Recombinant Vibrio vulnificus Phosphoribosylglycinamide formyltransferase 2 (purT)

What is the basic function of Phosphoribosylglycinamide formyltransferase 2 (purT) in Vibrio vulnificus?

Phosphoribosylglycinamide formyltransferase 2 (purT) in V. vulnificus is a critical enzyme in the de novo purine biosynthesis pathway, catalyzing the formylation of 5-phosphoribosylglycinamide (GAR) to produce 5-phosphoribosyl-N-formylglycinamide (FGAR). Unlike the folate-dependent purN pathway, purT utilizes formate and ATP to generate formyl phosphate as the formyl donor. This represents an alternative pathway for purine biosynthesis in V. vulnificus that may be particularly important under specific environmental conditions. Similar to other virulence factors in V. vulnificus, purT expression is likely regulated by growth phase and environmental conditions, potentially reaching maximum expression during early stationary phase .

How do I design primers for cloning Vibrio vulnificus purT gene?

When designing primers for V. vulnificus purT gene cloning, consider the following methodological approach:

• Obtain the complete gene sequence from genomic databases, noting that V. vulnificus has multiple biotypes with potential sequence variations

• Design primers with the following features:

  • 18-25 nucleotides length with 40-60% GC content

  • 5' extensions containing appropriate restriction sites compatible with your expression vector

  • 4-6 nucleotide overhangs before restriction sites to facilitate enzyme binding

  • Melting temperatures within 5°C of each other (typically 55-65°C)

  • Verification for absence of secondary structures using tools like OligoAnalyzer

This approach is similar to strategies used for cloning other V. vulnificus genes like plpA, which required careful primer design to ensure proper expression in recombinant systems .

What expression systems work best for producing recombinant V. vulnificus purT?

For optimal recombinant expression of V. vulnificus purT, consider these methodological insights:

Prokaryotic Systems:

• E. coli BL21(DE3) with pET vectors typically yields high expression levels

• Growth at lower temperatures (16-25°C) after induction prevents inclusion body formation

• Codon optimization may be necessary as V. vulnificus has different codon usage than E. coli

Expression Conditions Table:

What are the key structural features of V. vulnificus purT and how do they compare to homologs?

V. vulnificus purT possesses several distinctive structural features that influence its function:

The enzyme consists of two major domains: an N-terminal formyltransferase domain containing the active site and a C-terminal ATP-binding domain. The active site includes conserved residues for substrate binding and catalysis, notably arginine and histidine residues that coordinate the formate substrate. Compared to E. coli purT (the most well-characterized homolog), V. vulnificus purT shares approximately 65-70% sequence identity but may contain unique surface-exposed residues that affect protein stability under marine environmental conditions.

An important structural consideration in V. vulnificus proteins is adaptation to varying salinity and temperature conditions found in marine environments. This may be reflected in surface charge distribution and solvent-exposed residues that differ from terrestrial bacterial homologs. Similar adaptations have been observed in other V. vulnificus enzymes where environmental factors influence protein structure and function .

How can I determine the kinetic parameters of recombinant V. vulnificus purT?

For accurate kinetic characterization of V. vulnificus purT, a comprehensive methodological approach includes:

• Enzyme Activity Assay Setup:

  • Use a spectrophotometric coupled assay tracking ADP formation via pyruvate kinase and lactate dehydrogenase (monitoring NADH oxidation at 340nm)

  • Alternative: direct HPLC-based assay quantifying FGAR formation

  • Include controls with heat-inactivated enzyme

• Kinetic Parameter Determination:

  • Measure initial reaction velocities with varying concentrations of substrates (GAR, formate, ATP)

  • Plot data using Michaelis-Menten, Lineweaver-Burk, or non-linear regression analyses

  • Calculate Km and kcat values for each substrate

  • Assess potential cooperative binding effects

• Environmental Variable Testing:

  • Examine pH dependence (range 6.0-9.0)

  • Test temperature dependence (20-40°C)

  • Evaluate salt concentration effects (0-500mM NaCl)

  • Measure divalent cation requirements (Mg2+, Mn2+)

This approach parallels methodologies used for characterizing other V. vulnificus enzymes, where environmental factors significantly influence enzymatic activity .

What techniques are most effective for determining purT protein-protein interactions in V. vulnificus?

For investigating purT protein-protein interactions in V. vulnificus, employ these methodological approaches:

In vitro techniques:

• Pull-down assays using His-tagged recombinant purT with V. vulnificus cell lysates

• Surface plasmon resonance (SPR) to determine binding kinetics with candidate proteins

• Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions

In vivo approaches:

• Bacterial two-hybrid systems adapted for V. vulnificus

• Co-immunoprecipitation from bacterial lysates using anti-purT antibodies

• Proximity-dependent biotin identification (BioID) with purT as the bait protein

When designing protein interaction experiments, consider that purT may interact with other purine biosynthesis enzymes, forming a metabolic complex. Additionally, regulatory proteins like CRP (cAMP receptor protein) may interact with purT or its promoter region, as CRP has been shown to regulate various metabolic pathways in V. vulnificus, including the putAP operon .

How is the purT gene regulated in Vibrio vulnificus?

The regulation of purT in V. vulnificus likely involves multiple mechanisms based on patterns observed with other metabolic genes:

• Transcriptional Regulation:

  • Purine-responsive repression similar to PurR-mediated regulation in other bacteria

  • Growth phase-dependent expression, potentially peaking during early stationary phase

  • Involvement of global regulators such as cAMP receptor protein (CRP), which has been demonstrated to regulate metabolic operons in V. vulnificus

  • Possible regulation by the HlyU transcription factor, which activates various virulence factors in V. vulnificus

• Environmental Response Elements:

  • Temperature-responsive elements in the promoter region, given V. vulnificus' sensitivity to environmental temperatures

  • Oxygen-responsive regulation, potentially through FNR or similar regulators

  • Host environment sensing mechanisms, as seen with other V. vulnificus genes that show host-dependent induction

The purT gene may be co-regulated with other purine biosynthesis genes in a manner similar to the putAP operon, which shows cooperative binding of transcription factors to overlapping sites . Molecular biological analyses would be required to determine if purT expression is controlled via a specific promoter with binding sites for multiple regulators, as observed with plpA in V. vulnificus .

What are the best methods for studying purT expression under different environmental conditions?

To comprehensively analyze purT expression under varying conditions, implement this methodological framework:

Transcriptional Analysis:

• Construct purT promoter-reporter fusions (using lacZ or gfp) to monitor expression in real-time

• Perform quantitative RT-PCR to measure purT mRNA levels under different conditions

• Employ RNA-seq for global transcriptional analysis, comparing purT expression with other genes

Environmental Conditions to Test:

• Temperature variations (15-40°C) relevant to marine and host environments

• Oxygen levels (aerobic, microaerobic, anaerobic)

• Nutrient availability (carbon sources, purine precursors, amino acids)

• Host-mimicking conditions (serum exposure, epithelial cell co-culture)

• Various salinities relevant to estuarine environments

• Growth phases (logarithmic versus stationary)

Data Analysis Approach:

• Normalize expression against housekeeping genes stable under tested conditions

• Perform statistical analyses to identify significant changes

• Create correlation analyses between purT expression and physiological parameters

This approach parallels methods used for studying transcriptome changes in V. vulnificus after infection of human intestinal cells, which revealed induction patterns of various genes including plpA . The methodology should account for the potential growth phase-dependent nature of expression, as documented for other V. vulnificus genes .

How does purT expression correlate with V. vulnificus virulence?

The relationship between purT expression and V. vulnificus virulence requires investigation through these methodological approaches:

• Comparative Expression Analysis:

  • Compare purT expression levels between clinical (C-type) and environmental (E-type) V. vulnificus isolates

  • Analyze expression during human cell infection models using RT-qPCR

  • Correlate expression with established virulence factors like RtxA1 toxin

• Mutational Studies:

  • Generate purT deletion mutants and assess virulence in cell culture models

  • Measure cytotoxicity toward human epithelial cells (e.g., INT-407) with wild-type versus purT mutants

  • Evaluate bacterial survival in human serum and resistance to phagocytosis

• Host Response Evaluation:

  • Analyze neutrophil and macrophage responses to wild-type versus purT mutants

  • Measure inflammatory cytokine production in response to infection

  • Assess survival in mouse models of infection

While purT itself is not a classical virulence factor like RtxA1 or VvPlpA , its role in purine biosynthesis may be crucial for bacterial survival in the host environment where purines can be limited. The approach should consider that purine biosynthesis may be particularly important during systemic infection, where V. vulnificus must proliferate rapidly in the bloodstream .

What CRISPR-based approaches can be used to study purT function in V. vulnificus?

For CRISPR-based manipulation of V. vulnificus purT, consider these methodological strategies:

Gene Knockout and Modification:

• Design sgRNAs targeting purT with minimal off-target effects using V. vulnificus genome-specific tools

• Optimize CRISPR-Cas9 delivery using conjugation with an appropriate broad-host-range vector

• Employ homology-directed repair templates to introduce specific mutations in catalytic residues rather than complete gene deletion

• Create conditional knockdowns using CRISPRi (dCas9) systems when complete deletion may be lethal

Regulatory Element Analysis:

• Use CRISPR-Cas9 to delete or modify putative regulatory regions upstream of purT

• Create promoter fusions with reporter genes to monitor effects of CRISPR-mediated modifications

• Implement CRISPRa systems to artificially induce purT expression

Validation and Phenotypic Analysis:

• Confirm modifications using PCR, sequencing, and Western blotting

• Assess growth curves in minimal versus rich media to determine purine auxotrophy

• Analyze competitive fitness against wild-type strains in various conditions

• Evaluate virulence changes in cell culture and animal models

When designing CRISPR experiments, consider potential polar effects on adjacent genes and implement appropriate controls. The approach should be informed by molecular biological analyses similar to those used for studying other V. vulnificus genes like plpA, where precise genetic manipulations revealed regulatory mechanisms .

How can I develop specific inhibitors targeting V. vulnificus purT for research applications?

For developing research-grade inhibitors of V. vulnificus purT, implement this structure-based drug design approach:

• Target Identification and Validation:

  • Perform computational analysis of active site architecture

  • Identify unique structural features distinguishing V. vulnificus purT from human purine metabolism enzymes

  • Validate the essentiality of purT under relevant conditions

• Screening and Design Strategy:

  • Conduct in silico screening of compound libraries against homology models

  • Design transition-state analogs that mimic the formylation reaction

  • Focus on compounds targeting the ATP-binding pocket or formate-binding site

• Compound Testing Workflow:

  • Implement biochemical assays measuring purT activity inhibition

  • Determine IC50 and Ki values for promising compounds

  • Assess specificity by testing against related enzymes

  • Evaluate antibacterial activity against V. vulnificus and cytotoxicity to human cells

• Structural Optimization:

  • Utilize structure-activity relationship (SAR) studies to improve potency

  • Employ crystallography or cryo-EM to visualize inhibitor binding

  • Optimize physicochemical properties for research applications

This approach should consider that successful inhibitors may serve as chemical probes for investigating purT function in V. vulnificus pathogenesis, similar to how studies of other virulence factors like RtxA1 have enhanced understanding of V. vulnificus disease mechanisms .

What systems biology approaches can integrate purT function with global metabolic networks in V. vulnificus?

For integrating purT into the broader metabolic context of V. vulnificus, employ these systems biology methodologies:

• Genome-Scale Metabolic Modeling:

  • Develop or refine existing V. vulnificus metabolic models to accurately represent purine biosynthesis

  • Perform flux balance analysis to predict metabolic shifts during purT perturbation

  • Identify synthetic lethal interactions with purT through in silico gene deletions

• Multi-Omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and purT mutants

  • Analyze samples from various growth conditions and infection models

  • Use computational tools to construct regulatory networks connecting purT with other metabolic pathways

• Experimental Validation Approaches:

  • Validate key predictions through targeted metabolite quantification

  • Perform isotope labeling experiments to track metabolic flux through purT-dependent pathways

  • Create and phenotype double mutants of purT with predicted interacting genes

• Network Analysis:

  • Construct protein-protein interaction networks centered on purT

  • Perform pathway enrichment analysis to identify processes affected by purT dysfunction

  • Compare metabolic adaptations in clinical versus environmental isolates

This systems-level approach should account for the complex regulatory mechanisms in V. vulnificus, which often involve multiple transcription factors acting on the same promoter, as observed with the putAP operon regulation by CRP and PutR .

How does V. vulnificus purT differ structurally and functionally from homologs in other pathogenic bacteria?

The structural and functional differences of V. vulnificus purT compared to homologs in other pathogens reveal important evolutionary adaptations:

• Sequence and Structural Comparisons:

  • V. vulnificus purT shares approximately 80-85% amino acid identity with V. cholerae homologs, but only 65-70% with E. coli

  • Key differences may exist in the flexible loop regions that control substrate access to the active site

  • Marine bacterial purT enzymes like those in Vibrio species typically have adaptations for salt tolerance and temperature flexibility

• Functional Distinctions:

  • Kinetic parameters likely show adaptation to the marine environment with potentially broader temperature optima

  • Substrate specificity may differ slightly, particularly regarding alternative formyl donors

  • Allosteric regulation mechanisms may be unique to accommodate V. vulnificus metabolic networks

• Evolutionary Context:

  • purT represents an ATP-dependent alternative to the folate-dependent purN pathway

  • Different pathogens show varying reliance on purT versus purN pathways for purine biosynthesis

  • Gene arrangement and operon structure around purT varies between bacterial species, affecting co-regulation with other genes

These comparative insights align with observations about other V. vulnificus proteins, which often show adaptations to the dual lifestyle of this organism in both marine environments and human hosts .

What techniques are best for analyzing purT phylogeny across Vibrio species?

For robust phylogenetic analysis of purT across Vibrio species, implement this methodological framework:

• Sequence Acquisition and Alignment:

  • Collect purT sequences from diverse Vibrio species and strains using BLAST searches against genomic databases

  • Include purT sequences from clinical and environmental isolates of V. vulnificus to assess intra-species variation

  • Perform multiple sequence alignment using MUSCLE or MAFFT with iterative refinement

  • Clean alignments to remove poorly aligned regions using Gblocks or TrimAl

• Phylogenetic Tree Construction:

  • Implement maximum likelihood methods (RAxML or IQ-TREE) with appropriate evolutionary models

  • Perform Bayesian inference (MrBayes) as a complementary approach

  • Use bootstrapping (1000 replicates) to assess node support

  • Root trees with appropriate outgroups from other gamma-proteobacteria

• Evolutionary Analysis:

  • Calculate dN/dS ratios to detect selection pressure on purT

  • Identify sites under positive selection using methods like PAML

  • Perform reconciliation analysis comparing purT tree with species phylogeny to detect horizontal gene transfer events

  • Conduct comparative analysis of regulatory regions upstream of purT

This approach can reveal evolutionary patterns similar to those observed in V. vulnificus genotyping studies, where clinical isolates show distinct patterns from environmental isolates , potentially reflecting adaptation to different ecological niches.

How does horizontal gene transfer influence purT evolution in V. vulnificus populations?

To investigate horizontal gene transfer (HGT) of purT in V. vulnificus populations, employ these methodological strategies:

• Genomic Context Analysis:

  • Examine regions flanking purT for mobile genetic elements, insertion sequences, or abnormal GC content

  • Compare synteny of purT and surrounding genes across multiple V. vulnificus isolates

  • Identify potential recombination hotspots near the purT locus

• Population Genomics Approaches:

  • Analyze purT sequences from diverse geographical isolates and clinical versus environmental strains

  • Implement recombination detection programs (RDP, ClonalFrameML) to identify breakpoints

  • Calculate linkage disequilibrium between purT and neighboring genes

• Comparative Evolutionary Rates:

  • Compare evolutionary rates of purT with housekeeping genes to detect accelerated evolution

  • Analyze codon usage patterns for evidence of recent transfer events

  • Perform Bayesian dating analysis to estimate timing of potential HGT events

• Experimental Verification:

  • Test natural transformation frequencies using labeled purT alleles

  • Assess whether stress conditions increase HGT rates of metabolic genes

  • Determine if clinical isolates show different HGT patterns than environmental isolates