Recombinant Pseudomonas syringae pv. syringae Thiosulfate sulfurtransferase glpE (glpE)

What is the function of thiosulfate sulfurtransferase glpE in Pseudomonas syringae?

Thiosulfate sulfurtransferase glpE in P. syringae is believed to catalyze the transfer of a sulfur atom from thiosulfate to a suitable acceptor, typically glutathione (GSH), producing glutathione persulfide (GSSH). This reaction is part of cellular sulfur metabolism and may contribute to redox homeostasis. Based on studies in E. coli, glpE can convert thiosulfate to cellular sulfane sulfur, though with less efficiency than other rhodaneses like PspE . In P. syringae, this enzyme likely functions similarly but may have evolved specialized roles related to plant-pathogen interactions.

The proposed reaction mechanism is:
S₂O₃²⁻ + GSH → SO₃²⁻ + GSSH

What are the optimal conditions for recombinant expression of P. syringae glpE?

For optimal expression of recombinant P. syringae glpE, consider the following protocol:

• Clone the glpE gene into an expression vector containing an appropriate promoter (T7 or tac) and a purification tag (His6 or GST).

• Transform the construct into a suitable E. coli expression strain (BL21(DE3) or similar).

• Grow the culture at 37°C until OD600 reaches 0.6-0.8.

• Induce expression with 0.1-0.5 mM IPTG.

• Lower the temperature to 16-25°C for 4-16 hours post-induction to enhance protein solubility.

When working with rhodanese enzymes, including reducing agents (1-5 mM DTT or β-mercaptoethanol) in all buffers is crucial to maintain the catalytic cysteine in reduced form and prevent enzyme oxidation.

How can recombineering techniques be applied to modify glpE in the P. syringae genome?

Recombineering can be employed to modify the glpE gene directly in the P. syringae genome using the following approach:

• Generate linear DNA fragments containing your desired modification flanked by homology arms (50-1000 bp) to the target region.

• Introduce these fragments into P. syringae cells expressing recombination proteins similar to E. coli's RecET system .

• For P. syringae-specific recombineering, utilize the endogenous recombination machinery identified in P. syringae pv. syringae B728a, which shows functional similarity to RecET proteins from E. coli phages .

• Select for successful recombinants using appropriate antibiotic markers.

• Verify modifications through PCR, sequencing, or functional assays.

This methodology allows for precise genetic manipulations including point mutations, deletions, or insertions in the glpE gene.

What assay methods are recommended for measuring glpE activity in P. syringae extracts?

For measuring thiosulfate:glutathione sulfurtransferase activity of glpE in P. syringae extracts:

• Lead Acetate-Based H₂S Detection:

  • Prepare a reaction mixture containing 0.4 mM lead acetate, 0.5-150 mM thiosulfate, and 1-150 mM GSH in 100 mM HEPES buffer (pH 7.4) .

  • Add cell extract or purified enzyme to initiate the reaction.

  • Incubate at 37°C for 8 minutes.

  • Measure lead sulfide formation at 390 nm.

  • Calculate enzyme activity using an extinction coefficient of 5500 M⁻¹cm⁻¹ .

• Direct GSSH Quantification:

  • Extract cellular thiols and analyze by LC-MS to directly quantify GSSH formation.

  • Compare levels before and after thiosulfate treatment.

  • For bacterial samples, normalize to cell density (OD600).

• Cellular Sulfane Sulfur Measurement:

  • Extract cellular sulfane sulfur using the cold cyanolysis method.

  • Measure thiocyanate formation spectrophotometrically at 460 nm after reaction with ferric nitrate.

  • Convert measurements to cellular concentrations using the conversion factor: 1 mL of bacterial suspension at OD600 of 1 has a 3.6 μL cell volume .

How do environmental conditions affect glpE activity in Pseudomonas syringae?

Environmental conditions significantly affect glpE activity in P. syringae:

• pH Effects: Rhodanese enzymes typically show optimal activity between pH 7.0-8.0, with activity declining sharply below pH 6.0 or above pH 9.0.

• Temperature Influence: As a mesophilic bacterial enzyme, expect optimal activity between 25-37°C, with P. syringae variants potentially showing higher activity at lower temperatures that reflect their plant-associated lifestyle.

• Oxidative Stress: During plant infection, P. syringae encounters oxidative bursts as part of plant defense. Evidence from E. coli suggests that sulfurtransferase activity can provide protection against oxidative stress, as thiosulfate addition alleviates H₂O₂ toxicity in strains with functional rhodanese .

• Substrate Availability: Availability of thiosulfate and GSH in different cellular compartments influences activity. In E. coli, rhodanese activity varies between periplasmic and cytoplasmic compartments , suggesting similar compartmentalization effects may occur in P. syringae.

How does glpE from P. syringae compare functionally with E. coli rhodaneses?

Functional comparison between P. syringae glpE and E. coli rhodaneses reveals several key differences:

In E. coli, PspE is the most effective rhodanese for converting thiosulfate to glutathione persulfide, with a much lower Km value than glpE . The P. syringae glpE likely shows adaptations specific to its plant-pathogen lifestyle, potentially including modified substrate specificity or coupling to pathogenicity systems.

What role might glpE play in P. syringae virulence compared to other sulfurtransferases?

The potential role of glpE in P. syringae virulence likely involves several mechanisms:

• Oxidative Stress Protection: During plant infection, P. syringae faces oxidative bursts produced by host defenses. Sulfurtransferase activity can contribute to oxidative stress resistance, as demonstrated in E. coli where thiosulfate addition protected against H₂O₂ toxicity .

• Interaction with Virulence Systems: P. syringae relies on its type III secretion system (T3SS) for successful colonization . Sulfur metabolism enzymes like glpE might coordinate with these virulence systems, potentially supplying sulfur compounds needed for T3SS assembly or function.

• Phenotypic Heterogeneity Contribution: P. syringae populations show phenotypic heterogeneity during plant colonization . Differential expression of metabolic enzymes like glpE could contribute to this bacterial subpopulation specialization.

• Microcolony Formation: Sulfurtransferases may influence bacterial behavior during microcolony formation in planta, potentially coordinating with motility systems (flagella) that show heterogeneous expression patterns during infection .

How can high-throughput screening approaches be designed to identify inhibitors of P. syringae glpE?

A comprehensive high-throughput screening approach for P. syringae glpE inhibitors should include:

• Primary Enzyme-Based Screen:

  • Express and purify recombinant P. syringae glpE with a His-tag.

  • Establish a 384-well plate format assay using lead acetate-based detection of H₂S production.

  • Screen compound libraries at 10-20 μM against purified enzyme.

  • Define hit threshold as >50% inhibition.

• Secondary Assays:

  • Confirm hits with dose-response curves (IC50 determination).

  • Assess specificity by testing against other rhodaneses.

  • Determine inhibition mechanism (competitive, non-competitive, etc.) using varying substrate concentrations.

• Tertiary Cellular Assays:

  • Test effect of confirmed hits on cellular sulfane sulfur levels in P. syringae cultures.

  • Examine impact on bacterial growth in minimal media with thiosulfate as sole sulfur source.

  • Assess effects on P. syringae plant colonization using appropriate infection models.

• Structure-Activity Relationship Analysis:

  • Group confirmed inhibitors by chemical scaffolds.

  • Perform molecular docking studies if structural information is available.

  • Design and test analogs to improve potency and specificity.

What approaches should be used to investigate the role of glpE in P. syringae stress response and pathogenicity?

To investigate glpE's role in P. syringae stress response and pathogenicity:

• Generate and Characterize Mutant Strains:

  • Create glpE deletion mutants using recombineering approaches similar to those described for other P. syringae genes .

  • Construct complemented strains expressing wild-type glpE and catalytically inactive variants.

  • Develop reporter strains with transcriptional fusions (e.g., glpE::GFP) to monitor expression patterns.

• Oxidative Stress Response Assessment:

  • Compare wild-type and ΔglpE mutant growth under various H₂O₂ concentrations with and without thiosulfate supplementation.

  • Measure intracellular ROS levels using fluorescent probes.

  • Quantify expression of oxidative stress response genes to determine if glpE deletion alters global stress responses.

• Plant Infection Studies:

  • Evaluate bacterial growth curves in planta for wild-type, ΔglpE, and complemented strains.

  • Use confocal microscopy with fluorescent reporter strains to track single-cell behavior during infection.

  • Examine microcolony formation patterns similar to those studied for flagellar expression .

  • Assess plant defense responses triggered by different strains.

• Integration with Other Virulence Systems:

  • Investigate potential coordination between glpE expression and T3SS activity using dual reporter systems.

  • Create double mutants (ΔglpE Δhrp) to study genetic interactions.

  • Examine temporal expression patterns during infection using time-lapse microscopy approaches similar to those used for studying flagellar expression in P. syringae .

How might single-cell analysis techniques reveal heterogeneity in glpE expression during P. syringae infection?

Single-cell analysis of glpE expression during P. syringae infection can be approached through:

• Reporter Strain Construction:

  • Generate a glpE::fluorescent protein transcriptional fusion using methods similar to those described for fliC::GFP3 in P. syringae .

  • Ensure the reporter doesn't disrupt native glpE function.

  • Consider dual reporters to simultaneously monitor glpE and T3SS gene expression.

• Time-Lapse Microscopy Protocol:

  • Prepare agarose pads with plant apoplast-mimicking medium (HIM).

  • Inoculate reporter strains at low density (OD = 0.005).

  • Perform time-lapse imaging at 15-minute intervals for 24 hours at 25°C.

  • Acquire images using appropriate filter sets for your fluorescent proteins.

  • Example settings: GFP (470 nm excitation and 519 nm emission filters), 300 ms, 50% intensity .

• Flow Cytometry Analysis:

  • Recover bacteria from infected plant tissue at different time points.

  • Analyze population heterogeneity using flow cytometry.

  • Gate populations based on fluorescence intensity.

  • Isolate subpopulations through cell sorting for transcriptomic or proteomic characterization.

• Data Analysis Approaches:

  • Track individual cell lineages to determine if glpE expression states are inherited.

  • Quantify switching rates between expression states.

  • Correlate glpE expression with other virulence factors like T3SS components.

  • Perform spatial analysis to determine if expression patterns correlate with position within microcolonies.

This approach could reveal if glpE shows spatial or temporal heterogeneity similar to that observed for flagellar and T3SS expression in P. syringae , potentially indicating specialized roles within bacterial subpopulations during plant infection.