Recombinant Vibrio splendidus Probable ubiquinone biosynthesis protein UbiB (ubiB)

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

UbiB functions as a key component in the oxygen-dependent pathway of ubiquinone (UQ) biosynthesis in V. splendidus. It exhibits ATPase activity and is essential for UQ8 production under aerobic conditions. Research has demonstrated that deletion of the ubiB gene significantly impairs ubiquinone biosynthesis in aerobic environments, though residual production may still occur under anaerobic conditions through alternative pathways . UbiB is part of a complex enzymatic system that performs multiple modification reactions on the aromatic ring of 4-hydroxybenzoic acid (4-HB), the precursor molecule in UQ biosynthesis.

How does Vibrio splendidus synthesize ubiquinone across different oxygen conditions?

V. splendidus possesses two distinct and complementary pathways for ubiquinone biosynthesis that allow it to adapt to varying oxygen levels:

• Oxygen-dependent pathway: Utilizes UbiB and other enzymes including UbiH. This pathway requires molecular oxygen as a substrate for hydroxylation reactions .

• Oxygen-independent pathway: Relies on three proteins—UbiT, UbiU, and UbiV—which function in the absence of molecular oxygen. UbiU and UbiV form a heterodimer containing 4Fe-4S clusters that are essential for their activity as oxygen-independent hydroxylases .

Both pathways share some common enzymes (UbiA, UbiE, UbiG) while differing in others. This dual system provides metabolic plasticity that allows V. splendidus to synthesize ubiquinone across the entire oxygen range, which is critical for its survival in marine environments with fluctuating oxygen levels .

What experimental systems are available for genetic manipulation of ubiB in Vibrio splendidus?

Researchers can employ a suicide vector system based on the pir-dependent R6K replicative origin for efficient genetic manipulation of ubiB and other genes in V. splendidus. This system includes:

• Conjugation-based transfer of genetic constructs using RP4-based conjugation

• Counterselection using the ccdB gene of the Escherichia coli F plasmid under control of the arabinose PBAD promoter

• Markerless allelic replacement methodology that allows precise genetic modifications without introducing antibiotic resistance markers

This genetic system has been successfully used to create deletion mutants in V. splendidus and can be applied for constructing ubiB knockout strains to study its function . When working with V. splendidus, cultures should be maintained at 20°C in Luria Bertani medium supplemented with 0.5M NaCl (LBS), with appropriate antibiotics added for selection of recombinant strains .

How do the biochemical properties of UbiB differ between aerobic and anaerobic environments?

When examining UbiB function under anaerobic conditions, research indicates that deletion of ubiB does not completely eliminate UQ8 production . This suggests either:

• UbiB may retain limited functionality in low-oxygen environments through alternative biochemical mechanisms

• The oxygen-independent pathway (utilizing UbiT, UbiU, and UbiV) can compensate for UbiB absence

Research methodologies to investigate these differences should include:

• Protein activity assays under controlled oxygen concentrations

• ATP consumption measurements in purified recombinant UbiB preparations

• Structural analyses to identify potential oxygen-sensing domains

• Interaction studies with other Ubi proteins under varying oxygen conditions

What are the implications of persister cell formation in V. splendidus for studying UbiB function?

V. splendidus can form persister cells with reduced metabolic activity and multidrug resistance, which significantly impacts the study of UbiB and ubiquinone biosynthesis. Researchers investigating UbiB function must account for several considerations:

• Metabolic state variation: Persister cells likely have altered energy metabolism and potentially different requirements for ubiquinone compared to actively growing cells.

• Carbon source influence: Specific carbon sources can "wake up" V. splendidus persister cells, including:

  • Amino acids: L-glutamic acid (fastest revival at 17h), L-aspartic acid, L-arginine, L-phenylalanine, and L-leucine

  • Saccharides: Maltose, D-galactose, sorbitol, mannose, N-acetyl-D-glucosamine, D-glucose, and D-fructose

• Experimental design considerations: When studying UbiB function in persister populations, researchers should:

  • Control for the presence of persister cells in the population

  • Understand that antibiotic susceptibility changes in the presence of specific carbon sources

  • Consider using combinatorial approaches (e.g., adding L-glutamic acid, L-aspartic acid, L-phenylalanine, or D-glucose simultaneously with antibiotics like tetracycline) to eliminate V. splendidus completely

These factors necessitate careful experimental design when studying UbiB in V. splendidus populations that may contain persisters, as metabolic heterogeneity could confound results.

How does the structure-function relationship of UbiB relate to its role in the ubiquinone biosynthesis pathway?

The structure-function relationship of UbiB remains partially characterized, but current evidence suggests several key features:

• Protein domains: UbiB contains regions associated with ATPase activity that are essential for its function in the ubiquinone biosynthesis pathway .

• Evolutionary conservation: UbiB belongs to a family of proteins found across multiple proteobacterial species, indicating conserved structural elements critical for function.

• Interaction partners: UbiB likely functions as part of a multiprotein complex in the UQ biosynthetic pathway, potentially interacting with other enzymes like UbiA, UbiE, and UbiG that are common to both aerobic and anaerobic pathways .

Recommended experimental approaches to investigate this structure-function relationship include:

• Site-directed mutagenesis of conserved residues

• Protein-protein interaction studies using techniques such as bacterial two-hybrid systems or co-immunoprecipitation

• Structural analyses using X-ray crystallography or cryo-EM

• Complementation studies using recombinant UbiB variants in ubiB-deletion strains

How can recombinant UbiB be effectively expressed and purified for functional studies?

Expression and purification of recombinant V. splendidus UbiB presents several technical challenges that researchers should address:

Expression Systems:

• Bacterial expression: E. coli BL21(DE3) or similar strains can be used with pET-based vectors containing the V. splendidus ubiB gene under an inducible promoter.

• Alternative hosts: Consider using V. splendidus itself as an expression host for native folding, though this requires optimization of the genetic tools described previously .

Purification Strategy:

• Affinity tags: Incorporate a His6 or similar tag, preferably with a cleavable linker to allow tag removal.

• Membrane protein considerations: As UbiB is likely membrane-associated, include detergents during purification:

  • Initial screening with mild detergents (DDM, LMNG)

  • Optimization of detergent concentration to maintain protein stability

• Buffer optimization: Include components that preserve activity:

  • ATP or non-hydrolyzable analogs

  • Appropriate salt concentration (consider the halophilic nature of V. splendidus)

  • Reducing agents to maintain cysteine residues

Activity Verification:

• ATPase assay: Measure ATP hydrolysis using colorimetric or luminescent assays.

• Functional complementation: Test if the purified protein can restore UQ biosynthesis in ubiB deletion strains.

• Interaction studies: Verify binding to known partners in the UQ biosynthetic pathway.

When optimizing these protocols, researchers should be particularly attentive to maintaining the native conformation of UbiB, as improper folding could significantly impact its ATPase activity and functional studies.

What is the relationship between UbiB function and V. splendidus virulence in marine animals?

The relationship between UbiB function and V. splendidus virulence appears to be linked through energy metabolism and adaptation to environmental conditions. V. splendidus demonstrates variable virulence toward marine organisms, particularly oysters, with some populations being significantly less virulent (82.1% mean survival rate 24 hours post-infection) than others .

Ubiquinone biosynthesis, in which UbiB plays a crucial role, is essential for cellular bioenergetics and bacterial adaptation to environments with varying oxygen levels. This metabolic flexibility likely contributes to V. splendidus pathogenicity in several ways:

• Survival in host microenvironments: The ability to produce ubiquinone under both aerobic and anaerobic conditions enables V. splendidus to maintain energy production within different host tissues, which may experience variable oxygen tensions during infection.

• Stress response: Functional ubiquinone biosynthesis pathways help bacteria cope with oxidative stress and other host defense mechanisms.

• Persistence: The relationship between energy metabolism and persister cell formation suggests that UbiB-dependent ubiquinone production may influence the ability of V. splendidus to form persistent infections that are difficult to eliminate with antibiotics .

Research examining the direct relationship between UbiB function and virulence should consider experimental approaches such as:

• Infection models using ubiB knockout strains

• Comparative transcriptomics of ubiB expression during infection versus laboratory culture

• Analysis of ubiquinone levels in bacteria recovered from infected hosts

How do the oxygen-dependent and oxygen-independent ubiquinone biosynthesis pathways interact in V. splendidus?

The interaction between oxygen-dependent (UbiB-containing) and oxygen-independent (UbiT/U/V) ubiquinone biosynthesis pathways in V. splendidus represents a sophisticated metabolic network. Research has revealed several important aspects of their interaction:

• Complementary functionality: Both pathways contribute to ubiquinone production across different oxygen conditions, with partial pathway redundancy. Experimental evidence shows that deletion of ubiB impairs but does not completely eliminate UQ8 production under anaerobic conditions .

• Shared enzymatic components: Both pathways utilize common enzymes including UbiA (prenylation), UbiE and UbiG (methylation reactions), while employing different proteins for the hydroxylation steps .

• Regulatory crosstalk: The transition between pathways appears to be regulated in response to oxygen availability, though the precise regulatory mechanisms remain to be fully characterized.

A model of pathway interaction based on current research is presented below:

To study this interaction experimentally, researchers should consider approaches such as:

• Double and triple knockout strains targeting components of both pathways

• Metabolomic analysis of UQ intermediates under controlled oxygen conditions

• Transcriptional profiling to identify regulatory elements governing pathway switching

What experimental approaches can resolve contradictory findings about UbiB function?

Resolving contradictory findings about UbiB function requires systematic experimental approaches that address methodological variables and biological complexity:

• Standardized growth conditions:

  • Precisely control oxygen levels using specialized equipment (e.g., anaerobic chambers with oxygen sensors)

  • Document media composition, particularly carbon sources, as they influence metabolic state and can affect antibiotic susceptibility and persister cell formation

  • Monitor growth phase consistently, as UbiB activity may vary with bacterial growth stage

• Genetic complementation strategies:

  • Use inducible promoters with titratable expression to examine UbiB dosage effects

  • Perform cross-species complementation to identify functionally conserved domains

  • Create chimeric proteins to pinpoint functionally important regions

• Biochemical activity measurements:

  • Develop in vitro assays using purified components to directly measure UbiB activity

  • Employ isotopic labeling to track metabolic flux through the ubiquinone pathway

  • Quantify ubiquinone and intermediates using LC-MS/MS with appropriate internal standards

• Addressing persister cell phenomena:

  • Implement single-cell analyses to distinguish between population heterogeneity effects and genuine UbiB functional differences

  • Use specific carbon sources known to affect persister cell awakening (L-glutamic acid, L-aspartic acid) in experimental protocols

• Structure-based approaches:

  • Determine UbiB structure through crystallography or cryo-EM

  • Map conserved residues and create targeted mutations to test hypotheses about function

  • Model protein-protein interactions within the ubiquinone biosynthesis complex

By systematically applying these approaches, researchers can develop a more coherent understanding of UbiB function and resolve apparently contradictory findings that may arise from differences in experimental conditions or biological variability.

What are promising approaches for studying UbiB in the context of V. splendidus adaptation to marine environments?

Future research on UbiB in the context of V. splendidus environmental adaptation should focus on several promising directions:

• Environmental sampling and comparative genomics:

  • Collect V. splendidus strains from diverse marine microenvironments with varying oxygen levels

  • Sequence and compare ubiB and related genes across strains with different ecological niches

  • Correlate genetic variations with environmental parameters (oxygen levels, temperature, salinity)

• Experimental evolution studies:

  • Subject V. splendidus cultures to fluctuating or gradually changing oxygen conditions

  • Monitor genetic and expression changes in ubiB and related genes over multiple generations

  • Test evolved strains for fitness in original and new environments

• Host-pathogen interaction models:

  • Develop improved marine animal infection models (particularly oysters) with precise control of environmental conditions

  • Compare colonization efficiency of wild-type versus ubiB mutant strains under variable oxygen conditions

  • Examine UbiB function in the context of the aquatic microbiome and polymicrobial communities

• Metabolic integration analysis:

  • Apply flux balance analysis to model how UbiB-dependent pathways integrate with central metabolism

  • Investigate potential metabolic rewiring in response to oxygen fluctuations

  • Develop predictive models of V. splendidus metabolic adaptation in changing marine environments

These approaches would provide valuable insights into how UbiB contributes to the remarkable adaptability of V. splendidus across diverse marine environments and oxygen gradients, with implications for understanding both bacterial physiology and marine ecosystem dynamics.

How might targeting UbiB function contribute to controlling V. splendidus infections in aquaculture?

Targeting UbiB function represents a promising approach for controlling V. splendidus infections in aquaculture settings, with several potential intervention strategies:

• Rational inhibitor design based on UbiB structure:

  • Develop small molecule inhibitors that specifically target the ATPase activity domain of UbiB

  • Screen natural product libraries for compounds that selectively inhibit UbiB function

  • Design peptide-based inhibitors that disrupt UbiB interactions with other components of the ubiquinone biosynthesis machinery

• Environmental manipulation strategies:

  • Implement controlled oxygen fluctuation protocols in aquaculture systems that might disadvantage bacteria relying on oxygen-dependent ubiquinone biosynthesis

  • Combine specific carbon sources (like L-glutamic acid or L-aspartic acid) with appropriate antibiotics to enhance treatment efficacy against persister cells

  • Develop biofilm disruption approaches that might increase accessibility to UbiB-targeting compounds

• Resistance management considerations:

  • Investigate the likelihood of resistance development through mutations in the oxygen-independent pathway

  • Design combination therapies that target both oxygen-dependent and independent pathways

  • Develop cycling protocols to minimize selection pressure

• Delivery system innovations:

  • Create nanoparticle-based delivery systems for UbiB inhibitors that maximize efficacy in aquatic environments

  • Design time-release formulations appropriate for marine aquaculture settings

  • Develop feed-incorporated inhibitors with appropriate pharmacokinetics for target animals

Successful implementation of these approaches would require careful attention to aquaculture-specific factors including water quality parameters, host species physiology, and economic considerations related to treatment costs and efficacy.

What are the optimal conditions for heterologous expression of V. splendidus UbiB?

Optimizing heterologous expression of V. splendidus UbiB requires careful consideration of several parameters:

Expression System Selection:

• E. coli BL21(DE3) represents a standard starting point, but consider specialized strains like:

  • Rosetta(DE3) for rare codon optimization

  • C41(DE3) or C43(DE3) for membrane-associated proteins

  • SHuffle or Origami strains if disulfide bonds are present

Vector Design Elements:

• Promoter selection:

  • IPTG-inducible T7 promoter with tunable expression

  • Arabinose-inducible PBAD promoter for tighter regulation

  • Auto-induction compatible promoters for higher yields

• Fusion partners to enhance solubility:

  • MBP (maltose-binding protein)

  • SUMO

  • Thioredoxin

  • Include protease cleavage sites for tag removal

Expression Conditions:

• Temperature optimization:

  • Lower temperatures (16-20°C) often improve folding of complex proteins

  • This aligns with V. splendidus natural growth temperature of 20°C

• Media composition:

  • Consider supplementation with 0.5M NaCl to mimic V. splendidus natural environment

  • Add trace metals important for ATPase activity

  • Include ALA (5-aminolevulinic acid) if heme incorporation is suspected

• Induction parameters:

  • Induce at OD600 = 0.6-0.8 for optimal balance of cell density and metabolic activity

  • Use lower inducer concentrations (0.1-0.5 mM IPTG) for slower, more controlled expression

  • Extended expression times (overnight) at lower temperatures

Preliminary Experimental Design:

Optimizing these conditions through systematic testing will be crucial for obtaining functional recombinant UbiB protein suitable for downstream applications.

What analytical techniques are most effective for assessing UbiB activity in vitro?

Multiple complementary analytical techniques should be employed to comprehensively assess UbiB activity in vitro:

• ATPase activity assays:

  • Malachite green phosphate detection system for quantifying released phosphate

  • Luminescence-based ATP consumption assays (higher sensitivity)

  • Coupled-enzyme systems (PK/LDH) with spectrophotometric detection of NADH oxidation

• Ubiquinone and intermediate metabolite quantification:

  • HPLC-MS/MS analysis with multiple reaction monitoring (MRM)

  • Targeted metabolomic approaches using isotopically labeled standards

  • Rapid separation techniques optimized for hydrophobic quinone compounds

• Protein interaction studies:

  • Surface plasmon resonance (SPR) to measure binding kinetics with potential protein partners

  • Microscale thermophoresis for studying interactions in solution

  • Analytical ultracentrifugation to characterize complex formation

• Structural dynamics assessment:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify conformational changes upon substrate binding

  • Circular dichroism (CD) spectroscopy to monitor secondary structure changes

  • Fluorescence-based thermal shift assays to evaluate protein stability

An integrated analytical workflow might include:

Stage 1: Initial Activity Screening

• Rapid colorimetric ATPase assays to confirm functionality

• Thermal stability assessment to optimize buffer conditions

Stage 2: Detailed Biochemical Characterization

• Determination of kinetic parameters (Km, Vmax, kcat)

• Analysis of cofactor requirements and inhibitor sensitivity

Stage 3: Pathway Integration Studies

• Reconstitution assays with other ubiquinone biosynthesis components

• Detection of reaction intermediates using high-resolution MS techniques

These analytical approaches should be validated using appropriate controls, including catalytically inactive UbiB mutants (e.g., mutations in the ATPase domain) to establish assay specificity.

How can researchers effectively design mutations to probe UbiB structure-function relationships?

Designing mutations to probe UbiB structure-function relationships requires a strategic approach based on sequence conservation, structural predictions, and functional domains:

• Conservation-based mutation design:

  • Perform multiple sequence alignment of UbiB homologs across bacterial species

  • Identify invariant residues as primary targets for functional analysis

  • Create a conservation score map to prioritize mutations

• Domain-specific mutagenesis:

  • Target the ATPase domain with mutations in the Walker A and B motifs (e.g., lysine to methionine in the ATP-binding P-loop)

  • Examine putative protein-protein interaction surfaces through alanine scanning

  • Investigate potential membrane-interaction regions with hydrophobic to charged amino acid substitutions

• Structure-guided approaches:

  • Use homology modeling based on related proteins with known structures

  • If direct structural data is unavailable, employ protein structure prediction tools like AlphaFold

  • Focus on predicted active sites, substrate binding pockets, and interdomain interfaces

• Systematic mutation libraries:

  • Develop scanning mutagenesis libraries (alanine scanning across the protein)

  • Create domain swaps with functionally related proteins

  • Design chimeric proteins with the equivalent protein from the oxygen-independent pathway

Recommended Mutation Strategy Table: