Recombinant Salmonella paratyphi C Glucose-6-phosphate isomerase (pgi), partial

What is Glucose-6-phosphate isomerase and what function does it serve in Salmonella paratyphi C?

Glucose-6-phosphate isomerase (PGI) catalyzes the reversible isomerization between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), playing a critical role in both glycolysis and gluconeogenesis. In Salmonella paratyphi C, PGI functions as a key metabolic enzyme mediating carbon flow through central metabolic pathways, which is essential for bacterial energy production, growth, and potentially virulence. The enzyme impacts cellular carbon metabolic flow by directing intermediates through either the glycolytic or pentose phosphate pathways .

How does Salmonella paratyphi C PGI relate structurally to homologs in other Salmonella species?

While specific structural data for S. paratyphi C PGI is limited, we can draw inferences based on related species. The protein sequence of PGI is relatively conserved across Salmonella species, with essential catalytic residues and substrate-binding domains showing high conservation. For instance, Salmonella paratyphi A PGI contains key structural motifs including "MKNINPTQTS AWQALQKHYD EMKDVTIAEL" at the N-terminus, which are likely present in S. paratyphi C PGI with high sequence identity . Genetic analysis shows S. paratyphi C is more closely related to S. choleraesuis (sharing 4346 genes, covering 96.66% of the S. paratyphi C genome) than to S. typhi (sharing only 4008 genes) , suggesting that S. paratyphi C PGI might have higher sequence similarity to S. choleraesuis PGI.

What are the predicted kinetic properties of Salmonella paratyphi C PGI?

While specific kinetic parameters for S. paratyphi C PGI are not directly reported in the literature, insights can be drawn from studies of PGI in other organisms. PGI enzymes typically exhibit different affinities for their substrates G6P and F6P. For example, in plant systems, the Km values for G6P and F6P can differ significantly, with some PGI isozymes showing higher affinity for F6P than G6P . For bacterial PGIs, the catalytic efficiency (kcat/Km) is generally high, reflecting their essential role in central metabolism. A comprehensive kinetic characterization of recombinant S. paratyphi C PGI would involve determining Km, Vmax, and kcat values for both forward and reverse reactions under varying pH and temperature conditions.

What are the optimal storage conditions for recombinant Salmonella paratyphi C PGI?

For optimal stability and activity retention, recombinant Salmonella paratyphi C PGI should be stored according to these guidelines:

• Liquid form: -20°C to -80°C with an expected shelf life of approximately 6 months

• Lyophilized form: -20°C to -80°C with an extended shelf life of approximately 12 months

The shelf life is influenced by multiple factors including storage state, buffer composition, storage temperature, and the intrinsic stability of the protein itself. Working aliquots may be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can significantly compromise protein integrity and enzymatic activity .

How should reconstitution of lyophilized recombinant PGI be performed for experimental applications?

For optimal reconstitution of lyophilized recombinant PGI:

• Briefly centrifuge the vial prior to opening to ensure all material is at the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being standard for long-term storage)

• Prepare small working aliquots to minimize freeze-thaw cycles

• Store reconstituted protein at -20°C to -80°C for long-term storage

This protocol helps maintain protein stability and enzymatic activity for downstream applications while minimizing degradation.

What factors influence the stability and activity of recombinant PGI during experimental procedures?

Several critical factors affect the stability and activity of recombinant PGI during experiments:

• Temperature: Protein denaturation can occur at elevated temperatures, while excessively low temperatures may reduce enzymatic activity

• pH: Most PGI enzymes have a defined pH optimum; deviations can affect catalytic efficiency and stability

• Buffer composition: Presence of specific ions, chelators, or reducing agents can significantly impact activity

• Protein concentration: Dilute solutions may be more prone to activity loss through surface adsorption

• Presence of proteases: Contaminating proteases can degrade the enzyme during experiments

• Substrate concentrations: Excessive substrate concentrations may cause substrate inhibition

• Product inhibition: Accumulation of reaction products may inhibit enzyme activity in prolonged assays

Researchers should optimize these parameters for their specific experimental conditions to maintain >85% enzymatic activity .

How can enzyme activity assays be designed for Salmonella paratyphi C PGI?

Designing robust activity assays for S. paratyphi C PGI requires consideration of several methodological approaches:

Direct Spectrophotometric Assay:

• Couple PGI reaction with glucose-6-phosphate dehydrogenase (G6PDH)

• Monitor NADPH production at 340 nm as G6P is oxidized to 6-phosphogluconolactone

• Calculate PGI activity based on the rate of NADPH formation

Reaction conditions:

• Buffer: Typically Tris-HCl (50 mM, pH 7.4-8.0)

• Required components: G6P or F6P (substrate), NADP+ (cofactor for coupled reaction), MgCl₂, G6PDH (coupling enzyme)

• Temperature: 25-37°C (optimize based on experimental goals)

• Controls: Include no-enzyme and no-substrate controls

Data analysis:

• Initial velocity should be measured in the linear range of the reaction

• Specific activity calculated as μmol substrate converted per minute per mg protein

• For kinetic parameters, vary substrate concentration and analyze data using Michaelis-Menten or Lineweaver-Burk plots

This coupled assay approach provides sensitive, real-time measurement of PGI activity suitable for both basic characterization and inhibitor screening applications.

What approaches can be used to study the role of PGI in Salmonella paratyphi C metabolism?

Several complementary approaches can elucidate the role of PGI in S. paratyphi C metabolism:

• Gene knockout/knockdown studies:

  • Create pgi deletion mutants or conditionally express pgi

  • Analyze growth characteristics in different carbon sources

  • Assess virulence in infection models

• Metabolic flux analysis:

  • Use 13C-labeled glucose to trace carbon flow through glycolysis and related pathways

  • Compare flux distributions between wild-type and pgi-modified strains

  • Identify metabolic adaptations in response to PGI perturbation

• Comparative genomics and transcriptomics:

  • Compare pgi gene context and regulation across Salmonella serovars

  • Analyze transcriptional responses under different growth conditions

  • Investigate co-expression patterns with other metabolic genes

• Protein-protein interaction studies:

  • Identify potential interaction partners using pull-down assays or bacterial two-hybrid systems

  • Investigate whether PGI participates in metabolic enzyme complexes

• In vivo imaging:

  • Use fluorescently tagged PGI to study subcellular localization

  • Investigate potential dynamic relocalization under different stress conditions

These approaches would provide insights into both the canonical metabolic role of PGI and potential moonlighting functions in S. paratyphi C.

How can researchers verify the purity and integrity of recombinant Salmonella paratyphi C PGI preparations?

Comprehensive quality assessment of recombinant PGI preparations should include:

Purity Assessment:

• SDS-PAGE: Should demonstrate >85% purity with a single predominant band at the expected molecular weight

• Size exclusion chromatography: Confirms monodispersity and absence of aggregates or degradation products

• Mass spectrometry: Provides precise molecular weight determination and can detect minor contaminants

Functional Integrity:

• Specific activity measurement: Compare to theoretical maximum or reference standards

• Thermal shift assay: Evaluate protein stability and proper folding

• Circular dichroism: Assess secondary structure composition

Identity Confirmation:

• Western blotting with anti-PGI antibodies or anti-tag antibodies if the protein is tagged

• Peptide mass fingerprinting: Confirm protein identity through proteomic analysis

• N-terminal sequencing: Verify the correct start of the protein sequence

A comprehensive quality control report should include documentation of purity (≥85% by SDS-PAGE) , specific activity, and stability parameters to ensure experimental reproducibility.

How does the genetic context of pgi in Salmonella paratyphi C influence its expression and regulation?

The genetic context of the pgi gene in S. paratyphi C likely plays a significant role in its expression and regulation:

• Genomic organization: Analysis of S. paratyphi C RKS4594 genome (4,833,080 bp chromosome) would reveal whether pgi is part of an operon or independently transcribed, influencing its co-regulation with other genes.

• Regulatory elements: The promoter region of pgi may contain binding sites for transcription factors responding to carbon availability, oxygen status, or host environment signals.

• Evolutionary context: S. paratyphi C's divergence from a common ancestor with S. choleraesuis and convergence with S. typhi may have resulted in unique regulatory adaptations of metabolic genes like pgi.

• Chromosomal positioning: The location of pgi relative to the origin of replication (oriC is located at 4016 kb in RKS4594) could affect its expression level due to gene dosage effects during rapid growth.

• Impact of prophages: S. paratyphi C contains prophage regions that have mediated chromosomal rearrangements , potentially affecting the expression of nearby genes, though whether pgi is proximal to such regions would require specific sequence analysis.

Comparative analysis of pgi regulation across different Salmonella serovars could reveal serovar-specific adaptations related to host specificity and pathogenicity.

What are the implications of S. paratyphi C's evolutionary history for PGI function and adaptation?

The evolutionary history of S. paratyphi C has several potential implications for PGI function:

• Selective pressures during human adaptation: S. paratyphi C appears to have diverged from a common ancestor with S. choleraesuis (primarily a swine pathogen) relatively recently by adapting to a different niche . This adaptation may have involved selection on metabolic enzymes like PGI to optimize function in the human host environment.

• Convergent evolution with S. typhi: Despite being genetically closer to S. choleraesuis, S. paratyphi C has converged with S. typhi in pathogenicity , suggesting potential parallel adaptations in metabolic networks supporting typhoid-like disease.

• Pseudogene formation pattern: S. paratyphi C has a distinctive set of pseudogenes compared to related strains , reflecting specific gene inactivation events during adaptation. Whether this has altered metabolic network architecture affecting PGI function requires further investigation.

• Genomic stability impact: S. paratyphi C has an unbalanced genome structure that has undergone frequent rearrangements , which could potentially affect the expression context of metabolic genes including pgi.

These evolutionary considerations suggest that comparative biochemical characterization of PGI across different Salmonella serovars might reveal subtle adaptations reflecting their distinct ecological niches and pathogenic strategies.

How can structure-function analyses of Salmonella paratyphi C PGI contribute to understanding bacterial metabolism and pathogenesis?

Structure-function analyses of S. paratyphi C PGI would provide multiple insights:

• Catalytic mechanism elucidation:

  • Site-directed mutagenesis of predicted catalytic residues

  • Crystallography of enzyme-substrate complexes

  • Molecular dynamics simulations of the isomerization reaction

• Host adaptation signatures:

  • Comparative analysis with PGI from non-typhoid Salmonella

  • Identification of sequence variations in substrate binding regions

  • Assessment of kinetic parameters under conditions mimicking host environments

• Potential moonlighting functions:

  • Investigation of non-catalytic protein interactions

  • Localization studies under different growth conditions

  • Assessment of potential extracellular roles

• Drug target assessment:

  • Identification of structural features distinct from human PGI

  • Fragment-based screening for selective inhibitors

  • Evaluation of PGI essentiality in different infection stages

These approaches would connect the molecular properties of PGI to its role in S. paratyphi C metabolism and potentially pathogenesis, particularly in the context of this serovar's adaptation to the human host and its convergent evolution with S. typhi .

How does PGI from Salmonella paratyphi C compare with PGI from other enteric pathogens?

Comparative analysis of PGI across enteric pathogens reveals important functional and evolutionary patterns:

• Sequence conservation: PGI is generally highly conserved across enteric pathogens, reflecting its essential metabolic role. The core catalytic residues show particularly high conservation, while surface-exposed regions may exhibit greater variability.

• Phylogenetic relationships: PGI sequence comparison can complement whole-genome phylogenetic analyses. The close genetic relationship between S. paratyphi C and S. choleraesuis (sharing 96.66% of their genomes) is likely reflected in their PGI sequences.

• Substrate specificity: While the fundamental catalytic mechanism is conserved, subtle variations in substrate binding pocket residues may affect substrate preference and catalytic efficiency. These differences might reflect adaptation to specific host environments or nutritional niches.

• Regulatory differences: The regulation of PGI expression and activity may differ across enteric pathogens, reflecting their distinct ecological strategies and metabolic requirements.

• Structural stability: Variations in thermostability or pH optima of PGI across different enteric pathogens might correlate with their adaptation to specific host environments or exposure to environmental stresses.

This comparative approach can reveal how central metabolic enzymes have been fine-tuned during the evolution and host adaptation of different enteric pathogens.

What methodological challenges exist in comparing kinetic properties of PGI across different bacterial species?

Researchers face several methodological challenges when comparing PGI kinetics across species:

• Expression system consistency:

  • Different expression systems may introduce variable post-translational modifications

  • Codon optimization requirements differ across species

  • Tags or fusion partners may affect enzyme properties differently

• Purification protocol variables:

  • Buffer conditions may not be optimal across all species variants

  • Different contaminants may co-purify depending on the expression host

  • Varying susceptibility to denaturation during purification

• Assay standardization issues:

  • Optimal pH and temperature may vary between species

  • Coupled assay components may interact differently with PGI variants

  • Substrate purity and preparation can introduce variability

• Data analysis considerations:

  • Different kinetic models may be appropriate for different species variants

  • Allosteric effects may be present in some but not all species

  • Oligomerization state may vary and affect kinetic parameters

To address these challenges, researchers should implement strict standardization of expression, purification, and assay conditions when performing comparative studies, and consider validating findings using multiple complementary approaches.

How might differences in PGI contribute to the distinct pathogenicity profiles of Salmonella serovars?

PGI variations could contribute to pathogenicity differences among Salmonella serovars in several ways:

• Metabolic fitness in host environments:

  • Optimized kinetic parameters for specific host nutritional environments

  • Different efficiencies in glucose versus alternative carbon source utilization

  • Varied responses to host-imposed nutrient restriction

• Stress response coordination:

  • Differential regulation of pgi expression under host-relevant stresses

  • Variable integration with virulence gene expression networks

  • Serovar-specific metabolic adaptations to oxidative stress

• Host-specific adaptations:

  • S. paratyphi C and S. typhi have converged in human host adaptation

  • Metabolic enzyme adaptations may support typhoid-like disease presentation

  • Specialized metabolism may support persistence in different host tissues

• Interaction with host immune system:

  • Potential immunogenic differences between PGI variants

  • Variable exposure of PGI to host immune recognition

  • Possible moonlighting functions in host-pathogen interactions

Understanding these connections requires integrating biochemical characterization with infection models and systems biology approaches to map the relationship between metabolic functions and virulence properties across the Salmonella serovars.