Recombinant Vibrio vulnificus Fructose-1,6-bisphosphatase class 1 (fbp)

What is the role of Fructose-1,6-bisphosphatase in Vibrio vulnificus metabolism?

Fructose-1,6-bisphosphatase (Fbp) is a crucial enzyme in V. vulnificus that catalyzes the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate in gluconeogenesis. This reaction is essential for carbohydrate metabolism, particularly when the bacteria must synthesize glucose from non-carbohydrate precursors. In V. vulnificus, Fbp has been demonstrated to be indispensable for growth on certain carbon sources.

Research has shown that the deletion of the fbp gene significantly reduces bacterial cellulose production. In a comparative study, the BC (bacterial cellulose) yield of the wild-type strain reached 6.61 g/L after 7 days, while the Δfbp mutant strain produced only 3.72 g/L, demonstrating the enzyme's importance in carbohydrate metabolism . Complementation studies with recombinant fbp restored approximately 81% of the BC production capacity, confirming the critical role of this enzyme.

How is the fbp gene regulated in Vibrio species?

The regulation of fbp in Vibrio species appears to involve both carbon source-dependent and nitrogen-dependent regulatory mechanisms. In related Vibrio species such as V. cholerae, transcriptional regulation of metabolic genes is often controlled by global regulators. For instance, FruR acts as a transcriptional activator of the fru operon in V. cholerae and is essential for growth on fructose as a carbon source .

In V. vulnificus, the expression of fbp is influenced by different promoter systems:

These data indicate that optimizing promoter selection is crucial for recombinant expression studies of V. vulnificus fbp .

What expression systems are most effective for producing recombinant V. vulnificus Fbp?

The effectiveness of expression systems for V. vulnificus Fbp production depends on research objectives. Current evidence suggests:

• Heterologous Expression in E. coli: The pBD31 vector system has been successfully used for Fbp expression, with the enzyme effectively expressed as demonstrated by SDS-PAGE analysis showing a distinct band at 36.94 kDa .

• Inducible Promoter Systems: The PLacO1-lacIq promoter system demonstrates superior expression compared to the native promoter. This IPTG-inducible system allows for controlled expression timing and higher protein yields.

• Broad-Host-Range Plasmids: For expression within Vibrio species, broad-host-range plasmids containing replication origins from pBBR1 have been effective, allowing for both complementation studies and overexpression experiments.

The most efficient protocol involves:

• Cloning the fbp gene (GenBank: AMO50847.1) into an appropriate expression vector

• Transformation into the expression host

• Induction with 1 mM IPTG

• Cell harvesting after overnight induction

• Protein purification via affinity chromatography

This approach has yielded functional recombinant Fbp that can restore the defects observed in fbp deletion mutants.

How can recombinant V. vulnificus Fbp activity be accurately measured?

Accurate measurement of recombinant V. vulnificus Fbp activity requires specific assay conditions that account for its unique properties. A standard protocol includes:

• Spectrophotometric Assay: Coupling the Fbp reaction with phosphoglucose isomerase and glucose-6-phosphate dehydrogenase, monitoring NADPH formation at 340 nm.

• Optimal Reaction Conditions:

  • Temperature: 30-37°C (V. vulnificus optimal growth temperature)

  • pH: 7.0-8.0 (marine environment pH range)

  • Buffer: HEPES or Tris-HCl with Mg²⁺ as a cofactor

  • Substrate concentration: 0.1-1.0 mM fructose 1,6-bisphosphate

• Activity Calculation:

  • Enzyme activity (U) = (ΔA340/min) × reaction volume × dilution factor / (6.22 × enzyme volume)

  • Specific activity = U/mg protein

• Important Considerations:

  • Include phosphatase inhibitors to prevent non-specific hydrolysis

  • Test for allosteric regulation by AMP and fructose 2,6-bisphosphate

  • Assess the effects of salt concentration, as V. vulnificus is halophilic

Research has shown that inorganic phosphate can affect enzyme-substrate interactions in Vibrio species, as demonstrated in studies with related enzymes .

How can gene knockout and complementation studies be designed to investigate the role of fbp in V. vulnificus virulence?

To investigate fbp's role in V. vulnificus virulence, a systematic approach is necessary:

Knockout Strategy:

• Generate a precise fbp deletion mutant using homologous recombination or CRISPR-Cas9

• Confirm deletion via PCR and sequencing

• Verify protein absence via Western blot

Complementation Strategy:

• Clone the wild-type fbp gene into a broad-host-range vector (e.g., pBD31-fbp)

• Transform the construct into the Δfbp mutant

• Confirm expression via RT-PCR and Western blot

Phenotypic Analysis:

• Compare growth rates in different carbon sources

• Assess biofilm formation capacity

• Measure exopolysaccharide production

• Evaluate adherence to epithelial cells

• Test cytotoxicity using human cell lines

• Measure virulence in mouse models

This approach has been successful in related studies examining the role of NtrC-regulated exopolysaccharides in V. vulnificus biofilm formation and pathogenicity . Similarly structured experiments showed that mutations in genes involved in exopolysaccharide production resulted in decreased EPS production, attenuated biofilm formation, and lowered cytoadherence to human epithelial cells.

What approaches can be used to study the interaction between V. vulnificus Fbp and other metabolic enzymes?

Understanding the interaction between V. vulnificus Fbp and other metabolic enzymes requires multiple complementary approaches:

• Protein-Protein Interaction Studies:

  • Pull-down assays with tagged recombinant Fbp

  • Bacterial two-hybrid system

  • Co-immunoprecipitation followed by mass spectrometry

• Metabolic Flux Analysis:

  • ¹³C-labeled substrate tracing

  • Quantification of glycolytic and gluconeogenic intermediates

  • Comparison between wild-type and Δfbp strains

• Structural Studies:

  • X-ray crystallography of Fbp alone and in complex with interacting proteins

  • Molecular docking simulations

  • Site-directed mutagenesis of potential interaction sites

Research with related enzymes in V. vulnificus has revealed important metabolic interactions. For example, studies have shown that histidine phosphocarrier protein (HPr) interacts with pyruvate kinase A (PykA) in V. vulnificus, regulating glycolytic activity in response to glucose availability . This interaction was strictly dependent on inorganic phosphate, and only dephosphorylated HPr interacted with PykA. Similar mechanisms might regulate Fbp activity.

A particularly interesting experiment showed that dephosphorylated HPr decreased the Km of PykA for phosphoenolpyruvate by approximately fourfold without affecting Vmax, demonstrating how protein-protein interactions can fine-tune metabolic enzyme kinetics in Vibrio species .

How does V. vulnificus Fbp differ structurally and functionally from homologous enzymes in other pathogens?

V. vulnificus Fbp shows both conservation and divergence when compared to homologous enzymes in other pathogens:

Structural Comparisons:

• V. vulnificus Fbp is approximately 36.94 kDa , similar to other bacterial Fbps

• Sequence analysis reveals key catalytic residues are conserved

• Unique surface residues may reflect adaptation to marine environments

Functional Differences:

• Metal ion requirements may differ from terrestrial pathogens

• Allosteric regulation patterns show adaptation to fluctuating marine conditions

• Temperature and salt optima reflect V. vulnificus' halophilic nature

Evolutionary Context:
Phylogenetic analysis of Fbp sequences from various Vibrio species shows clustering that reflects evolutionary relationships within the genus. V. vulnificus Fbp is more closely related to those from other marine Vibrios than to enzymes from enteric pathogens like E. coli.

Studies of other metabolic enzymes in V. vulnificus provide context for these differences. For example, the fermentation-respiration switch (FrsA) protein in V. vulnificus was incorrectly reported to catalyze cofactor-independent decarboxylation of pyruvate, but subsequent quantum mechanical/molecular mechanical calculations demonstrated that its catalytic properties are distinct from those predicted . This highlights the importance of rigorous biochemical characterization of V. vulnificus enzymes rather than relying solely on homology-based predictions.

What role does Fbp play in V. vulnificus adaptation to different environmental conditions?

Fbp plays a critical role in V. vulnificus adaptation to changing environments, particularly in transitioning between nutrient-rich and nutrient-poor conditions:

Environmental Adaptation Mechanisms:

• Nutrient Switching: Fbp enables growth on non-carbohydrate carbon sources when glucose is unavailable, critical for survival in diverse marine and host environments.

• Biofilm Formation: Fbp contributes to exopolysaccharide production, which is essential for biofilm development. Studies have shown that EPS production by V. vulnificus is crucial for biofilm formation and pathogenic interaction .

• Host Interaction: The ability to utilize host-derived nutrients depends on gluconeogenic enzymes including Fbp. During infection, V. vulnificus must adapt to varying nutrient conditions within the host.

Experimental Evidence:
Research has demonstrated that NtrC-regulated exopolysaccharides, which depend on proper carbohydrate metabolism, are crucial for V. vulnificus biofilm formation. Biofilm formation and EPS production increased dramatically in media containing tricarboxylic acid cycle intermediates as carbon sources . Given Fbp's central role in gluconeogenesis, it likely contributes significantly to this phenotype.

Regulatory Networks:
The regulation of fbp gene expression appears to be integrated with sensing environmental nitrogen and carbon availability. This is similar to the regulation observed in the fru operon in V. cholerae, where FruR acts as a transcriptional activator in response to metabolic signals .

How can structural information about V. vulnificus Fbp be used for potential inhibitor design?

Structural information about V. vulnificus Fbp provides valuable insights for inhibitor design strategies:

Structure-Based Drug Design Approach:

• Catalytic Site Targeting:

  • Identify unique features of the V. vulnificus Fbp active site

  • Design transition-state analogs specific to V. vulnificus Fbp

  • Focus on competitive inhibitors that mimic substrate or product

• Allosteric Site Targeting:

  • Identify regulatory sites unique to V. vulnificus Fbp

  • Design molecules that lock the enzyme in inactive conformation

  • Target species-specific protein-protein interaction interfaces

• Computational Methods:

  • Homology modeling based on related Fbp structures

  • Virtual screening of compound libraries

  • Molecular dynamics simulations to identify flexible regions

Experimental Validation Pipeline:

• In vitro enzyme inhibition assays

• Cellular assays measuring effects on V. vulnificus growth

• Cytotoxicity testing against human cells

• Animal model efficacy testing

This approach has been successful with other V. vulnificus targets. For example, researchers have utilized structural information for the development of glycoconjugate vaccines against V. vulnificus, targeting the tetrasaccharide repeating units of V. vulnificus capsular polysaccharides . Similar principles could be applied to Fbp inhibitor design.

How should contradictory data on V. vulnificus Fbp function be interpreted and resolved?

When facing contradictory data on V. vulnificus Fbp function, a systematic approach to interpretation and resolution is essential:

Sources of Contradictions:

• Strain Variations: V. vulnificus is known to have significant genetic diversity. Studies have identified different biotypes and genotypes with varying virulence properties .

• Experimental Conditions: Different buffer compositions, salt concentrations, and pH can significantly affect enzyme activity measurements.

• Recombinant Protein Production Methods: Expression systems, purification protocols, and protein tags can influence enzyme properties.

Resolution Strategy:

• Standardize Methodologies:

  • Use the same buffer systems, pH, and temperature

  • Employ identical assay methods for enzyme activity

  • Standardize protein purification protocols

• Cross-Laboratory Validation:

  • Exchange strains and plasmids between laboratories

  • Perform blinded experiments to reduce bias

  • Collaborate on joint publications to resolve discrepancies

• Advanced Characterization:

  • Perform comprehensive kinetic analyses

  • Determine the effects of all potential regulators

  • Conduct structural studies to identify conformational states

Case Example:
A similar approach was used to resolve contradictions regarding V. vulnificus FrsA function. Initial reports suggested it functioned as a cofactor-independent pyruvate decarboxylase, but subsequent quantum mechanical/molecular mechanical calculations contradicted this assignment. Further experimental validation confirmed that the initial functional annotation was incorrect . This case demonstrates the importance of combining computational, structural, and kinetic evidence to resolve contradictory findings.

What bioinformatic approaches can identify potential regulatory elements controlling V. vulnificus fbp expression?

Comprehensive bioinformatic analysis can identify potential regulatory elements controlling V. vulnificus fbp expression:

Sequence-Based Approaches:

• Promoter Analysis:

  • Identify -10 and -35 regions upstream of the fbp gene

  • Search for binding sites of known transcription factors

  • Compare promoter sequences across Vibrio species

• Transcription Factor Binding Site Prediction:

  • Use position weight matrices of known bacterial regulators

  • Apply machine learning algorithms trained on validated binding sites

  • Perform comparative genomics across related species

• RNA Structure Analysis:

  • Predict secondary structures in the 5' UTR of fbp mRNA

  • Identify potential riboswitches or attenuators

  • Analyze for sRNA binding sites

Integration with Experimental Data:

• RNA-seq data to identify co-regulated genes

• ChIP-seq for direct identification of transcription factor binding

• EMSA to validate predicted binding sites

Example Analysis Workflow:
In V. cholerae, researchers identified that FruR acts as a transcriptional activator of the fru operon . A similar approach combining computational predictions with experimental validation could be applied to V. vulnificus fbp regulation. The native promoter of fbp (285 bp) has been successfully isolated and characterized in previous studies , providing a starting point for regulatory element analysis.

The recent advances in rapid detection methods for V. vulnificus, such as CRISPR-based approaches and real-time recombinant polymerase amplification , can be adapted to study gene expression in response to different environmental conditions, providing validation for bioinformatic predictions.