Recombinant Vibrio vulnificus Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the role of UbiB in ubiquinone biosynthesis in Vibrio vulnificus?

UbiB is a probable ubiquinone biosynthesis protein in V. vulnificus that likely functions as part of the electron transport chain involved in respiratory metabolism. It contributes to the biosynthetic pathway of ubiquinone (coenzyme Q), an essential lipid-soluble electron carrier in the respiratory chain. UbiB is hypothesized to function as a kinase or hydroxylase contributing to the early steps of ubiquinone synthesis. Unlike the better-characterized UbiU and UbiV proteins that form a heterodimer containing 4Fe-4S clusters for O₂-independent hydroxylation reactions, UbiB may operate through different mechanisms in the biosynthetic pathway . Current research suggests UbiB plays a crucial role in energy metabolism and potentially in virulence expression, given the importance of ubiquinone in bacterial bioenergetics across varying oxygen conditions.

How does UbiB expression relate to V. vulnificus pathogenicity?

Expression of UbiB in V. vulnificus appears to be linked to pathogenicity through its role in energy metabolism. While not directly characterized as a virulence factor like the MARTX(Vv) toxin, UbiB's contribution to ubiquinone biosynthesis supports bacterial survival and proliferation under varying environmental conditions, including during host infection .

V. vulnificus requires robust energy production systems during infection, and the ubiquinone biosynthesis pathway provides critical electron transport capabilities. The ability to synthesize ubiquinone across different oxygen tensions is particularly relevant for V. vulnificus as it transitions from marine environments to human hosts. This metabolic flexibility, supported by ubiquinone biosynthesis proteins including UbiB, likely contributes to the organism's capacity to adapt to host environments and express virulence factors. Recent studies indicate that metabolic adaptation mechanisms in V. vulnificus have been linked to antibiotic resistance and virulence expression, suggesting that UbiB may indirectly support pathogenicity through its metabolic function .

What are the optimal conditions for expressing recombinant V. vulnificus UbiB protein?

For optimal expression of recombinant V. vulnificus UbiB protein, a methodological approach incorporating the following parameters is recommended:

Expression System Recommendations:

• Host System: E. coli BL21(DE3) or E. coli C43(DE3) (particularly for membrane-associated proteins)

• Vector Selection: pET-based vectors with T7 promoter (pET28a for N-terminal His-tag or pET22b for periplasmic expression)

• Induction Conditions: 0.1-0.5 mM IPTG at mid-log phase (OD₆₀₀ = 0.6-0.8)

• Temperature: 16-18°C post-induction for 16-20 hours (to reduce inclusion body formation)

• Media: Terrific Broth supplemented with 0.5% glucose and appropriate antibiotics

Optimization Parameters:

• Start with small-scale expression trials testing multiple conditions

• For membrane-associated proteins, addition of 0.5% Triton X-100 during cell lysis improves solubility

• Consider codon optimization for E. coli if expression yields are low

• Supplement with iron sources if the protein contains Fe-S clusters similar to related ubiquinone biosynthesis proteins

Expression should be verified using SDS-PAGE and Western blotting with anti-His antibodies if using a His-tagged construct. Purification is typically achieved through nickel affinity chromatography followed by size exclusion chromatography to obtain pure protein for functional studies.

What methods are most effective for measuring UbiB activity in vitro?

Measuring UbiB activity requires specialized assays that account for its probable function in ubiquinone biosynthesis. Based on related ubiquinone biosynthesis proteins, the following methodological approaches are recommended:

Enzymatic Activity Assays:

• Hydroxylase Activity Measurement:

  • Substrate utilization assay using potential precursors (e.g., 4-hydroxybenzoate)

  • HPLC-based detection of reaction products

  • Spectrophotometric measurement of cofactor (NAD(P)H) oxidation at 340 nm

• Kinase Activity Assessment:

  • ³²P-ATP incorporation assay if UbiB functions as a kinase

  • Measurement of ADP formation using coupled enzyme assays

• Protein-Protein Interaction Studies:

  • Pull-down assays to identify interaction partners in the ubiquinone biosynthesis pathway

  • Surface plasmon resonance (SPR) to determine binding kinetics

Experimental Considerations:

• Include appropriate controls (heat-inactivated enzyme, no substrate controls)

• Optimize buffer conditions (pH 7.5-8.0, presence of divalent cations like Mg²⁺)

• Test activity under varying oxygen conditions to determine oxygen dependence

• Consider the potential requirement for Fe-S clusters or other cofactors

These methods should be adaptable based on emerging understanding of UbiB's specific function in the ubiquinone biosynthesis pathway, particularly in relation to the O₂-independent pathway characterized for UbiU-UbiV .

How can researchers effectively generate ubiB gene knockouts in V. vulnificus for functional studies?

Generating effective ubiB gene knockouts in V. vulnificus requires careful methodological consideration to ensure clean genetic manipulation while maintaining bacterial viability. The following protocol is recommended:

Gene Knockout Strategy:

• Homologous Recombination Approach:

  • Design primers to amplify 500-1000 bp flanking regions upstream and downstream of ubiB

  • Clone these regions into a suicide vector (e.g., pDM4 or pKAS32) flanking an antibiotic resistance cassette

  • Introduce the construct into V. vulnificus via conjugation or electroporation

  • Select for double crossover events using appropriate antibiotics and sucrose counter-selection

• CRISPR-Cas9 Approach (More Efficient):

  • Design sgRNA targeting ubiB with minimal off-target effects

  • Clone sgRNA into a CRISPR-Cas9 vector compatible with V. vulnificus

  • Include homology-directed repair template with antibiotic marker

  • Transform into V. vulnificus and select transformants

Verification Methods:

• PCR validation of the knockout using primers flanking the target region

• Sequencing to confirm precise genetic modification

• RT-PCR to verify absence of ubiB transcript

• Complementation studies to confirm phenotype is due to ubiB deletion

Critical Considerations:

• If UbiB is essential under normal conditions, consider conditional knockout strategies

• Use defined growth media supplemented with potential ubiquinone pathway intermediates

• Test growth under both aerobic and anaerobic conditions, as UbiB may be differentially important based on oxygen availability

• Include wild-type controls in all phenotypic analyses

This approach has been successfully applied to characterize genes involved in V. vulnificus pathogenicity and metabolism in previous studies .

How does UbiB function differ between O₂-dependent and O₂-independent ubiquinone biosynthesis pathways?

The function of UbiB in relation to oxygen-dependent and oxygen-independent ubiquinone biosynthesis pathways represents a complex aspect of V. vulnificus metabolism. Based on the current understanding of ubiquinone biosynthesis:

O₂-Dependent Pathway:

• In the canonical O₂-dependent pathway, UbiB likely functions as a kinase or accessory protein supporting monooxygenases that require molecular oxygen as a co-substrate

• May interact with O₂-dependent hydroxylases in the pathway to facilitate electron transfer

• Expression of UbiB may be regulated in response to oxygen availability, potentially through oxygen-sensing transcription factors

O₂-Independent Pathway:

• In contrast to the specialized UbiU-UbiV system that functions as an O₂-independent hydroxylase containing 4Fe-4S clusters , UbiB may play a supportive or alternative role

• Could function in parallel or sequentially with the UbiU-UbiV system depending on cellular redox state

• May contribute to pathway regulation under fluctuating oxygen conditions

Metabolic Integration:
The dual pathway system for ubiquinone biosynthesis represents an important adaptation mechanism for V. vulnificus to colonize environments with varying O₂ gradients. This metabolic plasticity has been linked to antibiotic resistance and virulence . UbiB likely contributes to this adaptability, allowing the bacterium to maintain energy production across diverse environmental conditions, including during host infection where oxygen availability can fluctuate significantly.

Current research supports that these pathways contribute to "optimizing bacterial metabolism over the entire O₂ range," with UbiB potentially serving as an adaptive node in this metabolic network.

How do structural variations in UbiB affect its function in different V. vulnificus biotypes?

Structural variations in UbiB across different V. vulnificus biotypes may significantly impact its function and contribute to biotype-specific metabolic adaptations:

Biotype Variations and Functional Implications:

V. vulnificus can be divided into three biotypes with varied host ranges and virulence characteristics . Analysis of UbiB structural variations requires:

• Sequence Comparison Analysis:

  • Alignment of ubiB gene sequences from all three biotypes to identify conserved and variable regions

  • Identification of single nucleotide polymorphisms (SNPs) that may affect protein structure

  • Analysis of selection pressure on different protein domains using dN/dS ratios

• Protein Structure-Function Correlations:

  • Homology modeling to predict structural differences in UbiB between biotypes

  • Identification of variations in catalytic sites or cofactor binding regions

  • Molecular dynamics simulations to predict how variants affect protein dynamics

• Experimental Validation Approaches:

  • Site-directed mutagenesis to replicate biotype-specific variations

  • Enzymatic activity assays to compare functional differences

  • Complementation studies using UbiB variants in knockout strains

Research Methodology Table:

This multifaceted approach would provide insights into how UbiB structural variations contribute to metabolic adaptations in different V. vulnificus biotypes, potentially relating to their distinct ecological niches and pathogenic potential.

How does oxygen availability affect ubiB gene expression and regulation in V. vulnificus?

Oxygen availability likely plays a critical role in ubiB gene expression and regulation in V. vulnificus, reflecting the bacterium's need to adapt its energy metabolism to environmental conditions:

Expression Regulation Mechanisms:

• Transcriptional Regulation:

  • Oxygen-responsive transcription factors (e.g., FNR-like or ArcA/ArcB homologs) likely regulate ubiB expression

  • Promoter analysis may reveal binding sites for these regulators

  • RNA-seq data under varying oxygen conditions would identify oxygen-dependent expression patterns

• Post-transcriptional Control:

  • Small regulatory RNAs may modulate ubiB mRNA stability in response to oxygen stress

  • RNA thermometers or riboswitches could fine-tune expression based on metabolic status

• Integration with Metabolic Networks:

  • Expression likely coordinated with other components of the respiratory chain

  • May interact with systems detecting electron transport chain status

  • Potentially co-regulated with the O₂-independent ubiquinone biosynthesis pathway proteins (UbiT, UbiU, UbiV)

Experimental Approaches to Investigate Regulation:

Researchers should employ:

• Promoter-reporter fusion assays to monitor expression under controlled oxygen conditions

• ChIP-seq to identify transcription factor binding

• Transcriptome analysis comparing aerobic, microaerobic, and anaerobic conditions

• Metabolic flux analysis to correlate expression with respiratory activity

V. vulnificus must colonize environments with large O₂ gradients or fluctuating O₂ levels, and this metabolic response has been linked to antibiotic resistance and virulence . Understanding ubiB regulation will provide insights into this adaptive process.

What genomic evidence supports horizontal gene transfer of ubiB among Vibrio species?

Investigating horizontal gene transfer (HGT) of ubiB among Vibrio species requires comprehensive genomic analysis. Current evidence suggests potential gene transfer events based on several genomic signatures:

Genomic Evidence for HGT:

• Sequence Homology and Phylogenetic Incongruence:

  • Comparative analysis of ubiB sequences across Vibrio species may reveal phylogenetic patterns inconsistent with species evolution

  • Similar to observed recombination in rtxA1 genes , ubiB might show evidence of inter-species transfer

  • Phylogenetic tree construction of ubiB compared to housekeeping genes can identify potential HGT events

• Mobile Genetic Element Association:

  • Proximity to known mobile genetic elements (transposons, insertion sequences)

  • Analysis of flanking regions for recombination hotspots

  • Investigation of genomic islands containing ubiB

• Nucleotide Composition Analysis:

  • Aberrant GC content or codon usage patterns compared to the core genome

  • Tetranucleotide frequency analysis to detect foreign origin

  • Calculation of codon adaptation index (CAI) to identify recent acquisitions

Methodological Approach:
Researchers should implement a multi-faceted analysis pipeline:

• Whole genome sequencing of diverse Vibrio isolates

• Bioinformatic detection of genomic islands using tools like IslandViewer

• Bayesian analysis of recombination events

• Molecular clock analysis to date potential gene transfer events

V. vulnificus is known to undergo significant genetic rearrangement of virulence factors as evidenced by rtxA1 gene recombination . Similar mechanisms may apply to metabolic genes like ubiB, potentially contributing to the bacterium's adaptive capacity across different environmental niches.

How can understanding UbiB function contribute to novel antimicrobial strategies against V. vulnificus?

Understanding UbiB function offers promising avenues for developing novel antimicrobial strategies against V. vulnificus, particularly given the concerning rise in antibiotic resistance observed in clinical isolates :

Therapeutic Target Potential:

• Metabolic Vulnerability Exploitation:

  • Targeting UbiB could disrupt ubiquinone biosynthesis, compromising cellular energy production

  • This approach may be particularly effective against V. vulnificus strains showing resistance to conventional antibiotics

  • Inhibition might be more effective under specific oxygen conditions where alternative pathways cannot compensate

• Rational Drug Design Approaches:

  • Structure-based design of small molecule inhibitors targeting UbiB active sites

  • Peptide mimetics that disrupt essential protein-protein interactions in the ubiquinone biosynthesis pathway

  • Allosteric inhibitors that prevent conformational changes required for function

• Combination Therapy Strategies:

  • UbiB inhibitors may sensitize resistant strains to existing antibiotics

  • Synergistic effects with drugs targeting other components of energy metabolism

  • Sequential therapy targeting aerobic and anaerobic metabolism components

Assessment of Target Validity:

Research Methodology:

• High-throughput screening of chemical libraries against recombinant UbiB

• Validation in whole-cell assays under varying oxygen conditions

• Assessment of resistance development potential through serial passage experiments

• In vivo efficacy testing in appropriate infection models

This approach aligns with growing interest in targeting bacterial metabolism as an alternative to conventional antibiotic targets, potentially addressing the significant public health concern posed by multidrug-resistant V. vulnificus .

What novel experimental approaches can enhance our understanding of UbiB interactions with other ubiquinone biosynthesis proteins?

Investigating UbiB interactions with other ubiquinone biosynthesis proteins requires sophisticated methodological approaches that can capture dynamic protein-protein interactions in a near-native environment:

Advanced Experimental Approaches:

• Proximity-based Proteomics:

  • BioID or APEX2 proximity labeling fused to UbiB to identify interaction partners in vivo

  • Spatial and temporal mapping of the UbiB interactome under varying oxygen conditions

  • Quantitative comparison between O₂-dependent and O₂-independent conditions

• Advanced Microscopy Techniques:

  • Single-molecule Förster resonance energy transfer (smFRET) to observe dynamic interactions

  • Super-resolution microscopy (STORM/PALM) to visualize UbiB localization and co-localization with other pathway components

  • Correlative light and electron microscopy (CLEM) to connect protein interactions with ultrastructural context

• Structural Biology Integration:

  • Cryo-electron tomography of cellular sections to visualize the ubiquinone biosynthesis complex in situ

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

  • Integrative structural modeling combining multiple experimental data sources

• Systems Biology Approaches:

  • Genetic interaction mapping using CRISPR interference (CRISPRi) to identify functional relationships

  • Metabolic flux analysis using isotope labeling to quantify pathway contributions

  • Mathematical modeling of the complete ubiquinone biosynthesis network

Data Integration Framework:

To maximize insights, researchers should implement a data integration framework that:

• Combines interaction data across multiple experimental scales

• Correlates protein interaction dynamics with metabolic outputs

• Incorporates evolutionary analysis of co-evolved protein interfaces

• Validates interaction models through targeted mutagenesis

This comprehensive approach would significantly enhance our understanding of how UbiB functions within the context of both O₂-dependent and O₂-independent ubiquinone biosynthesis pathways that allow V. vulnificus to "synthesize ubiquinone over the entire O₂ range" .

How do environmental stressors beyond oxygen availability influence UbiB function in V. vulnificus?

Beyond oxygen availability, various environmental stressors likely influence UbiB function in V. vulnificus, reflecting the bacterium's need to adapt to diverse ecological niches:

Environmental Stressors and Potential Impacts:

• Temperature Fluctuations:

  • V. vulnificus encounters temperature variations between marine environments and human hosts

  • UbiB enzymatic activity may have temperature optima aligned with host conditions

  • Protein stability and folding kinetics likely adapted to function across the temperature range encountered during infection

  • Experimental approach: Thermal shift assays and activity measurements across physiologically relevant temperatures

• Osmotic Stress:

  • Marine bacteria must adapt to changing salinity conditions

  • Membrane composition changes under osmotic stress may affect UbiB activity if membrane-associated

  • Potential interactions with osmoregulatory systems

  • Methodology: Compare UbiB expression and activity across salinity gradients relevant to estuarine environments

• pH Variation:

  • V. vulnificus faces acidic conditions during gastric passage and potentially in phagolysosomes

  • UbiB structure and function may be pH-dependent

  • Ubiquinone biosynthesis pathway activity could be regulated by pH to maintain redox balance

  • Approach: pH-dependent activity profiling and structural stability assessments

• Nutrient Limitation:

  • Iron restriction is a common host defense mechanism

  • If UbiB requires Fe-S clusters similar to UbiU-UbiV , function may be impaired during iron limitation

  • Carbon source availability may influence ubiquinone demand

  • Methodology: Nutrient limitation studies coupled with metabolomic analysis

Experimental Design Considerations:

For comprehensive assessment, researchers should implement:

• Multi-factorial experimental designs testing interactions between stressors

• Time-course studies to distinguish acute vs. adaptive responses

• Global approaches (transcriptomics, proteomics) to position UbiB responses within broader stress adaptation networks

• Genetic approaches using stress-responsive promoter fusions to ubiB to quantify environmental regulation

Understanding how UbiB function responds to these stressors will provide insights into V. vulnificus metabolic adaptation mechanisms that contribute to its pathogenicity and environmental persistence.