Recombinant Pseudomonas syringae pv. syringae Catalase-peroxidase (katG), partial

What is the function of catalase-peroxidase (katG) in Pseudomonas syringae?

KatG is a bifunctional enzyme that functions primarily to detoxify hydrogen peroxide (H₂O₂) by converting it to water and oxygen. In P. syringae, KatG serves as the major housekeeping catalase, providing approximately 95% of the total catalase activity during normal growth conditions. Research demonstrates that KatG offers significant protection against menadione-generated endogenous H₂O₂, helping maintain bacterial homeostasis under oxidative stress .

KatG also plays a nonredundant role in detoxifying exogenous H₂O₂, such as that produced by plants as part of their defense response. This ability to neutralize plant-derived reactive oxygen species is essential for bacterial survival in the host environment. Experimental evidence confirms that KatG is required for full virulence of P. syringae pv. tomato DC3000 in Arabidopsis thaliana, indicating its critical function in the host-pathogen interaction .

How does katG differ from other catalases in Pseudomonas syringae?

P. syringae possesses three distinct catalases with specialized functions:

These catalases demonstrate nonredundant roles in hydrogen peroxide detoxification. While KatG is the predominant catalase during normal growth, KatB rapidly and substantially accumulates in response to exogenous H₂O₂. This differential expression and function suggest a coordinated response system to various oxidative challenges encountered by the bacterium .

Why is katG considered essential for pathogenesis in Pseudomonas syringae?

KatG is essential for pathogenesis because:

• It enables bacterial survival in the oxidative plant environment by detoxifying plant-produced H₂O₂, which is a key component of plant defense responses.

• Research demonstrates that a P. syringae strain lacking all three catalases (KatB, KatE, and KatG) is severely compromised in its ability to grow in planta.

• Complementation studies show that expression of katG can partially rescue the growth of the catalase triple mutant in planta, confirming its direct role in virulence .

• The nonredundant ability of KatG and KatB to detoxify plant-produced H₂O₂ is essential for bacterial survival in plants, indicating specialized functions that cannot be fully compensated by other enzymes .

What expression systems are commonly used for recombinant katG production?

For the recombinant expression of P. syringae proteins including KatG, the following approach has proven effective:

• Cloning the gene into the pET28a vector, which incorporates a 6×His-tag for purification purposes.

• Expressing the recombinant protein in Escherichia coli BL21 cells, which lack certain proteases and are optimized for protein production.

• Utilizing PCR amplification with primers containing 20-bp sequences identical to the linearized plasmid sequence at the cutting site, followed by recombination to ensure proper gene orientation .

This expression system typically yields sufficient quantities of functional recombinant protein for biochemical and structural studies. The approach mirrors that used successfully for expressing multiple P. syringae transcription factors and could be readily adapted for KatG production .

How is katG expression regulated in Pseudomonas syringae at the transcriptional level?

The transcriptional regulation of katG in P. syringae involves complex regulatory networks. Recent high-throughput studies have identified binding motifs for 100 out of 301 transcription factors in P. syringae using HT-SELEX technology. While specific regulators of katG were not directly identified in the provided search results, the regulatory architecture of P. syringae suggests potential master regulators .

Many virulence-associated genes in P. syringae exhibit cross-regulation, suggesting katG might be under the control of virulence-associated master regulators. For instance, transcription factors such as MetR, PSPPH_1800, and other regulators involved in oxidative stress response pathways may influence katG expression. Additionally, ROS-responsive regulatory elements likely play a role in modulating katG expression under oxidative stress conditions .

The methodology to identify these regulatory interactions includes:

• HT-SELEX (High-Throughput Systematic Evolution of Ligands by Exponential Enrichment) to determine transcription factor binding motifs

• Genome scanning using position weight matrices (PWMs) to identify potential binding sites

• ChIP-seq and electrophoretic mobility shift assays (EMSA) to validate predicted interactions

• RT-qPCR and reporter assays to confirm functional regulation

What methodological approaches can quantify katG activity in experimental systems?

Research protocols to measure KatG activity include:

• Spectrophotometric Assays: Monitoring the decomposition of H₂O₂ at 240 nm, where the decrease in absorbance correlates with catalase activity. This can be performed with bacterial lysates or purified enzyme.

• Spot Dilution Assays: As described in research on P. syringae oxidative sensitivity:

  • Harvest overnight bacterial cultures by centrifugation

  • Wash cells twice with PBS

  • Adjust to OD₆₀₀ = 1

  • Prepare tenfold serial dilutions

  • Spot 5 μl of each dilution on plates containing different H₂O₂ concentrations (0, 0.25, 0.5 mM)

  • Incubate at 28°C for 2 days

  • Compare growth patterns between wild-type and mutant strains

• In Planta Growth Assays: These assess the contribution of KatG to bacterial survival under the oxidative stress encountered during plant infection:

  • Inoculate plant tissues with defined bacterial concentrations

  • Extract bacteria at various time points

  • Determine colony-forming units (CFUs) to quantify bacterial growth

  • Compare growth of wild-type, katG mutant, and complemented strains

How can recombineering techniques be applied to manipulate katG in Pseudomonas syringae?

Recombineering offers powerful approaches for precise genetic manipulation of katG in P. syringae:

• RecTE-mediated Recombination: P. syringae homologs of the lambda Red Exo/Beta and RecET proteins have been identified and characterized. These proteins can facilitate genomic recombination of linear DNA introduced by electroporation:

  • The RecT homolog alone is sufficient for recombination of single-stranded DNA oligonucleotides

  • Efficient recombination of double-stranded DNA requires expression of both RecT and RecE homologs

• Targeted Gene Disruption Protocol:

  • Design DNA fragments with homology to regions flanking the katG gene

  • Electroporate the linear DNA into P. syringae cells expressing RecT and RecE

  • Select for recombinants using appropriate markers

  • Confirm modifications by PCR and sequencing

• Allelic Replacement Strategy:

  • Create a construct containing the modified katG gene

  • Integrate the construct into the chromosome via recombination

  • Counter-select against the vector backbone

  • Confirm the desired modifications

This recombineering system provides a powerful tool for creating precise modifications to katG, including point mutations, deletions, and insertions, facilitating detailed structure-function studies.

What is the relationship between katG and the type III secretion system (T3SS) in Pseudomonas syringae pathogenesis?

The relationship between KatG and the T3SS represents a critical interface in P. syringae pathogenesis, although their interaction is complex and multifaceted. Research on P. syringae transcription factors revealed an intricate regulatory network connecting different virulence pathways .

In P. syringae, virulence depends largely on the T3SS, which delivers effector proteins into host cells. The T3SS component genes are clustered in a 25-kb pathogenicity-related island, while most effector genes are dispersed throughout the genome. Regulatory studies have identified 45 transcription factors that potentially control 46 T3SS genes .

While the direct regulatory link between katG and T3SS wasn't explicitly detailed in the search results, evidence suggests extensive crosstalk between different virulence systems. For example:

• Transcription factors like PilR bind promoter regions of genes associated with multiple virulence pathways, including T3SS and oxidative stress response.

• RhpR regulates genes involved in both T3SS and flagella-mediated motility, demonstrating the interconnected nature of these pathways.

• The metabolism-associated transcription factors TrpI and GntR directly regulate virulence factors, including ROS management systems and T3SS components .

This suggests that KatG likely functions within a coordinated virulence program, where its ability to detoxify ROS complements T3SS-mediated effector delivery, collectively enabling successful host colonization.

How do post-translational modifications affect katG function in Pseudomonas syringae?

While the search results don't provide specific information on post-translational modifications (PTMs) of KatG in P. syringae, bifunctional catalase-peroxidases are known to undergo several important modifications that affect their function. Based on general knowledge of catalase-peroxidases and related research:

• Heme Incorporation: KatG requires proper incorporation of heme groups for catalytic activity. The maturation process involves specific chaperones and cofactors.

• Oxidative Modifications: During catalytic cycling, KatG can undergo oxidative modifications that may either enhance or inhibit its activity. These modifications can serve as feedback mechanisms to regulate enzyme function during oxidative stress.

• Potential Phosphorylation Sites: Catalase-peroxidases may contain phosphorylation sites that influence activity, stability, or protein-protein interactions. Signal transduction pathways responsive to environmental stresses likely modulate KatG function through phosphorylation.

Methodological approaches to study these modifications include:

• Mass spectrometry to identify specific PTMs

• Site-directed mutagenesis to evaluate the functional significance of modified residues

• Activity assays comparing native and recombinant enzymes under various conditions

What are the optimal conditions for purifying active recombinant katG?

Based on successful approaches for purifying P. syringae proteins, the following protocol represents an effective strategy for obtaining active recombinant KatG:

• Expression System Selection:

  • Clone the katG gene into pET28a vector with an N-terminal 6×His-tag

  • Transform into E. coli BL21 cells for expression

  • Induce protein expression at lower temperatures (16-20°C) to enhance proper folding

• Lysis and Initial Purification:

  • Resuspend cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol

  • Add protease inhibitors to prevent degradation

  • Lyse cells using sonication or French press

  • Clarify lysate by centrifugation at 20,000×g for 30 minutes

• Affinity Chromatography:

  • Bind 6×His-tagged KatG to Ni-NTA resin

  • Wash with buffer containing 20-50 mM imidazole to remove non-specifically bound proteins

  • Elute KatG with buffer containing 250 mM imidazole

• Secondary Purification:

  • Further purify using size exclusion chromatography

  • Buffer exchange to remove imidazole and adjust to optimal storage conditions

• Quality Control:

  • Verify purity by SDS-PAGE

  • Confirm identity by Western blot and/or mass spectrometry

  • Measure specific activity using standard catalase assays

Critical considerations include maintaining reducing conditions throughout purification to protect the heme center and including glycerol in buffers to enhance protein stability.

How can researchers design experiments to distinguish between the catalase and peroxidase activities of katG?

Distinguishing between catalase and peroxidase activities of KatG requires targeted experimental approaches:

• Spectrophotometric Assays:

  • Catalase Activity: Measure the decomposition of H₂O₂ at 240 nm, where the decrease in absorbance corresponds to H₂O₂ breakdown.

  • Peroxidase Activity: Use chromogenic substrates like ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or o-dianisidine, which develop color in the presence of peroxidase activity and H₂O₂.

• Selective Inhibition:

  • Treat KatG with 3-amino-1,2,4-triazole to inhibit catalase activity

  • Use sodium azide at low concentrations to preferentially inhibit peroxidase activity

  • Compare activity profiles before and after inhibition

• Substrate Specificity Analysis:

  Activity	Substrate	Product	Detection Method
  Catalase	H₂O₂	H₂O + O₂	O₂ evolution or H₂O₂ consumption
  Peroxidase	H₂O₂ + electron donor	Oxidized donor + H₂O	Spectrophotometric detection of oxidized donor

• Site-Directed Mutagenesis:

  • Identify and mutate key residues predicted to affect either catalase or peroxidase activity

  • Measure both activities in mutant proteins to determine differential effects

• pH Profiling:

  • Determine the pH optima for both catalase and peroxidase activities

  • Use buffer conditions that favor one activity over the other

These methods collectively provide a comprehensive assessment of the bifunctional nature of KatG and enable researchers to investigate how each activity contributes to bacterial virulence and survival.

What are the main challenges in studying structure-function relationships in P. syringae katG?

Several significant challenges exist in studying structure-function relationships of P. syringae KatG:

• Protein Stability Issues: Catalase-peroxidases can be sensitive to oxidative damage and denaturation during purification and crystallization attempts. Maintaining the integrity of the heme center is particularly challenging.

• Functional Redundancy: The presence of multiple catalases in P. syringae (KatB, KatE, and KatG) complicates the interpretation of in vivo studies. While KatG contributes approximately 95% of catalase activity in vitro, the functional significance of each catalase varies depending on environmental conditions .

• Complex Regulation: P. syringae contains numerous transcription factors that potentially regulate katG expression. The study identifying binding motifs for 100 transcription factors highlights the complexity of gene regulation in this organism .

• Post-Translational Processing: Proper folding and incorporation of the heme cofactor are essential for activity but can be difficult to control in recombinant expression systems.

• Methodological Limitations: Techniques for genetic manipulation in P. syringae have been limited compared to model organisms like E. coli, though recent advances in recombineering offer new opportunities .

How can comparative genomics inform our understanding of katG evolution in Pseudomonas species?

Comparative genomics approaches can provide valuable insights into katG evolution and specialization:

• Phylogenetic Analysis: Construct phylogenetic trees based on katG sequences across Pseudomonas species to identify evolutionary relationships and potential horizontal gene transfer events.

• Synteny Analysis: Examine the genomic context of katG across species to identify conserved gene neighborhoods that might suggest functional associations or co-regulation.

• Selection Pressure Analysis: Calculate dN/dS ratios to identify regions under positive or purifying selection, indicating functional constraints or adaptive evolution.

• Structure Prediction Comparison: Use homology modeling to predict structural differences between KatG proteins from different Pseudomonas species, particularly focusing on active site residues.

• Transcription Factor Binding Site Analysis: Apply the position weight matrices (PWMs) generated from studies like the one described in search result to identify conserved regulatory elements in katG promoters across Pseudomonas species.

This comparative approach could reveal how KatG has evolved specialized functions in plant pathogens like P. syringae compared to other Pseudomonas species inhabiting different ecological niches.

What are the broader implications of understanding katG function for plant disease management?

Understanding KatG function in P. syringae has significant implications for developing novel strategies to manage plant diseases:

• Targeted Inhibitor Development: Detailed knowledge of KatG structure and function could enable the design of specific inhibitors that compromise bacterial survival in planta without affecting beneficial microorganisms.

• Host Resistance Engineering: Understanding how plants recognize and respond to bacterial catalases could inform breeding programs or genetic engineering approaches to enhance plant immune responses against P. syringae.

• Diagnostic Tools: KatG-based detection methods could provide rapid, specific diagnosis of P. syringae infections, enabling early intervention.

• Predictive Models: Knowledge of how environmental conditions affect katG expression and bacterial virulence could improve disease forecasting models.

• Biocontrol Strategies: Engineering competing microorganisms to interfere with KatG function could create effective biocontrol agents.