    • Memory B cell generation for long-term protection

  • Cell-mediated immunity:

    • CD4+ T helper cell responses (predominantly Th1-biased)

    • CD8+ cytotoxic T lymphocyte induction

    • Tissue-resident memory T cells at mucosal sites

• Response dynamics:

  • Initial innate immune activation (24-48 hours)

  • Peak adaptive responses typically 2-3 weeks after immunization

  • Memory responses persisting for months to years depending on strain design and dosing regimen

The balanced attenuation achieved through UbiB modification can optimize these responses by allowing sufficient replication to generate robust immunity while preventing disease symptoms .

What analytical methods are most effective for studying UbiB function in ubiquinone biosynthesis?

Several complementary analytical approaches provide comprehensive insights into UbiB function:

• Chromatographic analysis of ubiquinone intermediates:

  • HPLC with electrochemical or UV detection for quantification of ubiquinone and precursors

  • LC-MS/MS for structural identification and quantification of pathway intermediates

  • TLC methods for rapid screening of accumulated intermediates in mutant strains

• Genetic approaches:

  • Knockout/complementation studies to establish gene function

  • Site-directed mutagenesis to identify critical residues

  • Suppressor screens to identify functional partners

  • Conditional expression systems to study essentiality under different conditions

• Protein interaction studies:

  • Bacterial two-hybrid systems to identify interaction partners

  • Co-immunoprecipitation with tagged UbiB

  • Pull-down assays with recombinant proteins

  • Cross-linking mass spectrometry to map interaction interfaces

• Metabolic labeling:

  • Radioisotope or stable isotope labeling of precursors

  • Pulse-chase experiments to track ubiquinone synthesis kinetics

  • In vivo metabolic flux analysis

• Structural biology approaches:

  • X-ray crystallography (challenging due to potential membrane association)

  • Cryo-EM for complex assemblies

  • NMR for structure-function studies of specific domains

• Computational methods:

  • Homology modeling based on related structures

  • Molecular dynamics simulations to predict substrate binding and protein dynamics

  • Integrative structural modeling combining experimental data

How can researchers establish reliable assays to measure UbiB enzymatic activity?

Establishing reliable assays for UbiB activity presents challenges due to its potential membrane association and complex role. The following approaches provide complementary insights:

• Reconstituted in vitro assays:

  • Components needed:

    • Purified recombinant UbiB (with appropriate tags for solubility)

    • Substrate (2-octaprenylphenol or analogues)

    • Cofactors (likely including ATP)

    • Appropriate membrane mimetics (nanodiscs, liposomes, or detergent micelles)

    • Oxygen source (for aerobic pathway)

  • Detection methods:

    • HPLC-based product formation analysis

    • Coupled enzyme assays measuring ATP hydrolysis

    • Oxygen consumption monitoring for hydroxylation reactions

• Cell-based functional assays:

  • Complementation of ubiB-deficient strains with variants

  • Growth restoration on non-fermentable carbon sources

  • Ubiquinone production measurement in conditional expression strains

  • Reporter gene fusions to monitor pathway activity

• ATPase activity measurements:

  • Malachite green phosphate detection assay

  • Coupled enzyme assays (pyruvate kinase/lactate dehydrogenase)

  • Radiolabeled ATP hydrolysis assays

  • These provide insight into UbiB's potential role as an ATPase or kinase

• Binding assays:

  • Fluorescence-based ligand binding assays

  • Isothermal titration calorimetry

  • Surface plasmon resonance

  • Microscale thermophoresis for measuring substrate and cofactor interactions

Key considerations for assay optimization include:

• Buffer composition (pH, ionic strength, metal ions)

• Detergent selection for membrane protein solubilization

• Temperature and time optimization

• Product stability during analysis

• Controls to distinguish UbiB-specific activity from background reactions

What imaging techniques can be applied to study UbiB localization and dynamics?

Understanding UbiB's subcellular localization and dynamics provides critical insights into its function. Several imaging approaches are applicable:

• Fluorescence microscopy techniques:

  • Fusion protein approaches:

    • UbiB-GFP/mCherry fusions for live-cell imaging

    • Verification that fusions retain functionality via complementation assays

    • Photoactivatable or photoswitchable fluorescent proteins for pulse-chase studies

  • Super-resolution methods:

    • Structured illumination microscopy (SIM) for ~100 nm resolution

    • Stimulated emission depletion (STED) microscopy for ~30-50 nm resolution

    • Single-molecule localization microscopy (PALM/STORM) for ~20 nm resolution

    • These techniques can resolve UbiB distribution relative to membrane structures

• Immunolocalization approaches:

  • Immunogold electron microscopy for ultrastructural localization

  • Immunofluorescence with specific antibodies

  • Proximity ligation assays to detect protein-protein interactions in situ

• Dynamic analysis techniques:

  • Fluorescence recovery after photobleaching (FRAP) to measure mobility

  • Fluorescence correlation spectroscopy for diffusion analysis

  • Single-particle tracking of labeled UbiB

  • These methods reveal information about UbiB's membrane association dynamics

• Correlative microscopy:

  • Correlative light and electron microscopy (CLEM)

  • Combines fluorescence localization with ultrastructural context

  • Particularly valuable for membrane-associated proteins like UbiB

• Biosensor approaches:

  • FRET-based sensors to detect conformational changes

  • Split-GFP complementation to monitor protein-protein interactions

  • These approaches can provide real-time information about UbiB function

The studies of UbiJ-UbiK interactions with the membrane provide a model for how similar studies might be conducted with UbiB, as both are involved in the ubiquinone biosynthetic pathway and likely operate in similar membrane environments .

How should researchers interpret contradictory data regarding UbiB function?

When encountering contradictory results in UbiB research, a structured analytical approach is essential:

• Evaluate experimental conditions systematically:

  • Oxygen availability: UbiB function differs between aerobic and anaerobic conditions; inconsistent oxygenation during experiments can produce conflicting results .

  • Growth phase effects: UbiB activity and its impact may vary with bacterial growth phase.

  • Media composition: Different carbon sources can affect the relative importance of ubiquinone biosynthesis pathways.

  • Strain background differences: Genetic variations between laboratory strains can influence UbiB function and ubiquinone biosynthesis .

• Reconcile through mechanistic hypotheses:

  • Dual functionality model: UbiB may serve different roles under different conditions (e.g., regulatory versus catalytic).

  • Contextual activity framework: UbiB function may depend on the presence of specific interaction partners that vary between experimental systems.

  • Threshold effects: Contradictory results might reflect different UbiB expression levels, where function changes qualitatively above/below certain thresholds .

• Resolve technical discrepancies:

  • Analytical method limitations: Different detection methods for ubiquinone and intermediates have varying specificities and sensitivities.

  • Protein tag interference: Different fusion tags may differentially affect UbiB function.

  • Sample preparation artifacts: Ubiquinone is lipophilic and can be lost during extraction procedures.

  • Cross-reactivity issues: Antibodies may have different specificities when used in different contexts .

• Integration strategies:

  • Hierarchical evidence evaluation: Weight evidence based on methodological strengths.

  • Multiple hypothesis testing: Design experiments specifically to distinguish between competing models.

  • Systematic validation: Verify key findings using orthogonal methods .

What are common challenges in expressing recombinant UbiB and how can they be addressed?

Expression of recombinant UbiB presents several challenges typical of membrane-associated proteins. Here are common issues and their solutions:

Additional strategies include:

• Expressing truncated domains rather than full-length protein if membrane regions cause problems

• Using cell-free expression systems with lipid nanodiscs

• Exploring alternative expression hosts like Bacillus or yeast systems

• Implementing high-throughput construct screening to identify optimal expression conditions

How can researchers distinguish between direct and indirect effects of UbiB manipulation in experimental systems?

Distinguishing direct from indirect effects of UbiB manipulation requires multiple complementary approaches:

• Genetic complementation strategies:

  • Clean genetic systems: Use markerless deletion and complementation to avoid polar effects on neighboring genes.

  • Controlled expression: Utilize titratable expression systems to establish dose-dependency relationships.

  • Point mutations: Engineer catalytic site mutations that specifically affect UbiB function without altering protein levels or interactions.

  • Rescue experiments: Test if phenotypes can be rescued by ubiquinone supplementation or by expressing only specific domains of UbiB .

• Temporal analysis approaches:

  • Time-course studies: Monitor changes immediately following UbiB inactivation versus long-term adaptations.

  • Inducible systems: Use rapid induction/repression systems to distinguish primary from secondary effects.

  • Metabolic flux analysis: Track the immediate metabolic consequences of UbiB perturbation before compensatory changes occur .

• Biochemical discrimination methods:

  • In vitro reconstitution: Demonstrate direct biochemical activity with purified components.

  • Substrate trapping: Use catalytically inactive UbiB variants to trap and identify direct substrates.

  • Chemical complementation: Test if specific metabolic intermediates can bypass UbiB deficiency .

• Systems biology approaches:

  • Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics to distinguish primary from secondary responses.

  • Network analysis: Map changes onto metabolic and regulatory networks to identify direct connections.

  • Computational modeling: Use metabolic models to predict direct versus indirect consequences of UbiB perturbation .

• Control experiments:

  • Parallel manipulation: Compare UbiB perturbation with other ubiquinone pathway enzymes to distinguish UbiB-specific effects.

  • Alternative perturbation methods: Use both genetic and chemical inhibition approaches to confirm findings.

  • Multiple strain backgrounds: Test effects in different genetic backgrounds to identify context-dependent versus core functions .

What experimental approaches can elucidate the UbiB interactome in Salmonella?

Comprehensive characterization of the UbiB interactome requires multiple complementary approaches:

• Affinity-based methods:

  • Co-immunoprecipitation (Co-IP): Using antibodies against tagged UbiB to pull down interaction partners.

  • Tandem affinity purification (TAP): Employing dual tags for sequential purification to reduce non-specific interactions.

  • BioID or APEX proximity labeling: Fusing UbiB to a biotin ligase or peroxidase to biotinylate proximal proteins, capturing transient and weak interactions .

• Genetic and functional screening:

  • Bacterial two-hybrid screening: Systematic testing of potential interaction partners.

  • Suppressor screens: Identifying mutations that suppress ubiB mutant phenotypes, suggesting functional interactions.

  • Synthetic lethality analysis: Finding genes whose disruption is only lethal in combination with ubiB mutations .

• Structural approaches:

  • Crosslinking coupled with mass spectrometry (XL-MS): Capturing spatial relationships between interacting proteins.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Mapping interaction interfaces by monitoring solvent accessibility changes.

  • Cryo-electron microscopy: Visualizing complexes that include UbiB .

• In vivo visualization:

  • Bimolecular fluorescence complementation (BiFC): Detecting protein interactions through reconstitution of split fluorescent proteins.

  • Förster resonance energy transfer (FRET): Measuring proximity between fluorescently labeled proteins.

  • Split-protein complementation assays: Using reporter enzymes that regain activity when proteins interact .

• Integrative approaches:

  • Correlation analysis of -omics data: Identifying proteins with expression patterns that correlate with UbiB.

  • Co-evolution analysis: Detecting proteins that show coupled evolutionary changes, suggesting functional interaction.

  • Network modeling: Integrating multiple data types to predict functional associations .

How does the UbiB protein contribute to the stability of the ubiquinone biosynthetic complex?

UbiB likely plays several roles in stabilizing the ubiquinone biosynthetic complex, though specific evidence for S. schwarzengrund UbiB is limited. Based on studies of related proteins and pathways:

• Structural contributions:

  • UbiB may serve as a scaffold component within the metabolon, providing interaction surfaces for multiple proteins.

  • Its kinase-like domain could create a structural framework that helps organize pathway components spatially .

• Membrane anchoring:

  • Similar to other UbiB family proteins, it may facilitate interaction with the membrane, helping to localize the biosynthetic complex to appropriate membrane domains.

  • This membrane association could stabilize the complex in a specific orientation relative to the lipid bilayer .

• ATP-dependent stabilization:

  • As a member of the atypical kinase family, UbiB likely binds ATP, which could induce conformational changes that promote or stabilize protein-protein interactions.

  • ATP hydrolysis may drive dynamic rearrangements that maintain complex integrity during catalytic cycles .

• Regulatory phosphorylation:

  • UbiB might phosphorylate specific residues on partner proteins, altering their conformation or interaction properties.

  • Such modifications could serve as molecular "switches" that control assembly or disassembly of the complex .

• Metabolite channeling facilitation:

  • By bringing sequential enzymes into proximity, UbiB may create protected channels for intermediate transfer, preventing diffusion and side reactions.

  • This channeling function would enhance pathway efficiency and stability .