Recombinant Vibrio vulnificus Aspartate carbamoyltransferase regulatory chain (pyrI)

What is the role of the pyrI gene in Vibrio vulnificus compared to the extensively studied pyrH gene?

The pyrI gene in V. vulnificus encodes the regulatory chain of aspartate carbamoyltransferase, a key enzyme in pyrimidine biosynthesis, whereas the pyrH gene encodes UMP kinase that catalyzes UMP phosphorylation. While pyrH has been extensively characterized as essential for V. vulnificus in vivo survival and virulence , pyrI remains less studied. Methodologically, comparative genome analysis of clinical and environmental V. vulnificus strains reveals that both genes are part of the pyrimidine biosynthetic pathway, but they function at different steps. Researchers should approach pyrI characterization through complementation studies similar to those conducted with pyrH, where site-directed mutants can determine essential functional domains.

How does the structure of V. vulnificus pyrI compare to the characterized pyrI in V. cholerae?

Based on computational structure models available through resources like RCSB PDB (AF_AFC3LR52F1 for V. cholerae pyrI) , V. vulnificus pyrI likely maintains similar structural characteristics with high confidence regions (pLDDT >70). Methodologically, researchers should perform structural alignment analysis between the predicted V. vulnificus pyrI and the V. cholerae protein, focusing particularly on the allosteric binding sites and regulatory interfaces. Sequence identity clustering at various thresholds (30%, 50%, 70%, 90%, 95%, and 100%) can identify conserved domains that may be critical for function across Vibrio species.

What are the optimal conditions for heterologous expression of recombinant V. vulnificus pyrI?

For optimal expression of recombinant V. vulnificus pyrI, researchers should consider:

• Expression system selection: E. coli BL21(DE3) strains typically yield high expression of Vibrio proteins

• Vector optimization: Incorporate a 6×His-tag for purification, preferably at the N-terminus to avoid interfering with C-terminal functional domains

• Induction parameters:

  • IPTG concentration: 0.1-0.5 mM

  • Post-induction temperature: 18-25°C (lower temperatures often improve folding)

  • Duration: 4-16 hours

Importantly, iron availability in expression media should be controlled as V. vulnificus proteins often show iron-dependent expression patterns, similar to what has been observed in environmental versus iron-replete conditions for other Vibrio proteins .

What purification challenges are specific to V. vulnificus pyrI and how can they be addressed?

Purification of V. vulnificus pyrI presents several challenges:

Based on pyrI characterization in other Vibrio species, researchers should verify the quaternary structure post-purification, as functional pyrI typically forms dimers that associate with the catalytic subunits (pyrB) .

How does pyrI in V. vulnificus interact with the catalytic subunit pyrB to regulate aspartate carbamoyltransferase activity?

The interaction between pyrI (regulatory) and pyrB (catalytic) subunits in V. vulnificus likely follows the allosteric regulation model seen in other bacteria, but with pathogen-specific adaptations. To characterize this:

• Co-immunoprecipitation: Use anti-pyrI antibodies to pull down the complete enzyme complex

• Enzyme kinetics: Measure aspartate carbamoyltransferase activity with varying concentrations of:

  • Substrates (carbamoyl phosphate and aspartate)

  • Allosteric effectors (CTP as inhibitor, ATP as activator)

• Surface plasmon resonance: Quantify binding affinities between purified pyrI and pyrB

Researchers should compare these interactions between clinical and environmental isolates, as differences in pyrI regulation might contribute to strain-specific virulence, similar to observations made with the pyrH gene in V. vulnificus .

What experimental approaches are most effective for measuring pyrI-mediated allosteric regulation in V. vulnificus?

To effectively measure pyrI-mediated allosteric regulation:

• Colorimetric assays: Monitor carbamoyl aspartate formation using the colorimetric reaction with antipyrine and diacetyl monoxime

• Isothermal titration calorimetry (ITC): Determine thermodynamic parameters of nucleotide binding to pyrI

• Site-directed mutagenesis: Create mutations in predicted effector binding sites and measure changes in:

  • CTP inhibition constants

  • ATP activation profiles

  • Cooperativity (Hill coefficient)

Researchers should specifically investigate whether V. vulnificus pyrI shows altered regulatory responses in conditions that mimic the host environment (37°C, iron-rich, serum-containing media) compared to environmental conditions, as adaptive regulation may contribute to pathogenicity .

Is pyrI essential for V. vulnificus survival in vivo similar to pyrH?

While pyrH has been definitively shown to be essential for V. vulnificus in vivo survival , the essentiality of pyrI requires similar methodical investigation:

• Construction of conditional mutants: Use inducible promoters to control pyrI expression

• In vivo competition assays: Compare wild-type and pyrI-attenuated strains in mouse models (both normal and iron-overloaded)

• Complementation studies: Restore function with wild-type pyrI to confirm phenotype specificity

Key experimental considerations should include:

• Monitoring bacterial recovery from blood and tissues at multiple time points (3h, 6h, 9h, 24h)

• Measuring cytotoxicity in human cell lines

• Assessing growth in human serum and ascitic fluid

Based on pyrH studies, researchers should pay particular attention to the role of pyrI in iron-overloaded conditions, which significantly affect V. vulnificus virulence .

How does pyrI expression differ between clinical and environmental V. vulnificus strains?

To characterize pyrI expression differences:

• qRT-PCR analysis: Compare pyrI transcript levels between:

  • Clinical (C-genotype) versus environmental (E-genotype) strains

  • Various growth conditions (iron-replete/depleted, different temperatures, oxygen levels)

  • During infection versus environmental persistence

• Reporter gene assays: Construct pyrI promoter fusions with reporters like GFP or luciferase

• RNA-seq analysis: Identify co-regulated genes in the pyrI regulatory network

This approach is supported by genomic studies that have identified 278 genes specifically associated with clinical genotypes versus 167 genes with environmental genotypes in V. vulnificus . Understanding whether pyrI expression patterns correlate with these genotypic differences could provide insights into its role in pathogenicity.

How conserved is pyrI across Vibrio species and what does this suggest about its evolutionary importance?

To assess pyrI conservation:

• Phylogenetic analysis: Construct maximum-likelihood trees of pyrI sequences from:

  • Different V. vulnificus strains (clinical vs. environmental)

  • Related Vibrio species (V. cholerae, V. parahaemolyticus)

  • More distant gamma-proteobacteria

• Selection pressure analysis: Calculate dN/dS ratios to determine whether pyrI is under purifying or diversifying selection

• Synteny analysis: Examine conservation of the genomic context surrounding pyrI

The recA-pyrH metabarcoding approach has been successfully used to characterize Vibrio communities , suggesting that pyrH is sufficiently conserved for taxonomic purposes. Researchers should determine whether pyrI shows similar conservation patterns, which would indicate functional importance across Vibrio evolution.

What are the key structural differences in pyrI between clinical and environmental V. vulnificus strains?

To identify structural differences:

• Homology modeling: Generate structural models of pyrI from multiple strains using tools like AlphaFold

• Structural alignment: Compare predicted structures focusing on:

  • Allosteric binding pockets

  • Interfaces with pyrB (catalytic subunit)

  • Regions with highest sequence divergence

• Molecular dynamics simulations: Assess how strain-specific differences affect:

  • Protein stability

  • Nucleotide binding dynamics

  • Allosteric signal transmission

This comparative approach can reveal adaptations in pyrI that might contribute to the different ecological niches occupied by clinical versus environmental V. vulnificus strains, as observed in previous genomic studies .

What are the most effective genetic manipulation strategies for studying pyrI function in V. vulnificus?

For genetic manipulation of pyrI:

• CRISPR-Cas9 approach:

  • Design sgRNAs targeting conserved regions of pyrI

  • Include homology arms for precise gene editing

  • Incorporate counterselection markers

• Allelic exchange methods:

  • Use suicide vectors (e.g., pDM4) carrying pyrI variants

  • Select for double recombination events

  • Verify clean mutations by sequencing

• Conditional expression systems:

  • Arabinose-inducible promoters work effectively in Vibrio species

  • Temperature-sensitive replicons provide temporal control

Given the potential essentiality of pyrI, researchers should consider the approach used for pyrH studies, where site-directed mutants (e.g., R62H/D77N) affecting substrate binding were created rather than complete deletions .

How can researchers effectively validate the specificity of antibodies against V. vulnificus pyrI?

To validate antibody specificity:

• Cross-reactivity testing:

  • Test against recombinant pyrI from multiple Vibrio species

  • Include closely related proteins in the pyrimidine biosynthesis pathway

  • Use pyrI knockout/knockdown strains as negative controls

• Epitope mapping:

  • Identify immunodominant regions using peptide arrays

  • Generate antibodies against multiple epitopes for confirmation

  • Verify accessibility of epitopes in native protein

• Application-specific validation:

  • Western blot: Verify single band of correct molecular weight

  • Immunoprecipitation: Confirm pull-down of pyrI-interacting partners

  • Immunofluorescence: Compare localization patterns with tagged pyrI constructs

This rigorous validation is especially important when studying Vibrio species, as they often contain highly homologous proteins that can lead to antibody cross-reactivity.

How do post-translational modifications affect pyrI function in V. vulnificus under different environmental conditions?

Advanced investigation of post-translational modifications (PTMs) should include:

• Mass spectrometry analysis:

  • Compare PTM profiles between pyrI isolated from:

    • Bacteria grown at different temperatures (25°C vs. 37°C)

    • Iron-replete versus iron-depleted conditions

    • Bacteria recovered from infection models

• Site-directed mutagenesis:

  • Mutate identified PTM sites to non-modifiable residues

  • Create phosphomimetic mutations (S/T to D/E) to simulate phosphorylation

  • Assess effects on enzyme activity and regulation

• In vitro modification assays:

  • Identify kinases that phosphorylate pyrI

  • Test effects of oxidative stress on cysteine modifications

This approach is particularly relevant as V. vulnificus shows distinct metabolic adaptations in host versus environmental conditions , which may involve PTM-mediated regulation of key metabolic enzymes like pyrI.

What is the contribution of pyrI to metabolic adaptation during host infection compared to environmental persistence?

To investigate pyrI's role in metabolic adaptation:

• Metabolic flux analysis:

  • Compare pyrimidine pathway flux between wild-type and pyrI-attenuated strains

  • Measure incorporation of 13C-labeled precursors into pyrimidine nucleotides

  • Assess changes in flux under conditions mimicking:

    • Human serum

    • Ascitic fluid

    • Marine environments

• Transcriptome-metabolome integration:

  • Correlate pyrI expression levels with metabolite profiles

  • Identify metabolic networks affected by pyrI modulation

• In vivo imaging:

  • Use fluorescent biosensors to track pyrimidine metabolism in real-time during infection

This metabolic approach would complement findings on pyrH, which showed that mutants had significantly impaired growth in human serum and ascitic fluid, suggesting a critical role for the pyrimidine pathway during infection .

What approaches can be used to assess pyrI as a potential antimicrobial target against V. vulnificus?

To evaluate pyrI as a drug target:

• Target validation:

  • Determine essentiality through conditional knockdown

  • Assess virulence attenuation in pyrI-deficient strains

  • Verify conservation across clinical isolates

• High-throughput screening:

  • Develop assays measuring pyrI-dependent allosteric regulation

  • Screen compound libraries for inhibitors of:

    • pyrI-pyrB interaction

    • Nucleotide binding to regulatory site

    • Conformational changes upon effector binding

• Structure-based drug design:

  • Identify allosteric binding pockets unique to bacterial pyrI

  • Focus on regions divergent from human homologs

  • Use molecular docking to predict binding modes

This approach is supported by findings that pyrH is essential for V. vulnificus survival and has been proposed as "an attractive new target for the development of antibacterial drugs" .

How can researchers design pyrI inhibitors that are specific to pathogenic Vibrio species without affecting commensal bacteria?

For designing Vibrio-specific pyrI inhibitors:

• Comparative structural analysis:

  • Identify binding pockets unique to Vibrio pyrI

  • Focus on regions that differ from commensal bacteria

  • Target Vibrio-specific regulatory mechanisms

• Selectivity profiling:

  • Test candidate inhibitors against:

    • Multiple Vibrio species

    • Common gut microbiome species

    • Host cells

• In silico prediction of specificity:

  • Use machine learning to predict off-target binding

  • Model interactions with human metabolic enzymes

  • Assess potential for resistance development

This selective approach is crucial because broad-spectrum antibiotics can disrupt beneficial microbiota, and targeting pyrI in a Vibrio-specific manner could minimize this collateral damage while effectively treating V. vulnificus infections, which have mortality rates exceeding 50% for primary septicemia .