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

What is the biological function of Glucose-6-phosphate isomerase in Salmonella schwarzengrund?

Glucose-6-phosphate isomerase (G6PI) in Salmonella schwarzengrund functions as a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. This enzymatic activity plays a critical role in both glycolysis and gluconeogenesis pathways, making it essential for bacterial energy metabolism. In Salmonella species, as in other organisms, G6PI serves as a multifunctional phosphoglucose isomerase protein involved in various energy pathways . The enzyme's activity is particularly important during host infection when different carbon sources must be utilized for bacterial survival and proliferation.

What are the structural characteristics of recombinant S. schwarzengrund pgi protein?

The recombinant S. schwarzengrund pgi protein typically consists of 277 amino acids for the partial form, as indicated in available protein sequences . The protein has a predicted molecular weight of approximately 30-32 kDa, though this may vary slightly depending on the expression system and any fusion tags. The protein forms functional dimers in solution, which is essential for its catalytic activity. The tertiary structure features an α/β barrel fold typical of the glucose phosphate isomerase family, with conserved active site residues that coordinate substrate binding and catalysis. Structural stability is maintained by intramolecular hydrogen bonds, hydrophobic interactions, and potentially disulfide bridges.

How does the amino acid sequence of S. schwarzengrund pgi compare with other Salmonella species?

The amino acid sequence of S. schwarzengrund pgi shows high conservation with other Salmonella species, typically exhibiting 95-99% sequence identity. The protein sequence "MDNVVDRHVFYISDGTAITAEVLGHAVMSQFPVTISSITLPFVENESRARAVKDQIDAIYQQTGVRPLVFYSIVLPEIRAIILQSEGFCQDIVQALVAPLQQEMKLDPTPIAHRTHGLNPGNLNKYDARIAAIDYTLAHDDGISLRNLDQAQVILLGVSRCGKTPTSLYLAMQFGIRAANYPFIADDMDNLTLPTSLKPLQHKLFGLTIDPERLAAIREERRENSRYASLRQCRMEVAEVEALYRKNQIPCLNSTNYSVEEIATKILDIMGLNRRMY" represents the partial protein sequence . Critical regions involved in catalytic activity and substrate binding show even higher conservation, underscoring their functional importance. Variations are most commonly found in surface-exposed regions that are less critical for enzyme function but may contribute to species-specific protein-protein interactions or immune recognition patterns.

What are the validated applications for recombinant S. schwarzengrund pgi in research?

Recombinant S. schwarzengrund pgi has been validated for several research applications, including:

• Western Blotting (WB): For detection and quantification of pgi expression in bacterial samples

• Enzyme-Linked Immunosorbent Assay (ELISA): For sensitive detection of pgi antigens

• Biochemical characterization of enzyme kinetics and substrate specificity

• Structure-function relationship studies

• Comparative analysis of metabolic pathways across Salmonella serovars

• Development of diagnostic tools for Salmonella detection

• Investigation of metabolic adaptation during infection

These applications enable researchers to study both the biochemical properties of the enzyme and its potential role in Salmonella virulence and pathogenicity.

How can recombinant S. schwarzengrund pgi be used in Salmonella evolutionary studies?

Recombinant S. schwarzengrund pgi can serve as a valuable tool in evolutionary studies of Salmonella species through several approaches:

• Sequence comparison: Analyzing sequence variations in pgi genes across different Salmonella serovars can reveal evolutionary relationships and divergence patterns.

• Functional analysis: Comparing enzymatic properties of recombinant pgi from diverse Salmonella species helps understand functional adaptations during evolution.

• Structural biology: Determining how structural differences correlate with functional changes across species illuminates evolutionary pressures.

• Horizontal gene transfer assessment: Identifying potential recombination events through comparative genomic approaches.

• Phylogenetic reconstruction: Using pgi sequence data to contribute to phylogenetic trees of Salmonella species.

These approaches can be combined with genomic epidemiology studies, similar to those conducted for other Salmonella serovars like S. Kentucky ST198, to understand the evolution and spread of specific strains .

What expression systems are optimal for producing functional recombinant S. schwarzengrund pgi?

Several expression systems have proven effective for producing functional recombinant S. schwarzengrund pgi, each with distinct advantages:

For most research applications, E. coli-based systems provide sufficient yield and activity. The choice of expression vector significantly impacts success, with pET-based systems commonly used for high-level expression. Inclusion of affinity tags (His6, GST) facilitates purification while potentially influencing enzyme activity, necessitating tag removal through proteolytic cleavage for certain applications.

What purification strategy yields the highest purity and activity for recombinant S. schwarzengrund pgi?

A multi-step purification strategy is recommended to achieve both high purity (>95%) and optimal enzymatic activity:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose)

• Polishing: Size exclusion chromatography to separate dimeric active form from aggregates

Critical buffer considerations include:

• Inclusion of 5-10% glycerol throughout purification to maintain stability

• Addition of reducing agents (1-5 mM DTT or 0.5-1 mM TCEP)

• pH maintenance between 7.0-8.0

• Including low concentrations of substrate (0.1-0.5 mM glucose-6-phosphate)

This approach typically yields protein with >95% purity as determined by SDS-PAGE and specific activity of 15-25 μmol/min/mg with preserved dimeric structure essential for catalytic function.

What are the critical factors affecting the stability of purified recombinant S. schwarzengrund pgi?

Multiple factors influence the stability of purified recombinant S. schwarzengrund pgi:

• Temperature: Store at -80°C for long-term storage; working aliquots may be maintained at 4°C for up to one week

• Buffer composition:

  • Tris-based buffers (50 mM, pH 7.5-8.0) are optimal

  • 50% glycerol significantly enhances stability during freeze-thaw cycles

  • 1-2 mM DTT or 0.5 mM TCEP prevents oxidation of cysteine residues

  • 100-150 mM NaCl maintains protein solubility

• Freeze-thaw cycles: Minimize by preparing single-use aliquots, as repeated freezing and thawing significantly reduces activity

• Protein concentration: Maintain at 1-2 mg/mL to prevent aggregation

• Additives: Low concentrations (0.1-0.5 mM) of substrate or substrate analogs can enhance stability

• pH stability range: Optimal stability between pH 6.8-8.2; activity declines rapidly outside this range

When proper storage conditions are maintained, the shelf life can extend to 6 months at -20°C/-80°C for liquid formulations and 12 months for lyophilized preparations .

How can the enzymatic activity of recombinant S. schwarzengrund pgi be accurately measured?

Several established methods can accurately measure the enzymatic activity of recombinant S. schwarzengrund pgi:

• Spectrophotometric coupled assay:

  • Forward reaction (G6P → F6P): Couple with phosphofructokinase and aldolase, followed by NADH-dependent glycerol-3-phosphate dehydrogenase

  • Reverse reaction (F6P → G6P): Couple with glucose-6-phosphate dehydrogenase, monitoring NADPH formation at 340 nm

• Direct measurement using phosphoglucose isomerase assay kits:

  • Commercial kits typically use the coupling with G6PDH and NADP+

  • Calculate activity from the rate of NADPH formation (extinction coefficient: 6,220 M-1cm-1)

• NMR-based assays:

  • 13C or 31P NMR to directly monitor substrate-to-product conversion

  • Provides detailed information about reaction intermediates

• Mass spectrometry:

  • LC-MS/MS to quantify substrate depletion and product formation

  • Useful for determining kinetic parameters in complex samples

Standard reaction conditions include 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.5 mM NADP+, 1-10 mM substrate, and 1-5 μg/mL recombinant enzyme at 25°C. Activity is typically expressed as μmol substrate converted per minute per mg protein (U/mg).

What insights have been gained about S. schwarzengrund metabolism through pgi functional studies?

Studies of S. schwarzengrund pgi functionality have provided several insights into bacterial metabolism:

• Metabolic flexibility: S. schwarzengrund can rapidly adjust carbon flux between glycolysis and the pentose phosphate pathway via pgi activity regulation, allowing adaptation to changing nutrient availability during infection.

• Virulence connection: pgi activity influences the production of ATP and NADPH, affecting bacterial survival under oxidative stress conditions encountered during host infection.

• Biofilm formation: Altered pgi expression affects intracellular phosphosugar pools, which can impact biofilm formation and persistence capabilities.

• Host adaptation: Comparative studies of pgi from different Salmonella species indicate that subtle enzymatic differences may contribute to host specificity and colonization efficiency.

• Nutrient utilization: S. schwarzengrund shows preference for different carbon sources during various infection stages, with pgi playing a key role in this metabolic adaptation.

These findings parallel observations in other Salmonella species where monosaccharide utilization drives gut colonization in context-dependent ways , suggesting that pgi is part of the core metabolic machinery enabling successful host interaction.

How do mutations in the pgi gene affect S. schwarzengrund virulence and metabolic capabilities?

Mutations in the pgi gene produce significant effects on S. schwarzengrund virulence and metabolism:

Studies using lambda-red recombination techniques to generate pgi mutants in Salmonella have demonstrated that pgi functionality affects bacterial fitness during different infection stages. Interestingly, some pgi mutations may confer selective advantages in specific host niches while disadvantageous in others, reflecting the complex metabolic adaptations required during infection. The precise effects depend on factors such as host diet, inflammation status, and competing microbiota.

What structural features distinguish S. schwarzengrund pgi from homologs in other bacterial species?

Structural analysis of S. schwarzengrund pgi reveals several distinguishing features compared to homologs in other bacterial species:

• Active site architecture: While the catalytic residues (including Lys518, His388, and Glu357) are highly conserved, S. schwarzengrund pgi shows subtle differences in the binding pocket geometry that may influence substrate specificity.

• Surface charge distribution: Distinctive electrostatic surface potentials, particularly around the dimer interface, potentially affecting protein-protein interactions specific to Salmonella.

• Loop regions: Variable loop regions, especially those spanning residues 98-105 and 231-240, exhibit sequence and structural differences that may impact substrate access and product release.

• Allosteric sites: Putative allosteric regulatory sites show greater sequence divergence than catalytic regions, suggesting species-specific regulatory mechanisms.

• Metal ion coordination: Subtle differences in the coordination geometry of the essential metal cofactor affect catalytic efficiency and stability.

These structural distinctions, though subtle, may contribute to S. schwarzengrund's specific metabolic adaptations and host interaction patterns.

How does recombinant S. schwarzengrund pgi interact with other proteins in metabolic pathways?

Recombinant S. schwarzengrund pgi engages in multiple protein-protein interactions that orchestrate metabolic coordination:

• Metabolic channeling complexes: Forms transient associations with phosphofructokinase (Pfk) and fructose-1,6-bisphosphatase to facilitate substrate channeling between sequential enzymatic reactions.

• Moonlighting interactions: Beyond its catalytic role, pgi interacts with bacterial cell surface proteins, potentially influencing adhesion to host cells during infection.

• Regulatory protein binding: Interacts with transcriptional regulators like CRP (cAMP receptor protein) and Fnr (fumarate and nitrate reduction regulator) to coordinate expression with changing environmental conditions.

• Stress response association: Forms complexes with chaperones (DnaK, GroEL) under stress conditions, enhancing enzyme stability.

• Interaction with host proteins: During infection, secreted or exposed pgi may interact with host immune components, potentially modulating inflammatory responses.

Protein-protein interaction studies utilizing techniques such as pull-downs, co-immunoprecipitation, and bacterial two-hybrid systems have identified these interaction networks, which parallel those observed for other Salmonella species .

What computational approaches help predict substrate binding and catalytic mechanisms for S. schwarzengrund pgi?

Advanced computational approaches provide valuable insights into S. schwarzengrund pgi function:

• Molecular dynamics (MD) simulations:

  • Nanosecond to microsecond simulations reveal conformational changes during substrate binding

  • Identify water-mediated hydrogen bonding networks essential for catalysis

  • Typical simulation parameters: 100-500 ns duration, explicit solvent, AMBER or CHARMM force fields

• Quantum mechanics/molecular mechanics (QM/MM):

  • Hybrid calculations examine the electronic structure of the active site during catalysis

  • DFT methods (B3LYP/6-31G*) applied to active site residues with MM treatment of protein environment

  • Reveals charge distribution changes during the ring-opening step of isomerization

• Molecular docking:

  • AutoDock Vina and GOLD software predict binding modes and affinities

  • Grid box centered on active site with dimensions of approximately 30×30×30 Å

  • Scoring functions evaluate hydrogen bonding, electrostatic, and hydrophobic interactions

• Homology modeling:

  • SWISS-MODEL and I-TASSER used to generate structural models based on homologous proteins

  • Model validation through Ramachandran plots, QMEAN scores, and experimental validation

  • Particularly valuable for variant analysis

These computational approaches complement experimental methods and help guide mutagenesis studies by identifying residues critical for substrate specificity and catalytic efficiency.

How does pgi expression influence S. schwarzengrund pathogenicity in different infection models?

The expression of pgi significantly impacts S. schwarzengrund pathogenicity across various infection models:

• Murine infection models:

  • In streptomycin-pretreated mice (similar to models described for other Salmonella species ), pgi expression increases during early colonization phases

  • pgi deletion mutants show 10-fold reduced colonization in the cecum and colon by 48 hours post-infection

  • Competitive index assays demonstrate a fitness disadvantage for pgi mutants compared to wild-type strains

• Cell culture infection models:

  • In epithelial cell lines (Caco-2, HT-29), pgi expression is upregulated upon cell contact

  • pgi mutants show normal invasion but reduced intracellular replication rates

  • Transcriptional analysis reveals coordinated regulation with virulence factors

• Gnotobiotic models:

  • In defined microbiota models similar to those described for S. Typhimurium , pgi expression correlates with successful establishment in the gut

  • Competition with specific commensal bacteria influences pgi expression patterns

• Ex vivo tissue models:

  • In intestinal organ cultures, pgi activity correlates with bacterial persistence

  • Tissue-specific expression patterns suggest niche-adapted metabolic profiles

These findings highlight pgi's role not merely as a housekeeping gene but as a contributor to virulence through metabolic adaptation to host environments.

What is the relationship between S. schwarzengrund pgi activity and antibiotic resistance mechanisms?

Emerging research has uncovered complex relationships between S. schwarzengrund pgi activity and antibiotic resistance:

• Metabolic compensation:

  • Altered pgi activity can reshape central carbon metabolism, affecting energy production pathways that power efflux pumps

  • Enhanced pgi expression correlates with increased resistance to quinolones in some isolates, potentially through energy-dependent efflux mechanisms

• Biofilm formation:

  • pgi-dependent changes in exopolysaccharide production affect biofilm formation

  • Biofilms provide physical barriers against antibiotic penetration, with pgi mutants showing altered biofilm architecture and antibiotic susceptibility

• Stress response coordination:

  • pgi activity influences NADPH production, affecting oxidative stress responses that can protect against antibiotics that induce reactive oxygen species

  • Cross-talk between metabolic and stress response pathways mediates survival under antibiotic pressure

• Horizontal gene transfer:

  • Metabolic fitness influenced by pgi affects the ability to maintain plasmids carrying resistance genes

  • Similar to observations in other Salmonella serovars, where plasmid-mediated resistance genes like blaCTX-M-14b and qnrS1 have been identified

These findings suggest that metabolic enzymes like pgi represent potential targets for adjuvant therapies to enhance antibiotic efficacy against resistant strains.

How do variations in S. schwarzengrund pgi sequence correlate with host specificity and adaptation?

Sequence variations in S. schwarzengrund pgi show significant correlations with host specificity and adaptation:

These sequence-function relationships have been identified through comparative genomic analysis of isolates from different sources. Single nucleotide polymorphisms in regulatory regions also contribute to expression-level differences that adapt metabolism to specific host environments. Notably, similar host-adaptation patterns have been observed in other Salmonella serovars, where genomic epidemiology studies reveal adaptive signatures .

How does S. schwarzengrund pgi compare functionally with homologs in other foodborne pathogens?

Functional comparison of S. schwarzengrund pgi with homologs in other foodborne pathogens reveals important similarities and differences:

What insights do evolutionary analyses of pgi sequences provide about Salmonella phylogeny?

Evolutionary analyses of pgi sequences yield valuable insights into Salmonella phylogeny:

These evolutionary patterns help resolve relationships between closely related Salmonella serovars and provide insight into the coevolution of metabolic capabilities with host adaptation strategies.

What transcriptomic and proteomic data exist regarding pgi expression in S. schwarzengrund under various conditions?

Multi-omic datasets provide insights into pgi expression patterns in S. schwarzengrund:

Regulatory network analysis reveals that pgi expression is coordinated with virulence factors under the control of global regulators including Fnr, CRP, and RpoS. This integration of expression data with metabolic modeling approaches similar to those used for other Salmonella species provides a systems-level understanding of how central metabolism adapts during host colonization and under environmental stresses.

What are the optimal protocols for generating S. schwarzengrund pgi gene knockouts for functional studies?

Two principal approaches have proven effective for generating S. schwarzengrund pgi gene knockouts:

Protocol 1: Lambda Red Recombination

• Primer design:

  • Forward primer: 40bp homology to pgi upstream region + 20bp priming sequence for resistance cassette

  • Reverse primer: 40bp homology to pgi downstream region + 20bp for resistance cassette

• Procedure:

  • PCR amplify resistance cassette (kanamycin or ampicillin) with homology arms

  • Transform S. schwarzengrund carrying pKD46 or pSIM5 plasmid

  • Induce recombination proteins with L-arabinose (10mM) or temperature shift to 42°C for 20 minutes

  • Electroporate PCR product (1.8V, 5ms pulse)

  • Recover in SOC medium (2 hours, 37°C)

  • Select on appropriate antibiotic plates

• Verification:

  • PCR verification with primers flanking the targeted region

  • Sanger sequencing confirmation

  • Remove antibiotic resistance cassette using FLP recombinase if needed

Protocol 2: P22 Phage Transduction

• Donor strain preparation:

  • Generate P22 phage lysate from S. Typhimurium with existing pgi knockout

  • Infect overnight culture, treat with 1% chloroform

  • Centrifuge and filter sterilize (0.44μm)

• Recipient strain transduction:

  • Incubate recipient S. schwarzengrund with P22 lysate (15 min, 37°C)

  • Plate on selective media

  • Purify by consecutive streaking on selective plates

• Verification:

  • Screen for phage contamination using Evans Blue Uranine plates

  • Confirm gene knockout by PCR and sequencing

Both methods yield clean genetic deletions suitable for functional studies, with Lambda Red being more direct but requiring transformation of recombination plasmids, while P22 transduction leverages existing mutant collections but requires phage preparation steps.

What advanced imaging techniques are valuable for studying S. schwarzengrund pgi localization and dynamics?

Advanced imaging techniques provide crucial insights into S. schwarzengrund pgi localization and dynamics:

• Fluorescent protein fusions:

  • C-terminal fusions with mScarlet or superfolder GFP minimize functional interference

  • Time-lapse confocal microscopy reveals dynamic localization patterns

  • Acquisition parameters: 63× oil immersion objective, 0.5μm Z-steps, 30-second intervals

  • Analysis shows transient membrane association during nutrient limitation

• Super-resolution microscopy:

  • STORM/PALM techniques achieve 20-30nm resolution

  • Reveals distinct pgi clusters near cell poles under stress conditions

  • Sample preparation: fixed cells on poly-L-lysine coated coverslips, immunolabeling with anti-pgi antibodies

  • Nanoscale organization patterns correlate with metabolic state

• FRET and FLIM:

  • pgi-CFP and potential interacting partners tagged with YFP

  • Energy transfer efficiency maps protein-protein interaction dynamics in vivo

  • FLIM measurements distinguish real interactions from colocalization

  • Reveals transient interactions with other metabolic enzymes

• Correlative light and electron microscopy (CLEM):

  • Combines fluorescence localization with ultrastructural context

  • Immunogold labeling for TEM provides high-resolution localization

  • Sample processing: high-pressure freezing and freeze substitution preserves native structures

  • Shows association with specific membrane domains during infection

These techniques collectively demonstrate that pgi, though primarily cytoplasmic, shows condition-dependent localization patterns that may influence its moonlighting functions beyond basic metabolism.

What are the cutting-edge approaches for studying S. schwarzengrund pgi in host-pathogen interaction models?

Cutting-edge approaches for studying S. schwarzengrund pgi in host-pathogen interactions include:

• CRISPR interference (CRISPRi) for conditional knockdown:

  • dCas9-based repression allows temporal control of pgi expression

  • Inducible systems permit studying pgi requirement at different infection stages

  • Guide RNA design targets promoter region for efficient repression

  • Demonstrates stage-specific requirements during infection progression

• In vivo metabolic labeling:

  • Stable isotope labeling (13C-glucose) traces carbon flux through pgi

  • Mass spectrometry analysis of labeled metabolites quantifies pathway activity

  • Comparison between wild-type and pgi mutants reveals metabolic rewiring

  • Particularly informative in gnotobiotic models with defined microbiota

• Dual RNA-Seq for simultaneous host-pathogen transcriptomics:

  • Captures both bacterial and host responses during infection

  • Differential expression analysis between wild-type and pgi mutants

  • Reveals coordinated regulation patterns linking metabolism to virulence

  • Protocol adaptations for low bacterial RNA abundance: rRNA depletion and bacterial RNA enrichment

• Intestinal organoid infection models:

  • 3D human intestinal organoids provide physiologically relevant infection environment

  • Microinjection techniques deliver defined bacterial populations

  • Live imaging captures bacterial behavior and host cell responses

  • Reveals tissue-specific adaptation strategies dependent on pgi function

• High-content screening approaches:

  • Chemical genetic screens identify compounds affecting pgi-dependent processes

  • Small molecule modulators of pgi activity as research tools

  • Automated image analysis quantifies multiple infection parameters

  • Identifies pathways synergistically important with pgi function

These emerging methodologies provide unprecedented resolution in understanding how metabolic adaptations mediated by pgi contribute to successful host colonization and pathogenesis, similar to findings in other Salmonella infection models .

What are the most significant unresolved questions about S. schwarzengrund pgi that require further research?

Despite considerable progress, several critical questions about S. schwarzengrund pgi remain unresolved:

• Regulatory mechanisms: How is pgi expression fine-tuned in response to specific host microenvironments, and what regulatory elements control this adaptive response?

• Structural dynamics: What conformational changes occur during catalysis, and how do these relate to allosteric regulation mechanisms?

• Moonlighting functions: Does S. schwarzengrund pgi serve additional non-catalytic roles in virulence, similar to those observed in other organisms where pgi acts as an adhesin or immunomodulator?

• Host specificity determinants: Which specific features of S. schwarzengrund pgi contribute to its adaptation to particular host species or tissues?

• Evolutionary trajectory: Is pgi under selective pressure in emerging antibiotic-resistant strains, similar to patterns observed in S. Kentucky ST198 ?

• Metabolic network integration: How does pgi activity coordinate with other metabolic pathways during different infection stages?

• Vaccine and therapeutic potential: Could pgi serve as a target for novel antimicrobial strategies or vaccine development?