Recombinant Glutaredoxin-2 (grxB)

What is the molecular structure of Glutaredoxin-2 and how does it differ from other glutaredoxins?

Glutaredoxin-2 (Grx2) represents a distinct structural class compared to other glutaredoxins. In E. coli, the grxB gene spans 645 base pairs, located between the rimJ and pyrC genes, encoding a protein of 215 amino acid residues with a molecular mass of 24.3 kDa . This makes Grx2 substantially larger than Grx1 and Grx3, which are both approximately 9-kDa proteins .

Functionally, Grx2 contributes up to 80% of total Grx activity under normal physiological conditions . Unlike Grx1 and Grx3, Grx2 is not a hydrogen donor for ribonucleotide reductase, suggesting divergent functional roles within cellular metabolism . Catalytically, Grx2 maintains similar apparent Km values for GSH (2-3 mM) as other glutaredoxins but possesses the highest apparent kcat (554 s-1), indicating superior catalytic efficiency .

What primary cellular functions does Glutaredoxin-2 perform?

Glutaredoxin-2 participates in multiple essential cellular processes through its thiol-disulfide exchange activity. The protein plays critical roles in:

• Thiol-disulfide exchange reactions: Grx2 catalyzes GSH-disulfide oxidoreductions via its redox-active cysteine residues, contributing to cellular redox homeostasis .

• DNA synthesis: As part of the glutaredoxin system, Grx2 contributes to the formation of deoxyribonucleotides, though not through the same mechanism as Grx1 and Grx3 .

• Redox sensing: Grx2 helps cells sense cellular reduction-oxidation potentials, allowing appropriate responses to redox changes .

• Protein structure maintenance: Through its role in controlling protein folding and protecting against oxidative damage, Grx2 helps maintain proper protein structure .

• Stress responses: Grx2 contributes significantly to acid tolerance responses in bacteria, with research showing increased expression during acid stress and compromised acid tolerance in grxB deletion mutants .

• Biofilm formation: Studies in C. sakazakii have demonstrated that grxB affects biofilm formation, surface hydrophobicity, and auto-aggregation under both normal and acid stress conditions .

• Signal transduction: Grx2 participates in various signal transduction pathways, particularly those involved in cellular responses to environmental stresses .

Collectively, these functions position Grx2 as a crucial component of cellular stress response systems, particularly in maintaining redox balance and facilitating adaptation to environmental challenges.

How is grxB gene expression regulated under different environmental conditions?

The regulation of grxB expression demonstrates significant responsiveness to environmental stressors, particularly acid stress. Research has shown that the relative expression of grxB increases during acetate-induced acid tolerance response in E. coli . Both proteomic analysis using 2-D electrophoresis and real-time fluorescence quantitative PCR have confirmed the up-regulation of the GrxB protein and grxB gene under acidic environmental conditions .

The protein interactions of GrxB provide additional insights into its regulatory context. Grx2 interacts with several proteins including GrxA, Dps, TrxB, GapA, PfkA, PfkB, OsmC, YdiZ, and YiaG, which are also related to acid stress and biofilm formation . These interacting partners are primarily associated with cellular homeostasis and glycolysis pathways, suggesting that grxB regulation is integrated with broader metabolic and stress response networks .

The functional significance of this regulated expression becomes apparent in studies of grxB deletion mutants, which show decreased resistance to acid stress compared to wild-type strains . This indicates that the induction of grxB expression under acidic conditions is part of an adaptive response that enhances bacterial survival in challenging environments.

While specific transcriptional regulators controlling grxB expression have not been fully characterized in the provided search results, the coordinated expression with other stress response genes suggests integration into broader stress response regulatory networks.

What reliable methods exist for measuring Glutaredoxin-2 activity in vitro?

The standard method for measuring Glutaredoxin-2 activity employs a spectrophotometric assay that monitors the oxidation of NADPH, providing a quantitative measure of enzyme activity. The protocol includes:

• Preparation of Grx reaction buffer containing:

  • 0.5 mM glutathione (GSH)

  • 0.5 mM 2-hydroxyethyl disulfide (HED)

  • 0.5 mM NADPH

  • 0.5 U/ml glutathione reductase (GR)

  • 50 mM Tris-HCl

• Formation of a mixed disulfide containing both HED and GSH by allowing approximately 2 minutes for this reaction .

• Initiation of the Grx reaction by adding bacterial crude protein extract containing Glutaredoxin-2 .

• Measurement of the decrease in absorbance at 340 nm, which correlates with the oxidation of NADPH during the reaction .

• Calculation of activity where one unit of Grx activity is defined as the amount required to oxidize 1 μmol NADPH per minute at 25°C .

This method enables quantitative comparison of Grx activity between different strains or under varying experimental conditions. Table 1 shows representative results comparing wild-type and ΔgrxB strains under different pH conditions:

Values represent mean ± SD from three independent experiments. ∗P < 0.05; ∗∗P < 0.01 (ΔgrxB vs. WT)

For more precise characterization, this basic assay can be adapted by varying substrate concentrations to determine kinetic parameters, including Km and kcat values. Previous research has established that Grx2 maintains similar apparent Km values for GSH (2-3 mM) as other glutaredoxins but possesses superior catalytic efficiency with an apparent kcat of 554 s-1 .

How can researchers generate and validate a grxB gene knockout model?

Construction of a grxB knockout model can be accomplished using the high-efficiency bacterial conjugation method. The following protocol has been successfully implemented:

• PCR Amplification of Flanking Fragments:

  • Amplify two fragments flanking the grxB gene independently by PCR

  • Use primers containing attB sequences (outside primers) and linking sequences (inside primers)

  • Use genomic DNA as the template

• Fusion of Flanking Fragments:

  • Join the two PCR fragments by fusion PCR (overlap extension reaction)

  • Use outside primers to amplify the fused product

  • Prepare a 50 μl reaction with appropriate DNA polymerase mix

  • Run PCR with suitable thermal cycling conditions

  • Purify the fusion product using a PCR purification kit

• Vector Construction:

  • Transform the fusion homology arm into an appropriate plasmid (e.g., PHGM01)

  • Use a recombination reaction with BP clonase II enzyme mix

  • Incubate for at least 1 hour or overnight at 25°C

• Conjugation and Selection:

  • Introduce the mutagenesis vector into an appropriate E. coli strain

  • Confirm the correct destination vector by DNA sequencing

  • Transfer the vector by conjugation into the target bacterial strain

  • Select for mutants using appropriate antibiotics

• Validation of Knockout:

  • Confirm the deletion mutant by sequencing the mutated regions

  • Verify the absence of the grxB gene using specific primers

  • Assess Grx activity to confirm functional deletion (expect reduced activity)

Additional validation should include phenotypic assays comparing wild-type and knockout strains:

• Acid tolerance assessment

• Cellular morphology analysis

• Surface hydrophobicity tests

• Auto-aggregation assays

• Biofilm formation analysis

The effectiveness of this knockout approach is demonstrated by the observation that ΔgrxB strains possess diminished Grx activity compared to wild-type strains, with approximately 78% of wild-type activity under normal conditions and 81% under acid stress . These reductions confirm successful functional deletion while suggesting some compensatory activity from other glutaredoxins.

What phenotypic assays provide valuable insights when characterizing grxB mutants?

Several phenotypic assays have proven particularly informative for characterizing the functional consequences of grxB deletion:

• Acid Tolerance Assessment:

  • Subject wild-type and ΔgrxB strains to acidic conditions (e.g., pH 4)

  • Monitor survival rates at different time points

  • Observe adaptive responses through extended cultivation under acid stress

  • Deletion of grxB results in decreased resistance to acid stresses, providing a quantifiable phenotype

• Cellular Morphology Analysis via Transmission Electron Microscopy (TEM):

  • Prepare samples using negative staining with 3% phosphotungstic acid

  • Observe cells cultured under normal and acidic conditions (pH 4) at various time points

  • Document morphological changes, which are typically more pronounced in ΔgrxB mutants

  • Under acid stress, cells initially change from slender rhabditiform to thick claviform shapes, with ΔgrxB cells showing more severe bulge deformations

• Cell Surface Hydrophobicity (CSH) Test:

  • Mix bacterial suspension with xylene

  • Measure optical density of the aqueous phase

  • Calculate the percentage of cells migrating to the xylene phase

  • ΔgrxB strains typically show reduced hydrophobicity under both normal and acid stress conditions

• Auto-Aggregation (AAg) Assay:

  • Prepare standardized bacterial suspensions

  • Store at room temperature for 20 hours

  • Measure the decrease in optical density

  • Calculate AAg percentage using the formula: [(A₀ - A)/A₀] × 100

  • Wild-type cells precipitate faster than ΔgrxB cells, indicating stronger auto-aggregation ability

• Biofilm Formation Analysis:

  • Culture cells in appropriate media

  • Assess biofilm formation quantitatively using crystal violet staining

  • ΔgrxB strains exhibit weaker biofilm formation under both normal and acid stress conditions

• Motility Assays:

  • Assess swimming motility on soft agar plates (0.3% agar)

  • Evaluate swarming motility on appropriate plates (0.5% agar)

  • Compare motility under different pH conditions

  • Interestingly, motility remains largely unaffected by grxB deletion, distinguishing it from other affected phenotypes

These assays collectively provide a comprehensive characterization of the functional roles of Grx2 in bacterial physiology, particularly in relation to stress responses and community behaviors.

What experimental variables require optimization when working with recombinant Glutaredoxin-2?

When working with recombinant Glutaredoxin-2, several critical parameters require careful optimization:

• Protein Expression Considerations:

  • Expression system selection: E. coli systems are commonly used for recombinant Grx2 expression

  • Vector design: Consider solubility-enhancing tags and appropriate promoters

  • Expression conditions: Temperature, inducer concentration, and duration significantly impact protein folding

  • Monitor protein solubility as truncated forms of Grx2 (1-114 and 1-133) have been shown to form inclusion bodies

• Protein Structure Preservation:

  • The COOH-terminal half of the molecule is essential for GSH-disulfide oxidoreductase activity

  • Expression of truncated forms lacking this region results in inactive protein

  • Preserve the integrity of the active site (C9PYC12) which contains crucial cysteine residues

• Activity Assay Optimization:

  • Buffer composition: Optimize GSH concentration (Km ≈ 2-3 mM), HED, NADPH, and glutathione reductase concentrations

  • Temperature: Standard assays are conducted at 25°C

  • pH: Consider the optimal pH for enzyme activity

  • Reaction timing: Allow sufficient time (approximately 2 minutes) for the formation of the mixed disulfide before adding enzyme

• Storage Conditions:

  • Temperature: Store purified protein at -20°C as recommended

  • Buffer composition: Include appropriate stabilizers and reducing agents

  • Aliquoting: Prepare small aliquots to avoid repeated freeze-thaw cycles

• Functional Studies Preparation:

  • Include appropriate controls (wild-type, complemented mutant)

  • Account for the influence of growth phase on Grx2 activity

  • For acid stress studies, carefully control pH conditions and exposure duration

  • Consider potential compensatory mechanisms from other glutaredoxins in knockout studies

These optimization considerations are crucial for obtaining reliable and reproducible results when working with recombinant Glutaredoxin-2. Particular attention should be paid to protein folding and the preservation of catalytic activity, as the structural integrity of Grx2 is closely linked to its functional properties.

How does Glutaredoxin-2 contribute to bacterial acid tolerance mechanisms?

Glutaredoxin-2 plays a significant role in bacterial acid tolerance through multiple mechanisms:

• Regulated Expression Under Acid Stress:

  • The expression of grxB increases during acetate-induced acid tolerance responses in E. coli

  • Both protein and gene expression levels are up-regulated under acidic conditions, as confirmed by proteomic analysis and real-time PCR

  • This regulated expression suggests a programmed role in acid adaptation

• Morphological Adaptation Facilitation:

  • Under acid stress (pH 4), both wild-type and ΔgrxB cells undergo morphological changes from slender rhabditiform to thick claviform shapes with reduced flagella

  • ΔgrxB cells display more pronounced bulge deformations with rough and fusiform characteristics

  • Over time (24-36 hours), cells gradually regain normal morphologies, with wild-type cells adapting more effectively

  • By 48 hours, both strains appear morphologically normal despite continued acid stress

• Cellular Redox Balance Maintenance:

  • Grx2 contributes to maintaining protein structure and function under acidic conditions

  • The protein's thiol-disulfide exchange activity may protect cellular proteins from acid-induced oxidative damage

  • This protection helps preserve cellular functions essential for acid tolerance

• Integration with Broader Stress Response Networks:

  • GrxB interacts with several proteins involved in acid stress responses, including GrxA, Dps, TrxB, and others

  • These interactions suggest that Grx2 contributes to a comprehensive network of acid tolerance mechanisms

  • The associated proteins are enriched for cellular homeostasis and glycolysis pathways, linking metabolic adaptation to acid tolerance

• Quantifiable Impact on Acid Resistance:

  • Deletion of grxB results in measurably decreased resistance to acid stresses

  • The ΔgrxB strain shows altered surface properties and cellular morphology under acid stress

  • These changes correlate with reduced survival under acidic conditions

The contribution of Grx2 to acid tolerance appears to be part of a multifaceted adaptation strategy that involves maintaining cellular redox homeostasis, protecting protein structure and function, and facilitating morphological adaptations necessary for survival in acidic environments.

What is the relationship between Glutaredoxin-2 activity and biofilm formation?

Research has established a clear relationship between Glutaredoxin-2 activity and biofilm formation, particularly through effects on cellular surface properties and intercellular interactions:

• Diminished Biofilm Formation in grxB Mutants:

  • Deletion of grxB notably leads to weaker biofilm formation under both normal and acid stress conditions

  • This effect is consistent and quantifiable, establishing a direct link between Grx2 and biofilm development

• Altered Cell Surface Properties:

  • The ΔgrxB mutant exhibits decreased cell surface hydrophobicity (CSH)

  • Surface hydrophobicity is a critical factor in initial attachment during biofilm formation

  • This reduced hydrophobicity occurs under both normal and acidic conditions

  • The consistent reduction in CSH suggests a direct role for Grx2 in maintaining cell surface properties conducive to biofilm formation

• Reduced Auto-Aggregation Capacity:

  • The ΔgrxB mutant shows significantly reduced auto-aggregation ability

  • Wild-type cells precipitate faster than ΔgrxB cells when suspended in PBS

  • This reduced capacity for cell-to-cell interaction directly impacts the ability to form robust biofilms

  • The same phenomenon is observed under both normal and acidic conditions

• Unchanged Motility:

  • Interestingly, the deletion of grxB does not affect bacterial motility (both swimming and swarming)

  • This suggests that the influence of Grx2 on biofilm formation is not mediated through changes in motility

  • Rather, the effects appear to be primarily through alterations in surface properties and cell-to-cell interactions

• Potential Molecular Mechanisms:

  • Grx2 interacts with several proteins also related to biofilm formation

  • These interactions may influence pathways that regulate cell surface properties

  • The protein's role in maintaining redox homeostasis may protect surface proteins necessary for biofilm development

  • The glutaredoxin system may serve as a link between environmental sensing and biofilm regulation

This relationship positions Grx2 as a potential target for strategies aimed at controlling biofilm formation in both industrial and medical contexts, particularly for bacteria that pose contamination or infection risks through biofilm production.

How does Glutaredoxin-2 participate in cellular redox homeostasis networks?

Glutaredoxin-2 serves as a key component in maintaining cellular redox homeostasis through several coordinated mechanisms:

The comprehensive integration of Grx2 in redox homeostasis networks positions it as both a sensor and effector in cellular responses to changing redox conditions. This multifunctional role enables cells to adapt their redox management strategies in response to environmental challenges, maintaining essential cellular functions despite fluctuating conditions.

What structural and functional differences exist in Glutaredoxin-2 across bacterial species?

While comparative studies of Glutaredoxin-2 across diverse bacterial species remain limited, several important structural and functional differences have been identified:

These differences highlight the evolutionary adaptation of Glutaredoxin-2 to meet the specific needs of different bacterial species. While maintaining core catalytic functions, Grx2 appears to have acquired specialized roles and regulatory mechanisms tailored to the ecological and physiological context of each organism.

How should researchers interpret variable results in Glutaredoxin-2 activity assays?

When encountering variable or inconsistent results in Glutaredoxin-2 activity assays, researchers should consider multiple factors that may influence the measurements:

• Assay Condition Variables:

  • Buffer composition variations: Ensure consistent concentrations of GSH, HED, NADPH, and glutathione reductase

  • pH fluctuations: Standardize pH conditions as enzyme activity is pH-sensitive

  • Temperature inconsistencies: Maintain constant temperature (typically 25°C) during measurements

  • Reaction timing: Ensure consistent time for mixed disulfide formation before enzyme addition

• Protein Quality Factors:

  • Storage degradation: Verify protein integrity before assays

  • Oxidation state of active site cysteines: Reducing agents may be necessary to maintain catalytic activity

  • Protein concentration accuracy: Use reliable protein quantification methods

  • Purification method differences: Standardize purification protocols across experiments

• Biological Sample Variations:

  • Growth phase differences: Standardize culture conditions and harvesting time

  • Stress exposure history: Prior exposure to stressors may alter enzyme activity

  • Strain background effects: Genetic background may influence measured activity

  • Compensatory mechanisms: Other glutaredoxins may partially compensate for Grx2, especially in knockout studies

• Technical Interpretation Approaches:

  • Statistical rigor: Ensure sufficient replicates (minimum three independent experiments)

  • Positive controls: Include purified Grx2 with known activity

  • Negative controls: Include heat-inactivated samples and buffer-only reactions

  • Standard curves: Develop standard curves using purified enzyme when possible

• Comparative Analysis:

  • Relative vs. absolute measurements: Consider reporting relative activity between experimental and control conditions

  • Multiple measurement techniques: Validate using alternative activity measurement methods

  • Literature comparison: Compare results with published values while considering methodological differences

When interpreting variability, it's important to note that even in ΔgrxB strains, some Grx activity remains (approximately 78-81% of wild-type activity) . This residual activity likely comes from other glutaredoxins and must be accounted for when interpreting results. Careful standardization of experimental conditions and inclusion of appropriate controls are essential for obtaining reliable and reproducible measurements of Glutaredoxin-2 activity.

What common challenges arise in recombinant expression of Glutaredoxin-2?

Recombinant expression of Glutaredoxin-2 presents several challenges that researchers should anticipate and address:

• Protein Folding and Solubility Issues:

  • Research has shown that truncated forms of Grx2 (1-114 and 1-133) form inclusion bodies

  • The full-length protein may also be prone to misfolding and aggregation

  • Implementation strategies:

    • Optimize expression conditions (lower temperature, reduced inducer concentration)

    • Consider solubility-enhancing fusion tags

    • Explore refolding protocols if inclusion bodies form

• Catalytic Activity Preservation:

  • The active site cysteines (C9PYC12) are susceptible to oxidation during purification

  • Improper folding can result in structurally intact but catalytically inactive protein

  • Implementation strategies:

    • Include reducing agents in purification buffers

    • Minimize exposure to oxidizing conditions

    • Verify activity at each purification step

• Structural Integrity Maintenance:

  • The COOH-terminal half of Grx2 is required for activity

  • Proteolytic degradation during purification could result in loss of this essential region

  • Implementation strategies:

    • Include protease inhibitors

    • Minimize processing time

    • Verify protein integrity by SDS-PAGE

• Expression Host Considerations:

  • Endogenous glutaredoxins from the expression host may interfere with characterization

  • Codon usage may affect expression efficiency

  • Implementation strategies:

    • Consider expression in glutaredoxin-deficient hosts

    • Optimize codon usage for the expression system

    • Use tagged constructs for specific purification

• Storage Stability:

  • Purified Grx2 may lose activity during storage

  • Implementation strategies:

    • Store at -20°C as recommended

    • Add stabilizers if necessary

    • Avoid repeated freeze-thaw cycles by preparing aliquots

• Activity Measurement Challenges:

  • Buffer components can artificially influence Grx activity

  • Non-physiological concentrations of GSH or other thiols may affect measurements

  • Implementation strategies:

    • Carefully control buffer composition

    • Include appropriate controls

    • Validate activity under different conditions

Awareness of these common challenges allows researchers to implement appropriate strategies for successful recombinant expression and characterization of Glutaredoxin-2, ensuring they obtain properly folded, active protein suitable for their specific research applications.

How can researchers confirm that observed phenotypes specifically result from grxB deletion?

To establish that observed phenotypes genuinely result from grxB deletion rather than from secondary effects, researchers should implement a comprehensive validation strategy:

• Genetic Complementation:

  • Reintroduce the wild-type grxB gene into the deletion mutant

  • Express the gene under control of its native promoter or an inducible promoter

  • Verify that the complemented strain reverts to the wild-type phenotype

  • This restoration of function provides strong evidence for the specific role of grxB

• Multiple Independent Mutants:

  • Create and characterize multiple independent grxB deletion mutants

  • Confirm consistent phenotypes across all mutants

  • This approach reduces the likelihood that secondary mutations are responsible for observed effects

• Quantitative Functional Assessment:

  • Measure Grx activity in wild-type, mutant, and complemented strains

  • Use the established NADPH-coupled spectrophotometric assay

  • Verify that activity decreases in the mutant and is restored upon complementation

  • The search results show ΔgrxB strains exhibit approximately 78% of wild-type Grx activity under normal conditions and 81% under acid stress

• Molecular Verification:

  • Confirm absence of the grxB gene by PCR

  • Verify absence of Grx2 protein by Western blot if antibodies are available

  • Check that other glutaredoxins (Grx1, Grx3) are unaffected

  • Sequence the mutated region to confirm precise deletion

• Phenotypic Specificity Analysis:

  • Compare with other glutaredoxin deletion mutants (e.g., grxA, grxC)

  • Identify phenotypes unique to grxB deletion versus those common to all glutaredoxin mutants

  • This helps distinguish Grx2-specific functions from general glutaredoxin functions

• Control for Polar Effects:

  • Ensure that deletion of grxB does not affect expression of adjacent genes

  • The search results indicate that grxB is located between rimJ and pyrC genes in E. coli

  • Measure expression of these flanking genes in wild-type and ΔgrxB strains

• Dose-Dependent Response:

  • Express grxB at different levels in the deletion background

  • Establish a correlation between Grx2 expression level and phenotype severity

  • This quantitative relationship provides evidence for direct causality

What control experiments should be included when investigating Glutaredoxin-2 in stress response studies?

When investigating the role of Glutaredoxin-2 in stress responses, particularly acid stress as highlighted in the search results, several essential control experiments should be included:

• Strain Control Panel:

  • Wild-type strain (positive control for normal Grx2 function)

  • ΔgrxB strain (experimental condition lacking Grx2)

  • Complemented ΔgrxB strain (to verify phenotype specificity)

  • Strains with deletions in other redox or stress response systems to distinguish specific effects

• Stress Condition Controls:

  • Untreated conditions (baseline control)

  • Dose-response evaluation with varying stress intensity (e.g., pH range from 7 to 4)

  • Time-course assessment to distinguish immediate vs. adaptive responses

  • Recovery conditions to assess reversibility of effects

• Enzyme Activity Measurements:

  • Measure total Grx activity in all strains under test conditions

  • Include positive control (purified Grx2) in activity assays

  • Include negative control (heat-inactivated lysate) in activity assays

  • Compare activity under stress and non-stress conditions

  • Activity measurements should follow the established NADPH-coupled spectrophotometric assay

• Morphological Analysis Controls:

  • Examine cell morphology at multiple time points (12, 24, 36, and 48 hours)

  • Include both stress and non-stress conditions

  • Use consistent sample preparation techniques (e.g., negative staining with 3% phosphotungstic acid)

  • Analyze multiple fields to ensure representative observations

• Surface Property Assessments:

  • Measure cell surface hydrophobicity under both normal and stress conditions

  • Perform auto-aggregation assays with standardized protocols

  • Include appropriate blanks and calibration controls

  • These measurements provide insight into the physiological impact of stress and grxB deletion

• Biofilm Formation Controls:

  • Assess biofilm formation under both normal and stress conditions

  • Use quantitative methods (crystal violet staining)

  • Include uninoculated media controls

  • These assays reveal functional consequences of stress response mechanisms

• Cross-stress Response Controls:

  • Compare responses to the target stress with responses to other stresses

  • This helps distinguish specific stress responses from general stress effects

  • Particularly relevant for comparing acid stress with oxidative stress, given Grx2's redox functions

These control experiments collectively provide a robust framework for interpreting the role of Glutaredoxin-2 in stress responses, allowing researchers to distinguish Grx2-specific effects from broader stress responses and to identify the mechanisms through which Grx2 contributes to stress tolerance.