Recombinant Aeromonas hydrophila subsp. hydrophila Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the UbiB protein in Aeromonas hydrophila and what is its primary function?

The UbiB protein in Aeromonas hydrophila belongs to the UbiB family of proteins that are crucial for coenzyme Q (ubiquinone) biosynthesis. UbiB proteins represent a subfamily that includes kinases like COQ8B and pseudo-kinases like COQ8A in humans. Despite being classified in the kinase structural family, many UbiB proteins perform non-canonical functions through their ATP-binding domains. The primary recognized biological function of UbiB proteins is their requirement for coenzyme Q biosynthesis, which has been extensively demonstrated in yeast models .

How does Aeromonas hydrophila ubiB differ from human COQ8 proteins?

While both are involved in ubiquinone biosynthesis pathways, A. hydrophila UbiB and human COQ8 proteins (COQ8A and COQ8B) show evolutionary divergence. Human COQ8 proteins include both a functional kinase (COQ8B) and a pseudo-kinase (COQ8A), whereas bacterial UbiB proteins typically maintain different structural characteristics while serving similar metabolic functions. Human COQ8 mutations have been directly linked to multiple human diseases, including autosomal recessive cerebellar ataxia and steroid-resistant nephrotic syndrome . The bacterial UbiB, including the one from A. hydrophila, serves as an evolutionary ancestor to these more specialized eukaryotic proteins.

Why is ubiquinone biosynthesis important in bacterial systems?

Ubiquinone (coenzyme Q) is an essential component of the electron transport chain in both prokaryotic and eukaryotic organisms. In bacterial systems like A. hydrophila, ubiquinone plays critical roles in:

• Energy metabolism through oxidative phosphorylation

• Adaptation to different environmental conditions

• Resistance to oxidative stress

• Potential virulence expression in pathogenic strains

The biosynthesis pathway, mediated partly by UbiB, allows bacteria to maintain cellular respiration under various growth conditions, contributing to their survival and pathogenicity. In A. hydrophila specifically, the ability to adapt to both aquatic environments and host systems may be partly dependent on efficient ubiquinone production .

What are the optimal conditions for expressing recombinant A. hydrophila UbiB protein in laboratory settings?

For optimal expression of recombinant A. hydrophila UbiB protein, researchers should consider the following methodological approach:

• Expression system selection: E. coli BL21(DE3) typically yields high expression levels for bacterial proteins. Alternative systems include specialized E. coli strains designed for membrane protein expression if UbiB shows membrane association properties.

• Vector optimization: Vectors containing T7 promoters with His-tag or GST-tag systems facilitate purification. The tag position (N- or C-terminal) should be evaluated for minimal interference with protein functionality.

• Growth conditions:

  • Temperature: 18-25°C after induction (lower temperatures reduce inclusion body formation)

  • Media: Enriched media such as Terrific Broth supplemented with glucose

  • Induction: IPTG concentration of 0.1-0.5 mM at mid-logarithmic phase (OD600 0.6-0.8)

  • Duration: Extended expression periods (16-24 hours) at lower temperatures

• Protein extraction and purification:

  • Cell lysis via sonication or pressure-based methods in buffers containing 20-50 mM Tris-HCl, 100-300 mM NaCl, pH 7.5-8.0

  • Addition of 10-15% glycerol and 1-5 mM β-mercaptoethanol to maintain protein stability

  • Affinity chromatography followed by size exclusion chromatography

These conditions should be optimized based on preliminary small-scale expression trials, as the specific properties of A. hydrophila UbiB may necessitate modifications to this general protocol .

What are the appropriate methods for assessing UbiB functionality in A. hydrophila mutants?

Assessing UbiB functionality in A. hydrophila mutants requires multiple complementary approaches:

• Growth phenotype analysis:

  • Comparison of wild-type and ubiB-knockout strains under aerobic vs. anaerobic conditions

  • Growth kinetics in media with different carbon sources

  • Survival rates under oxidative stress conditions (H₂O₂ exposure)

• Biochemical analysis:

  • Ubiquinone quantification via HPLC-MS/MS

  • Measurement of respiratory chain activity using oxygen consumption assays

  • ATP production quantification under various growth conditions

• Genetic complementation:

  • Restoration of wild-type phenotype through plasmid-based expression of UbiB

  • Cross-species complementation with homologous genes to assess functional conservation

• Protein interaction studies:

  • Co-immunoprecipitation to identify protein partners in the ubiquinone biosynthesis pathway

  • Bacterial two-hybrid systems to map interaction networks

• Transcriptional analysis:

  • qRT-PCR to measure expression levels of UbiB and related genes

  • RNA-seq to identify compensatory mechanisms in UbiB-deficient strains

This multi-faceted approach provides comprehensive assessment of UbiB functionality, distinguishing between direct biochemical effects and secondary adaptive responses in the bacterial system .

How can researchers effectively incorporate UbiB as a target in A. hydrophila vaccine development?

For incorporating UbiB as a target in A. hydrophila vaccine development, researchers should follow this methodological framework:

• Epitope mapping and antigenicity assessment:

  • In silico prediction of B-cell and T-cell epitopes within the UbiB sequence

  • Synthesis of predicted epitope peptides for immunological testing

  • ELISA-based validation of antibody response against recombinant UbiB

• Construct design strategies:

  • Development of DNA vaccines encoding full-length UbiB or immunogenic fragments

  • Creation of recombinant protein vaccines with appropriate adjuvants

  • Design of chimeric constructs combining UbiB epitopes with known immunogenic bacterial proteins

• Delivery system optimization:

  • For fish vaccines: evaluation of bath immersion vs. injection routes

  • Novel carriers such as single-walled carbon nanotubes (SWCNTs) which have shown promise in A. hydrophila vaccine delivery

  • Microencapsulation technologies for oral delivery systems

• Immunological evaluation protocol:

  • Quantification of specific antibody titers (IgM in fish models)

  • Analysis of immune-related gene expression (IFN-I, TNF-α, CRP, IL-8, IgM, MHC I, CD8α)

  • Challenge studies with virulent A. hydrophila strains

  • Assessment of relative percent survival (RPS)

• Cross-protection analysis:

  • Testing vaccine efficacy against multiple A. hydrophila strains

  • Evaluation of protection duration through time-course studies

When designing these vaccines, researchers should account for the genetic diversity observed within A. hydrophila strains, as the species shows high intraspecific genetic diversity that could affect vaccine efficacy across different isolates .

How should researchers interpret conflicting results regarding UbiB functionality across different Aeromonas strains?

When encountering conflicting results regarding UbiB functionality across different Aeromonas strains, researchers should employ the following analytical framework:

• Genetic context analysis:

  • Compare the genomic organization around the ubiB gene in different strains

  • Assess presence of compensatory pathways or redundant genes

  • Evaluate horizontal gene transfer events that might introduce strain-specific differences

• Phylogenetic interpretation:

  • Construct phylogenetic trees based on UbiB sequence alignments

  • Correlate functional differences with evolutionary distance

  • Group strains by clade to identify pattern-specific functionalities

• Experimental variable identification:

  • Systematically review experimental conditions across studies (temperature, media, oxygen levels)

  • Standardize growth phases used for functional assays

  • Control for expression levels when comparing recombinant proteins

• Methodological reconciliation:

  • Directly compare assay sensitivities and limitations

  • Conduct side-by-side experiments with multiple strains under identical conditions

  • Develop strain-specific reference standards for quantitative comparisons

• Statistical approach:

  • Implement meta-analysis techniques for data integration

  • Use multivariate analysis to identify factors contributing to variability

  • Establish confidence intervals for functional parameters

This approach acknowledges that A. hydrophila shows high intraspecific genetic diversity with different pathogenicity potential for humans and other animals . The distribution of virulence-related genes indicates it is a genetically heterogeneous species, which likely extends to metabolism-related genes like ubiB .

What statistical approaches are most appropriate for analyzing differential expression of UbiB under various environmental conditions?

For analyzing differential expression of UbiB under various environmental conditions, researchers should employ the following statistical methodologies:

• Normalization techniques specific to experimental platform:

  • For qRT-PCR: Use multiple reference genes (minimum 3) validated for stability under experimental conditions

  • For RNA-seq: Apply TMM (Trimmed Mean of M-values) or DESeq2 normalization

  • For proteomics: Implement total spectral count normalization or NSAF (Normalized Spectral Abundance Factor)

• Appropriate statistical tests based on experimental design:

  • Two-condition comparisons: Paired t-test (parametric) or Wilcoxon signed-rank test (non-parametric)

  • Multiple condition comparisons: ANOVA with post-hoc tests (Tukey's HSD or Dunnett's test)

  • Time course experiments: Mixed-effects models or repeated measures ANOVA

  • Account for multiple testing using Benjamini-Hochberg procedure

• Environmental factor correlation analysis:

  • Principal Component Analysis (PCA) to identify major sources of variation

  • Hierarchical clustering to group similar environmental responses

  • Regression models to quantify relationships between environmental parameters and expression

  • Interaction term analysis for complex environmental combinations

• Visualization and interpretation approaches:

  • Heat maps showing expression across conditions with hierarchical clustering

  • Volcano plots highlighting statistical significance and fold change

  • Network analysis connecting UbiB expression with related metabolic genes

• Validation strategy:

  • Cross-platform verification (e.g., RNA-seq findings confirmed by qRT-PCR)

  • Biological replication (minimum n=3) with power analysis to ensure adequate sample size

  • Bootstrapping or jackknife resampling for robust confidence interval estimation

This statistical framework accounts for the observation that A. hydrophila strains can endure various environmental conditions, including low temperatures and starvation, by reshaping virulence factor content, which likely involves metabolic adaptation through genes like ubiB .

How can researchers determine if UbiB genetic variations contribute to virulence differences in clinical A. hydrophila isolates?

To determine if UbiB genetic variations contribute to virulence differences in clinical A. hydrophila isolates, researchers should implement this comprehensive analytical framework:

• Genomic comparison methodology:

  • Whole genome sequencing of multiple clinical isolates with varying virulence

  • SNP and indel identification in ubiB and flanking regulatory regions

  • Structural variant detection affecting ubiB expression or function

  • Correlation of specific ubiB variants with virulence phenotypes

• Functional genetics approach:

  • Site-directed mutagenesis to introduce observed variants into reference strains

  • Allelic exchange experiments swapping ubiB variants between high and low virulence strains

  • Complementation studies in ubiB knockout backgrounds

  • CRISPR-Cas9 precision editing to test specific polymorphisms

• Expression-virulence correlation analysis:

  • Quantitative RT-PCR measuring ubiB expression levels across isolates

  • Western blot analysis of UbiB protein abundance

  • Creation of transcriptional fusions (ubiB promoter-reporter) to track expression in vivo

  • Correlation of expression patterns with virulence in animal models

• Metabolic impact assessment:

  • LC-MS measurement of ubiquinone levels in variant strains

  • Respiratory capacity quantification through oxygen consumption rates

  • Growth kinetics under oxidative stress conditions

  • ATP production efficiency in various host-mimicking environments

• Statistical association framework:

  • Multiple regression models connecting genetic variants to virulence metrics

  • Machine learning approaches (Random Forest, Support Vector Machines) to identify predictive variants

  • Population structure correction to account for clonal relationships between isolates

  • Odds ratio calculations for specific variants in high vs. low virulence groups

This approach recognizes that A. hydrophila exhibits high intraspecific genetic diversity with different pathogenicity potentials, and strains demonstrate different capabilities in adapting to various environmental conditions, which may be influenced by metabolic variations in the ubiquinone biosynthesis pathway .

How does inhibition of UbiB in A. hydrophila compare to inhibition of COQ8 in eukaryotic systems regarding metabolic consequences?

The inhibition of UbiB in A. hydrophila compared to COQ8 inhibition in eukaryotic systems reveals fundamental differences in metabolic consequences that reflect their evolutionary divergence:

• Primary metabolic impact comparison:

  • A. hydrophila UbiB inhibition: Primarily affects aerobic respiration, with limited direct impact on anaerobic metabolic pathways. Bacteria can often shift to alternative electron acceptors or fermentative metabolism.

  • Eukaryotic COQ8 inhibition: Disrupts both mitochondrial respiration and extra-mitochondrial functions of ubiquinone, including its antioxidant properties in cellular membranes.

• Compensatory mechanism differences:

  • Bacterial systems: Demonstrate rapid adaptation through alternative respiratory quinones (menaquinone, demethylmenaquinone) and modulation of central carbon metabolism.

  • Eukaryotic systems: Show limited metabolic plasticity with compensation primarily through increased glycolysis and mitochondrial structural remodeling.

• Tissue/organelle specificity:

  • A. hydrophila: Effects are system-wide but may impact energy-intensive processes like motility and secretion systems first.

  • Eukaryotes: High-energy demanding tissues (brain, muscle, heart) are disproportionately affected, with tissue-specific COQ8A vs. COQ8B dependencies.

• Temporal dynamics comparison:

  • Bacterial response: Rapid metabolic reprogramming occurs within minutes to hours.

  • Eukaryotic response: Slower compensatory responses over hours to days, with progressive cellular damage.

• Inhibitor specificity considerations:

  • The first potent COQ8 inhibitor (TTP-UNC-CA157) demonstrates high specificity for human COQ8 proteins .

  • Bacterial UbiB inhibitors would likely require different chemical scaffolds due to structural differences.

This comparative analysis has significant implications for developing targeted antimicrobials that could inhibit bacterial UbiB while sparing human COQ8 proteins, potentially offering a new strategy against antibiotic-resistant A. hydrophila strains .

What are the structural and functional relationships between bacterial UbiB and the broader family of atypical kinases?

The structural and functional relationships between bacterial UbiB and the broader family of atypical kinases reveal important evolutionary and mechanistic insights:

• Structural architecture comparison:

  • Conserved elements: Bacterial UbiB proteins maintain the core kinase fold with ATP-binding motifs (P-loop, catalytic loop) but often lack substrate-binding domains present in conventional kinases.

  • Divergent features: Unique insertions within the kinase domain that likely confer specificity for ubiquinone biosynthesis rather than protein phosphorylation.

  • Active site modifications: Altered catalytic residues that enable ATP binding but channel energy toward different chemical reactions than phosphoryl transfer.

• Functional mechanistic spectrum:

  • ATP utilization: Unlike conventional kinases that transfer phosphate groups to substrates, UbiB proteins may use ATP binding and hydrolysis for conformational changes or as energy input for other chemical reactions.

  • Substrate specificity: While conventional kinases typically target proteins, UbiB proteins interact with metabolic intermediates in ubiquinone biosynthesis.

  • Regulatory mechanisms: UbiB activity appears to be modulated by metabolic state rather than the phosphorylation cascades that regulate conventional kinases.

• Evolutionary relationship analysis:

  • UbiB represents an early branch of the kinase superfamily that maintained ATP-binding capability but evolved distinct catalytic functions.

  • In eukaryotes, this lineage expanded to include both catalytically active members (COQ8B) and pseudo-kinases (COQ8A) .

  • The preservation of ATP-binding without conventional kinase activity suggests strong selective pressure for this biochemical property.

• Interaction network differences:

  • Unlike signaling kinases that participate in extensive protein-protein interaction networks, UbiB proteins appear to function within more limited metabolic complexes.

  • The ATP-binding domain likely serves as a structural scaffold for assembly of ubiquinone biosynthetic machinery.

How can multi-omics approaches be integrated to fully characterize the role of UbiB in A. hydrophila metabolism and pathogenicity?

A comprehensive multi-omics integration strategy for characterizing UbiB's role in A. hydrophila metabolism and pathogenicity should implement the following methodological framework:

• Sequential multi-omics experimental design:

  • Genomics: Whole genome sequencing of multiple A. hydrophila strains with varying virulence

  • Transcriptomics: RNA-seq under diverse conditions (varying oxygen levels, host-mimicking environments, stress conditions)

  • Proteomics: Quantitative proteomics with emphasis on membrane protein enrichment

  • Metabolomics: Targeted analysis of ubiquinone intermediates and global metabolic profiling

  • Phenomics: High-throughput phenotypic characterization (Biolog, growth curves, virulence assays)

• Integrative computational pipeline:

  • Data normalization and preprocessing specific to each omics layer

  • Multi-omics factor analysis (MOFA) to identify latent factors driving variation across datasets

  • Network integration using algorithms like WGCNA (Weighted Gene Co-expression Network Analysis) adapted for multi-omics

  • Bayesian network modeling to infer causal relationships

  • Genome-scale metabolic models with UbiB constraints to predict systemic effects

• Validation experimental framework:

  • CRISPR-dCas9 modulation of ubiB expression for controlled perturbation

  • Isotope labeling experiments to track metabolic flux through ubiquinone pathway

  • Infection models with wild-type and ubiB-modified strains

  • Chemical genetic approaches using UbiB inhibitors at sub-lethal concentrations

• Hierarchical data interpretation strategy:

  • Layer 1: Direct UbiB interaction partners and immediately affected metabolites

  • Layer 2: Metabolic pathway adjustments and compensatory mechanisms

  • Layer 3: Global physiological adaptations and virulence factor expression

  • Layer 4: Host interaction changes and immune response modulation

• Visualization and knowledge representation:

  • Interactive multi-dimensional visualizations integrating all omics layers

  • Mechanistic models showing UbiB's position in cellular response networks

  • Comparative analysis with known virulence regulators

This comprehensive approach would address the high intraspecific genetic diversity observed in A. hydrophila while accounting for the complex relationships between metabolism and virulence, ultimately providing a systems-level understanding of how UbiB influences both bacterial physiology and pathogenicity .

What are the most effective protein purification strategies for obtaining functionally active recombinant A. hydrophila UbiB?

For obtaining functionally active recombinant A. hydrophila UbiB, researchers should implement the following optimized purification strategy:

• Expression system selection and optimization:

  • Recommended system: E. coli C41(DE3) or C43(DE3) strains specifically designed for membrane-associated proteins

  • Vector design: pET series with tunable promoter strength and C-terminal His-tag (N-terminal tags may interfere with membrane interactions)

  • Co-expression considerations: Include chaperones (GroEL/GroES) to enhance proper folding

• Solubilization and extraction protocol:

  • Membrane fraction isolation: Differential centrifugation (40,000×g for 1 hour) following French press or sonication lysis

  • Detergent screening panel:

    Detergent	Concentration	Protein Recovery	Activity Retention
    DDM	1%	High	Moderate-High
    LMNG	0.1%	Moderate	High
    Digitonin	1%	Low-Moderate	Very High
    Triton X-100	1%	High	Low

  • Solubilization conditions: 4°C incubation for 2-3 hours in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol

• Multi-step purification strategy:

  • IMAC optimization: Cobalt resin often provides higher purity than nickel for membrane proteins

  • Buffer optimization: Include 0.05% detergent in all buffers to prevent aggregation

  • Salt gradient: Step-wise reduction from 300 mM to 150 mM NaCl improves purity

  • Size exclusion chromatography: Final polishing step using Superdex 200 in buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 0.02% detergent

  • ATP addition: Including 1 mM ATP in purification buffers helps maintain native conformation

• Activity preservation techniques:

  • Lipid supplementation: Addition of E. coli lipid extract (0.01-0.05 mg/ml) to purification buffers

  • Cryoprotectants: 10% glycerol plus 5% sucrose for freeze-thaw stability

  • Storage optimization: Aliquot and flash-freeze in liquid nitrogen, store at -80°C

  • Thawing protocol: Rapid thawing at 25°C followed by immediate placement on ice

• Quality control assessment:

  • SEC-MALS: To confirm monodispersity and oligomeric state

  • Thermal shift assay: To verify protein stability and ligand binding

  • ATP binding assay: To confirm functional capacity

  • Activity reconstitution: In liposomes or nanodiscs for functional studies

This comprehensive approach addresses the challenges associated with membrane-associated proteins and has been adapted specifically for UbiB family proteins, which require special consideration to maintain their association with the lipid environment for proper function .

What bioinformatic pipelines are most suitable for analyzing UbiB conservation across Aeromonas species and strains?

For analyzing UbiB conservation across Aeromonas species and strains, researchers should implement the following specialized bioinformatic pipeline:

• Sequence acquisition and database construction:

  • Genome retrieval: Extract complete genomes from NCBI GenBank, focusing on diverse Aeromonas species

  • Local database creation: Build BLAST-searchable database of Aeromonas genomes

  • Annotation verification: Implement Prokka or RAST re-annotation to ensure consistent gene calling

  • Pipeline automation: Use Snakemake or Nextflow to create reproducible workflows

• Multi-level comparative analysis framework:

  • Primary sequence comparison:

    Analysis Level	Tools	Parameters	Output
    Sequence alignment	MAFFT (G-INS-i)	--maxiterate 1000	Multiple sequence alignment
    Conservation scoring	ConSurf	Bayesian method	Position-specific conservation scores
    Selective pressure	PAML (codeml)	Site models M1a vs M2a	dN/dS ratio for each codon
    Domain architecture	InterProScan	All available databases	Functional domain mapping

  • Structural conservation prediction:

    • AlphaFold2 modeling of representative UbiB proteins

    • ConSurf-DB mapping of conservation onto predicted structures

    • PyMOL visualization with conservation heat-mapping

• Taxonomic and phylogenetic integration:

  • Species tree construction: Using 16S rRNA and multi-locus sequence typing

  • UbiB gene tree: Maximum-likelihood phylogeny with IQ-TREE (best-fit model)

  • Reconciliation analysis: Gene-species tree reconciliation using GeneRax

  • Horizontal transfer detection: Using HGTector or similar algorithms

• Functional motif and regulatory element analysis:

  • Motif discovery: MEME suite for conserved sequence motifs

  • Regulatory region analysis: Promoter prediction and comparative analysis

  • RNA structural conservation: For potential post-transcriptional regulation

  • Codon usage analysis: To detect translation optimization patterns

• Visualization and interpretation tools:

  • Conservation mapping: Jalview with custom coloring schemes

  • Phylogenetic visualization: iTOL with metadata integration

  • Structure-function mapping: PyMOL scripts for conservation projection

  • Interactive dashboards: Using R Shiny or Python Dash for data exploration

This pipeline would effectively capture the high intraspecific genetic diversity observed within Aeromonas hydrophila, which has been documented through methods such as random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers . The approach also accounts for potential horizontal gene transfer events that might influence UbiB evolution within this genus .

What are the current technological limitations in studying UbiB-protein interactions and how can they be overcome?

Current technological limitations in studying UbiB-protein interactions present significant challenges that require innovative methodological approaches to overcome:

• Membrane protein interaction detection limitations:

  • Current limitations: Traditional yeast two-hybrid systems fail with membrane-associated proteins; co-immunoprecipitation disrupts native membrane environments; in vitro reconstitution may not reflect physiological interactions.

  • Advanced solutions:

    • Membrane yeast two-hybrid systems specifically designed for membrane protein interactions

    • Proximity labeling techniques (BioID, APEX) to capture transient interactions in native environments

    • Single-molecule pulldown (SiMPull) for direct visualization of protein complexes from cell lysates

    • Nanobody-based extraction of membrane protein complexes in their native lipid environment

• Structural characterization challenges:

  • Current limitations: Crystallization difficulties with membrane proteins; cryo-EM resolution limitations for smaller proteins; NMR signal complexity for membrane-embedded proteins.

  • Advanced solutions:

    • Lipid cubic phase crystallization optimized for UbiB-family proteins

    • Cryo-EM with fragment antigen-binding (Fab) enhancement to increase effective particle size

    • Integrative structural biology combining cross-linking mass spectrometry, SAXS, and computational modeling

    • Solid-state NMR approaches optimized for membrane proteins

• Interaction dynamics measurement constraints:

  • Current limitations: Limited temporal resolution of conventional techniques; difficulty distinguishing direct vs. indirect interactions; poor sensitivity for weak or transient interactions.

  • Advanced solutions:

    • Förster resonance energy transfer (FRET) with optimized fluorophore pairs for membrane environments

    • Single-molecule fluorescence microscopy to capture interaction kinetics

    • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for mapping interaction interfaces

    • Microfluidic-based surface plasmon resonance for real-time kinetic measurements

• In vivo interaction verification challenges:

  • Current limitations: Limited genetic manipulation tools for Aeromonas; difficulty in distinguishing physiologically relevant interactions; host environment effects on bacterial protein interactions.

  • Advanced solutions:

    • CRISPR-Cas9 genome editing optimized for Aeromonas to create tagged proteins at endogenous loci

    • Split fluorescent protein complementation assays adapted for bacterial systems

    • Bacterial protein interaction reporter systems based on antibiotic resistance or fluorescence

    • Ex vivo infection models with imaging capabilities to study interactions during host colonization

• Computational prediction limitations:

  • Current limitations: Poor performance of standard protein-protein interaction prediction algorithms for membrane proteins; limited training data for bacterial membrane protein interactions.

  • Advanced solutions:

    • Deep learning approaches trained specifically on membrane protein interactions

    • Molecular dynamics simulations in mixed lipid bilayers to predict interaction energetics

    • Coevolutionary analysis across multiple Aeromonas species to identify coevolving residues

    • Graph neural networks incorporating both sequence and structural features

By implementing these advanced methodological approaches, researchers can overcome the current technical barriers to studying UbiB protein interactions, potentially revealing critical insights into how this protein functions within the ubiquinone biosynthesis pathway and potentially contributes to bacterial pathogenicity .

How can insights from A. hydrophila UbiB research be applied to develop novel antimicrobial strategies?

Translating A. hydrophila UbiB research into novel antimicrobial strategies requires a systematic approach:

• Target validation and druggability assessment:

  • Essentiality confirmation: Generation of conditional ubiB mutants to verify growth dependency

  • Comparative analysis: Structural differences between bacterial UbiB and human COQ8 proteins that can be exploited for selectivity

  • Vulnerability mapping: Identification of critical residues and regions through alanine scanning and directed evolution

  • Microbial community impact: Assessment of collateral effects on beneficial microbiota

• Inhibitor development pipeline:

  • Screening strategy options:

    Approach	Advantages	Disadvantages	Technical Requirements
    High-throughput biochemical assay	Direct measurement of activity	Requires purified protein	ATPase or specific activity assay
    Phenotypic screening	Identifies compounds with cellular activity	Target confirmation needed	Growth inhibition assay under respiratory conditions
    Structure-based design	Rational approach to selectivity	Requires structural data	Computational docking and MD simulations
    Fragment-based screening	Identifies novel chemical scaffolds	Low initial potency	Biophysical detection methods (STD-NMR, SPR)

  • Chemical space exploration: Focus on scaffolds distinct from existing kinase inhibitors for novelty

  • Lead optimization strategy: Prioritize bacterial selectivity over absolute potency

• Resistance risk assessment and mitigation:

  • Resistance mechanism prediction: In vitro selection of resistant mutants followed by whole genome sequencing

  • Frequency of resistance: Determination of spontaneous resistance rates

  • Collateral sensitivity patterns: Identification of increased susceptibility to other antimicrobials in ubiB mutants

  • Combination strategy: Development of dual-targeting approaches to reduce resistance emergence

• Delivery system innovation:

  • Bacterial penetration enhancement: Siderophore conjugation for iron uptake pathway exploitation

  • Biofilm targeting: Integration with biofilm-disrupting agents

  • Selective delivery: Microbiome-sparing formulations

  • Environmental application: Controlled release systems for aquaculture settings

• Translational development path:

  • In vitro validation: Activity against diverse clinical isolates of A. hydrophila

  • Ex vivo models: Tissue-based infection models

  • In vivo efficacy: Animal models of A. hydrophila infection

  • Safety assessment: Mammalian cell toxicity and selectivity index determination

This approach leverages insights from recent advances in understanding UbiB proteins, including the development of the first potent COQ8 inhibitor (TTP-UNC-CA157), which could serve as a starting point for developing selective bacterial UbiB inhibitors . Given that A. hydrophila exhibits wide spectra of antibiotic resistance profiles and multi-resistant strains have been reported, targeting the metabolically essential UbiB represents a promising alternative antimicrobial strategy .

What potential applications exist for engineering UbiB to enhance recombinant protein production in biotechnology?

Engineering UbiB for enhanced recombinant protein production in biotechnology presents several innovative applications:

• Metabolic engineering for improved cellular energetics:

  • UbiB overexpression: Strategic upregulation to enhance ubiquinone production, potentially increasing ATP generation capacity

  • Controlled expression systems: Development of inducible UbiB expression cassettes that can be activated during high-demand production phases

  • Subcellular localization optimization: Targeting UbiB to specific membrane regions to enhance local ubiquinone concentrations near respiratory complexes

  • Engineered UbiB variants: Creation of hyperactive mutants through directed evolution to maximize ubiquinone biosynthesis

• Stress resistance enhancement in production strains:

  • Oxidative stress protection: Leveraging ubiquinone's antioxidant properties to shield production organisms from reactive oxygen species

  • Application frameworks:

    Production System	UbiB Engineering Approach	Expected Benefits	Validation Metrics
    Bacterial fermentation	Codon-optimized UbiB overexpression	Increased biomass, extended production phase	Cell density, ATP levels, protein yield
    Insect cell culture	UbiB-enhanced energy metabolism	Higher protein yields, improved glycosylation	Specific productivity, product quality
    Mammalian cell production	COQ8-UbiB chimeras for enhanced function	Extended culture longevity, reduced apoptosis	Cell viability, culture duration
    Yeast expression systems	UbiB variants with temperature stability	Robust performance at high-density fermentation	Growth rate at elevated temperatures

• Bioprocess optimization through UbiB modulation:

  • Dynamic regulation: Linking UbiB expression to production phase transitions

  • Feedback-responsive systems: Creating regulatory circuits that adjust UbiB levels based on cellular energy status

  • Scale-up considerations: Engineering UbiB expression systems that maintain consistency from laboratory to industrial scale

  • Process intensification: Using UbiB enhancement to support high-cell-density cultivation

• Co-production applications:

  • Ubiquinone (CoQ10) co-production: Dual harvesting of recombinant protein products and CoQ10 as a valuable bioproduct

  • Redox balance engineering: Manipulating UbiB to optimize intracellular redox conditions for proper protein folding

  • Secretion enhancement: Using improved energetics to drive efficient protein secretion

  • Chaperone coupling: Coordinating UbiB enhancement with chaperone expression for improved protein quality

• Implementation strategy for production platforms:

  • Chassis-specific optimization: Tailoring UbiB engineering to match the metabolic background of production organisms

  • Integration with existing enhancement technologies: Combining with traditional approaches (media optimization, feeding strategies)

  • Monitoring systems: Development of biosensors for real-time tracking of ubiquinone levels and energy status

  • Regulatory considerations: Addressing potential regulatory concerns for products from metabolically engineered organisms

This approach represents a novel strategy for addressing one of the fundamental limitations in recombinant protein production—cellular energy capacity—by targeting the ubiquinone biosynthesis pathway through UbiB engineering . The approach could be particularly valuable for production of challenging proteins that impose high metabolic burdens on host cells.

How might CRISPR-Cas technologies be optimized for studying UbiB function in A. hydrophila?

Optimizing CRISPR-Cas technologies for studying UbiB function in A. hydrophila requires addressing specific challenges associated with this bacterial system:

• CRISPR-Cas system adaptation for A. hydrophila:

  • Cas9 codon optimization: Tailoring codon usage for efficient expression in A. hydrophila

  • Promoter selection: Screening native promoters for optimal Cas9 and gRNA expression levels

  • Delivery optimization: Development of species-specific transformation protocols

    • Electroporation parameters: 2.5 kV/cm, 25 μF, 200 Ω in 10% glycerol

    • Alternative: Conjugation using RP4-based systems from E. coli donors

  • Temperature considerations: Adapting protocols for the mesophilic growth temperature of A. hydrophila (25-30°C)

• Guide RNA design strategy for UbiB targeting:

  • Target site selection considerations:

    Target Region	Advantages	Scientific Questions Addressed
    Coding sequence	Direct functional disruption	Essential nature of UbiB protein
    Promoter region	Modulation of expression	Regulatory control and expression thresholds
    ATP-binding motif	Specific functional domain targeting	Role of ATP binding in UbiB function
    C-terminal region	Potential regulatory domain disruption	Structure-function relationships

  • Off-target minimization: Implementation of A. hydrophila-specific scoring algorithms

  • PAM site availability analysis: Mapping all potential target sites in ubiB genomic context

  • Multiplex targeting: Simultaneous targeting of ubiB and related genes for pathway analysis

• Advanced CRISPR applications beyond knockout:

  • CRISPRi implementation: dCas9-based transcriptional repression for tunable ubiB downregulation

  • CRISPRa development: Adaptation of activation systems for controlled overexpression

  • Base editing optimization: Precision modification of specific codons without DSB formation

  • CRISPR-scanning: Systematic functional mapping of UbiB domains through targeted mutations

  • Prime editing: Precise introduction of specific mutations to study structure-function relationships

• Phenotypic characterization frameworks:

  • High-throughput growth analysis: Automated growth curve generation under various conditions

  • Microfluidic single-cell analysis: Monitoring phenotypic heterogeneity in CRISPR-modified populations

  • Metabolic profiling: Targeted metabolomics focusing on ubiquinone and respiratory intermediates

  • In vivo fitness assessment: Competition assays between wild-type and CRISPR-modified strains

• Integration with complementary technologies:

  • CRISPR-Seq: Combining CRISPR modification with RNA-seq for transcriptomic impact assessment

  • CUT&Tag adaptation: Chromatin profiling to identify UbiB-associated genomic regions

  • CRISPR interference screens: Genome-wide screening for genetic interactions with ubiB

  • In vivo CRISPR delivery: Phage-based systems for studying UbiB function during infection

These optimized CRISPR-Cas approaches would address the challenge of studying UbiB function in A. hydrophila, accounting for the genetic diversity observed across strains and the need for precise genetic manipulation to understand the role of this protein in ubiquinone biosynthesis and potentially in pathogenicity .

What role might UbiB play in A. hydrophila biofilm formation and how can this be investigated?

The potential role of UbiB in A. hydrophila biofilm formation represents an unexplored frontier requiring specialized investigative approaches:

• Hypothesis framework for UbiB-biofilm connections:

  • Energetic basis: Ubiquinone biosynthesis affects ATP availability for initial attachment and matrix production

  • Redox signaling: Ubiquinone-derived redox signals potentially regulate biofilm-specific gene expression

  • Structural contribution: Possible direct role of UbiB in biofilm matrix organization

  • Stress response integration: Ubiquinone's antioxidant properties may protect biofilm cells from oxidative stress

• Genetic manipulation strategy:

  • Expression modulation approach:

    Genetic Modification	Technique	Expected Biofilm Phenotype if Hypothesis Correct
    UbiB knockout	CRISPR-Cas9 deletion	Reduced biofilm formation, altered architecture
    UbiB conditional expression	Inducible promoter system	Tunable biofilm characteristics based on expression level
    UbiB overexpression	Strong constitutive promoter	Enhanced initial attachment, accelerated maturation
    Site-directed UbiB mutations	Base editing	Domain-specific effects on biofilm properties

  • Reporter fusion construction: Creation of ubiB-gfp translational fusions to visualize expression in biofilms

  • Complementation testing: Rescue analysis with wild-type and mutant UbiB variants

• Biofilm phenotyping methodology:

  • Quantitative assessment techniques:

    • Crystal violet staining with spectrophotometric quantification

    • Confocal laser scanning microscopy with 3D reconstruction

    • Atomic force microscopy for nanoscale architecture analysis

    • Rheological measurements of biofilm mechanical properties

  • Dynamic formation analysis: Flow cell systems with time-lapse imaging

  • Matrix composition analysis: Extraction and quantification of exopolysaccharides, proteins, and eDNA

  • Spatial expression profiling: Analysis of ubiB expression across biofilm regions

• Metabolic investigation framework:

  • Metabolic imaging: Utilization of fluorescent redox sensors to visualize ubiquinone-related activity

  • Microelectrode analysis: Oxygen and redox gradient mapping within biofilms

  • Metabolomic profiling: Comparison of ubiquinone and related metabolites in planktonic vs. biofilm cells

  • Respiratory activity assessment: CTC (5-cyano-2,3-ditolyl tetrazolium chloride) staining for respiratory activity

• Translational applications exploration:

  • Anti-biofilm strategy development: Targeted inhibition of UbiB to disrupt biofilm formation

  • Biofilm engineering: Controlled manipulation of UbiB to create biofilms with desired properties

  • Interspecies interactions: Investigation of how UbiB-dependent metabolism affects multispecies biofilms

  • Host-pathogen interface: Analysis of biofilm-specific UbiB activity during infection