Recombinant Shigella boydii serotype 4 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the UbiB protein and what is its role in Shigella boydii?

UbiB is classified as a probable ubiquinone biosynthesis protein in Shigella boydii serotype 4. It plays a critical role in the ubiquinone (coenzyme Q) biosynthetic pathway. Ubiquinone functions as an essential electron carrier in the bacterial respiratory chain during oxidative phosphorylation . The protein is part of the terpenoid-quinone biosynthesis pathway, which is critical for energy metabolism in this pathogenic bacterium. Methodologically, researchers can confirm UbiB's role through knockout studies and complementation assays, where eliminating the ubiB gene would result in impaired ubiquinone production and respiratory deficiencies.

What is the amino acid sequence and structural characteristics of the UbiB protein?

The complete amino acid sequence of UbiB from Shigella boydii serotype 4 consists of 546 amino acids. The primary sequence begins with MTPGEVRRLYFIIRTFLSYGLDELIPKMRITLPLRLWRYSLFWMPNRHKDKLLGERLRLA and continues through the full sequence as detailed in the UniProt database (Q31UF1) . Structural analysis would typically involve predictive modeling and experimental approaches such as X-ray crystallography or cryo-EM to determine key functional domains. Researchers should note that the protein contains regions that suggest membrane association, consistent with its role in the ubiquinone biosynthetic pathway which occurs near the membrane.

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

For recombinant UbiB protein expression, Escherichia coli BL21 has proven to be an effective expression system, especially when the chimeric gene is codon-optimized for E. coli expression . The methodological approach involves:

• Sequence optimization using the codon usage of E. coli to enhance expression

• Utilization of appropriate expression vectors (typically pET series)

• Induction with IPTG under optimized conditions (temperature, time, concentration)

• Purification using affinity chromatography based on the added tag

The expression results in a protein of approximately 60.6 kDa that can be confirmed via SDS-PAGE and Western blot techniques .

How does the UbiB protein from Shigella boydii compare with homologous proteins in other bacteria?

Comparative analysis of UbiB from Shigella boydii serotype 4 with homologous proteins in other bacterial species reveals important evolutionary relationships and functional conservation. Researchers should employ the following methodological approach:

• Multiple sequence alignment using CLUSTAL Omega or similar tools

• Phylogenetic analysis to determine evolutionary relationships

• Structural homology modeling to identify conserved domains

• Functional complementation studies across species

The table below summarizes key comparative features:

This comparative approach can identify critical conserved residues that may serve as targets for structure-function studies or antimicrobial development.

What are the critical functional domains of UbiB and how do mutations affect its activity?

Based on sequence analysis and structural prediction, UbiB contains several functional domains that are critical for its role in ubiquinone biosynthesis. Researchers investigating this question should implement site-directed mutagenesis studies targeting:

• The ATP-binding motif (likely located within the N-terminal region)

• Putative ubiquinone precursor binding sites

• Membrane interaction domains

• Protein-protein interaction sites for complex formation with other ubiquinone biosynthetic enzymes

The amino acid sequence indicates potential critical regions such as "VGIPALVRAFKEK" and "VAALEKNGTNMKLLAERG" that might be involved in substrate binding or catalytic activity . Methodologically, researchers could create a library of point mutations and assess their impact on:

How does oxygen availability affect UbiB expression and activity in Shigella boydii?

This question explores the regulatory mechanisms controlling UbiB in different oxygen conditions. Shigella boydii, like many facultative anaerobes, may modulate ubiquinone biosynthesis based on oxygen availability. The KEGG pathway database indicates that there are both O2-dependent and O2-independent ubiquinone biosynthesis pathways in prokaryotes (modules M00117 and M00989, respectively) .

Methodological approach:

• Culture S. boydii under varying oxygen concentrations (aerobic, microaerobic, anaerobic)

• Perform RT-qPCR to quantify ubiB transcript levels

• Conduct Western blot analysis to measure UbiB protein abundance

• Assess ubiquinone production using HPLC

• Conduct RNA-seq to identify co-regulated genes in the pathway

Expected findings would show differential regulation of UbiB under aerobic versus anaerobic conditions, with potential shifts between the O2-dependent and O2-independent pathways.

What are the optimal conditions for expressing and purifying recombinant UbiB protein?

Optimization of expression and purification conditions is critical for obtaining high-quality recombinant UbiB protein. The methodological approach should include:

Expression optimization:

• Test multiple E. coli strains (BL21, BL21(DE3), Rosetta)

• Evaluate expression vectors with different promoters and fusion tags

• Optimize induction parameters:

  • IPTG concentration (0.1-1.0 mM)

  • Induction temperature (16°C, 25°C, 37°C)

  • Induction duration (3h, 6h, overnight)

• Conduct small-scale expression tests before scaling up

Purification protocol:

• Cell lysis: Sonication or French press in Tris-based buffer with protease inhibitors

• Clarification: Centrifugation at 15,000 × g for 30 minutes

• Affinity chromatography: Based on fusion tag (His, GST, MBP)

• Optional secondary purification: Ion exchange or size exclusion chromatography

• Buffer optimization: Tris-based buffer with 50% glycerol for storage at -20°C

The purified protein should be stored at -20°C for short-term use or -80°C for extended storage, with the addition of 50% glycerol to prevent freeze-thaw damage. Working aliquots can be stored at 4°C for up to one week .

How can researchers assess the enzymatic activity of recombinant UbiB protein in vitro?

Assessment of UbiB enzymatic activity presents challenges due to its role in a multi-enzyme pathway. Researchers should consider these methodological approaches:

• ATP hydrolysis assay:

  • Measure ATP consumption using malachite green phosphate detection

  • Control experiments with catalytically inactive mutants

• Coupled enzyme assays:

  • Reconstitute partial or complete ubiquinone biosynthesis pathway

  • Use purified enzyme components and synthetic substrates

  • Monitor formation of intermediates by HPLC or LC-MS/MS

• Membrane incorporation studies:

  • Assess UbiB integration into artificial liposomes

  • Measure effects on membrane potential using fluorescent probes

• Substrate binding assays:

  • Isothermal titration calorimetry with potential substrates

  • Fluorescence-based binding assays using intrinsic tryptophan fluorescence

Data from these assays should be analyzed using appropriate enzyme kinetics models (Michaelis-Menten, allosteric models) to determine parameters such as Km, Vmax, and substrate specificity.

What approaches can be used to study protein-protein interactions involving UbiB in the ubiquinone biosynthetic pathway?

Understanding the protein-protein interactions of UbiB is essential for elucidating its role in the ubiquinone biosynthetic complex. Researchers should employ multiple complementary approaches:

• Co-immunoprecipitation:

  • Generate specific antibodies against UbiB or use tag-based pull-down

  • Identify interaction partners by mass spectrometry

  • Validate interactions with reciprocal co-IP experiments

• Bacterial two-hybrid system:

  • Create fusion constructs with UbiB and other pathway components

  • Screen for interactions using reporter gene activation

  • Quantify interaction strength through β-galactosidase assays

• Bimolecular Fluorescence Complementation (BiFC):

  • Create split-fluorescent protein fusions with UbiB and potential partners

  • Visualize interactions through reconstituted fluorescence

  • Perform subcellular localization studies

• Surface Plasmon Resonance (SPR):

  • Immobilize purified UbiB on sensor chips

  • Measure binding kinetics with purified partners

  • Determine association/dissociation constants

These approaches should be applied to investigate interactions with known ubiquinone biosynthesis proteins including UbiA, UbiC, UbiD, UbiE, UbiF, UbiG, and UbiH.

How should researchers interpret discrepancies between in vitro and in vivo studies of UbiB function?

Discrepancies between in vitro biochemical assays and in vivo functional studies of UbiB are common and require careful interpretation. The methodological approach to resolving such discrepancies includes:

• Systematic comparison of experimental conditions:

  • Document differences in protein concentration, buffer composition, pH, and temperature

  • Assess the presence/absence of membrane components or cellular extracts

  • Consider the impact of tag locations and protein modifications

• Complementary approaches:

  • Conduct in-cell studies using UbiB variants with site-specific mutations

  • Perform complementation studies in ubiB knockout strains

  • Use conditional expression systems to titrate UbiB levels

• Data integration framework:

  • Create a comprehensive model incorporating both in vitro and in vivo data

  • Identify parameters that may explain discrepancies (post-translational modifications, protein-protein interactions)

  • Design experiments to directly test these parameters

• Statistical validation:

  • Apply appropriate statistical tests to determine significance of discrepancies

  • Consider biological versus technical variability in the data

  • Calculate effect sizes to quantify the magnitude of differences

By systematically addressing these factors, researchers can develop a more complete understanding of UbiB function in its native context.

What bioinformatic tools are most useful for analyzing the evolutionary significance of UbiB across bacterial species?

To analyze the evolutionary significance of UbiB, researchers should employ a comprehensive suite of bioinformatic tools and methodologies:

• Sequence analysis tools:

  • BLAST/PSI-BLAST for homolog identification

  • MUSCLE or CLUSTAL for multiple sequence alignments

  • MEGA or PhyML for phylogenetic tree construction

  • ConSurf for identifying conserved regions

  • PAML for detecting sites under selective pressure

• Structural bioinformatics:

  • I-TASSER or AlphaFold for protein structure prediction

  • CATH or SCOP for structural classification

  • PyMOL or Chimera for visualization and analysis of conserved domains

• Genomic context analysis:

  • Examine gene neighborhood conservation using tools like SyntTax or GeConT

  • Analyze operonic structures and gene fusion events

  • Investigate horizontal gene transfer patterns using BLAST-based methods

• Data visualization and integration:

  • Gephi or Cytoscape for network analysis of protein families

  • R or Python for custom evolutionary analyses and visualization

  • Integrated databases like STRING for functional association networks

When applying these tools, researchers should focus on questions such as:

• Does UbiB show evidence of positive selection in pathogenic versus non-pathogenic species?

• Are there lineage-specific adaptations in UbiB sequence or structure?

• Does genomic context suggest functional divergence of UbiB homologs?

How can researchers differentiate between direct and indirect effects when studying UbiB function through gene knockout or silencing?

Differentiating between direct and indirect effects in UbiB functional studies requires a careful experimental design and comprehensive analysis. Researchers should implement this methodological framework:

• Genetic approach optimization:

  • Create clean deletion mutants (vs. insertional inactivation)

  • Use inducible systems for temporal control of gene silencing

  • Implement complementation studies with wild-type and mutant variants

  • Consider polar effects on adjacent genes in the ubiquinone biosynthesis operon

• Multi-omics characterization:

  • Transcriptomics: RNA-seq to identify affected pathways

  • Proteomics: Global protein expression changes

  • Metabolomics: Focus on ubiquinone precursors and related metabolites

  • Fluxomics: Isotope labeling to track metabolic rewiring

• Time-course experiments:

  • Monitor early vs. late changes after UbiB depletion

  • Establish temporal sequence of metabolic effects

  • Identify primary vs. secondary responses

• Compensation analysis:

  • Investigate upregulation of alternate pathways

  • Identify suppressor mutations in evolved strains

  • Test epistatic relationships with other ubiquinone biosynthesis genes

• Direct biochemical validation:

  • In vitro reconstitution of specific reactions

  • Substrate accumulation analysis

  • Enzyme activity assays with cell extracts

By implementing this comprehensive approach, researchers can build a causality network that distinguishes the direct biochemical functions of UbiB from secondary effects caused by ubiquinone deficiency.

What controls should be included when studying recombinant UbiB protein function?

Proper experimental controls are essential for rigorous analysis of UbiB function. Researchers should implement the following control strategy:

Positive controls:

• Well-characterized enzymes with similar functions (e.g., UbiB from E. coli)

• Purified ubiquinone standards for quantification

• Known functional mutants with characterized phenotypes

Negative controls:

• Catalytically inactive UbiB mutants (e.g., ATP-binding site mutations)

• Empty vector controls for expression studies

• Unrelated proteins of similar size and folding properties

Specificity controls:

• Substrate analogs to test binding specificity

• Competitive inhibitors for enzymatic assays

• Non-specific binding controls for interaction studies

System controls:

• Experiments in presence/absence of oxygen to test pathway switching

• Tests across different growth phases

• Media composition controls (carbon source, iron availability)

How can researchers overcome challenges in structural studies of membrane-associated proteins like UbiB?

Membrane-associated proteins like UbiB present unique challenges for structural studies. Researchers should consider these methodological approaches:

By combining these approaches, researchers can overcome the challenges inherent in studying membrane-associated proteins and generate valuable structural insights into UbiB function.