Recombinant Pseudomonas syringae pv. tomato Sulfoxide reductase catalytic subunit YedY (yedY)

What is YedY and what is its function in Pseudomonas syringae pv. tomato?

YedY, now more commonly referred to as MsrP, is a periplasmic methionine sulfoxide reductase. Based on homology with the well-characterized MsrP in Escherichia coli, we can infer that in Pseudomonas syringae pv. tomato, this enzyme functions to reduce methionine sulfoxide residues in proteins located in the periplasmic space. MsrP works in conjunction with MsrQ, an inner membrane heme-binding quinol dehydrogenase, to carry out this reduction reaction .

The reaction catalyzed can be generally described as:

Protein-L-methionine-(R)-S-oxide + electron-transfer quinol → Protein-L-methionine + electron-transfer quinone + H₂O

and

Protein-L-methionine-(S)-S-oxide + electron-transfer quinol → Protein-L-methionine + electron-transfer quinone + H₂O

This enzyme plays a crucial role in protecting bacterial proteins from oxidative damage, particularly in the periplasmic space where exposure to reactive oxygen species from the environment is more likely during plant-pathogen interactions.

How does YedY expression differ across Pseudomonas syringae pathovars?

While specific expression data for YedY across different P. syringae pathovars is not directly addressed in the search results, we can draw inferences from the evolutionary relationships between these pathovars. P. syringae pv. tomato strain DC3000 (PtoDC3000) is phylogenetically distinct from typical P. syringae pv. tomato strains and more closely related to P. syringae pv. maculicola isolates from Brassicaceae species .

This phylogenetic distinction suggests that YedY expression patterns might differ between:

• Typical P. syringae pv. tomato strains (isolated from tomato, pathogenic only on tomato)

• PtoDC3000-like strains (isolated from various Brassicaceae and Solanaceae, with broader host range)

The different host ranges and environmental adaptations of these pathovars likely influence the expression patterns of stress-response proteins like YedY. Researchers should consider these phylogenetic relationships when studying YedY expression in different P. syringae strains, as the regulation might reflect adaptation to specific host environments and oxidative stress conditions.

What techniques are recommended for purifying recombinant YedY?

Purification of recombinant YedY from Pseudomonas syringae pv. tomato requires specialized techniques due to its periplasmic localization. While the search results don't provide specific purification protocols for this enzyme, the following methodological approach is recommended based on general principles for periplasmic protein purification:

Step-by-step purification protocol:

• Periplasmic extraction:

  • Perform osmotic shock by resuspending cells in a hypertonic solution (20% sucrose, 30 mM Tris-HCl, pH 8.0, 1 mM EDTA)

  • Incubate on ice for 10 minutes

  • Collect cells by centrifugation and rapidly resuspend in ice-cold 5 mM MgSO₄

  • Gently agitate for 10 minutes on ice

  • Centrifuge to separate periplasmic fraction (supernatant) from spheroplasts

• Initial purification:

  • Apply periplasmic extract to an appropriate affinity column (if recombinant YedY contains an affinity tag)

  • For His-tagged constructs, use Ni-NTA or IMAC chromatography

  • Include 5-10 mM imidazole in binding buffer to reduce non-specific binding

• Secondary purification:

  • Pool YedY-containing fractions and apply to ion-exchange column

  • Anion exchange chromatography (e.g., Q-Sepharose) is typically suitable

  • Elute with a linear NaCl gradient (0-500 mM)

• Final polishing:

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Store purified YedY in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, with 10% glycerol for stability

Each step should be optimized for the specific recombinant YedY construct, with activity assays performed to monitor purification efficiency and protein stability.

How does recombination influence the evolution of YedY in Pseudomonas syringae populations?

Recombination plays a significant role in the evolution of Pseudomonas syringae, including genes like YedY. Analysis of P. syringae pv. tomato and related isolates revealed that recombination contributed more than mutation to genetic variation between closely related isolates .

The ratio of recombination to mutation (ρ/θ) for various genes in P. syringae showed that recombination was approximately five times more influential than mutation in generating genetic diversity . This has important implications for YedY evolution:

These data suggest that YedY in P. syringae likely experiences significant recombination events, particularly between closely related isolates. This may facilitate:

• Transfer of adaptive variants between pathovars

• Acquisition of substrate specificity modifications

• Adaptation to different oxidative stress conditions in various plant hosts

Researchers studying YedY evolution should employ multilocus sequence typing (MLST) approaches similar to those used in broader P. syringae genomic studies to detect recombination events specifically affecting this gene .

What is the relationship between YedY function and phage resistance in Pseudomonas syringae?

The relationship between YedY function and phage resistance represents an unexplored but potentially significant research area. Recent studies on Pseudomonas syringae pathovar syringae (Pss) show that bacteria rapidly develop resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways .

Since both LPS biosynthesis and YedY function occur in the periplasmic/membrane interface, there may be functional interactions between these systems:

• Potential mechanistic connections:

  • LPS modifications might alter the periplasmic environment affecting YedY function

  • Oxidative stress during phage infection could increase demand for YedY activity

  • YedY might protect LPS biosynthesis enzymes from oxidative damage

• Experimental approaches to investigate this relationship:

  • Generate YedY knockout mutants and assess phage susceptibility patterns

  • Examine YedY expression during phage infection using qRT-PCR or reporter constructs

  • Perform comparative proteomics of wild-type and ΔyedY strains during phage infection

  • Analyze the LPS profiles of strains with varying YedY expression levels

• Coevolutionary implications:

  • Phage pressure might select for variants of YedY with altered activity or regulation

  • YedY function might influence the fitness costs associated with phage resistance mutations

This represents an important intersection between oxidative stress responses, periplasmic protein function, and phage-bacterial coevolution that could yield insights into bacterial adaptation mechanisms .

What methodological challenges exist in assaying YedY activity in vitro?

Assaying YedY activity presents significant methodological challenges due to its periplasmic localization and dependence on membrane-associated components. Several technical considerations must be addressed:

• Reconstitution of the complete enzyme system:

  • YedY (MsrP) requires its partner protein MsrQ, which is membrane-bound

  • A functional assay requires both components and appropriate electron donors

  • The MsrP-MsrQ system uses quinols as electron donors, which are hydrophobic and require special handling

• Substrate selection challenges:

  • Natural substrates are periplasmic proteins with oxidized methionine residues

  • Both R and S diastereomers of methionine sulfoxide must be considered

  • Synthetic peptide substrates may not fully recapitulate natural activity

• Detection methods:

  • Direct monitoring of methionine sulfoxide reduction is challenging

  • Coupled assays monitoring quinone formation are possible but complex

Recommended activity assay protocol:

Researchers should validate their assay system using controls including heat-inactivated enzyme, known inhibitors, and comparison with E. coli MsrP activity where possible .

How does horizontal gene transfer affect YedY distribution across bacterial species?

Horizontal gene transfer (HGT) likely influences the distribution and evolution of YedY across bacterial species, particularly within the Pseudomonas genus. The search results indicate that recombination plays a significant role in P. syringae evolution, suggesting that genes like yedY might be subject to HGT events .

• Phylogenetic analysis approach:

  • Researchers should construct phylogenetic trees based on YedY sequences from diverse bacterial species

  • Compare YedY trees with species trees based on conserved markers

  • Incongruence between trees may indicate HGT events

  • Apply computational methods to detect recombination breakpoints within or near yedY

• Genomic context analysis:

  • Examine the genomic neighborhood of yedY across species

  • Look for signatures of mobile genetic elements (insertion sequences, transposons)

  • Analyze GC content and codon usage patterns as indicators of recent HGT

• Functional implications of HGT:

  • Transfer between species might introduce variants with altered substrate specificity

  • Acquisition of yedY might contribute to adaptation to new ecological niches

  • Co-transfer with other oxidative stress response genes could provide selective advantages

The high rate of recombination observed in P. syringae (ρ/θ > 5) suggests that HGT events affecting yedY might be relatively common, particularly between closely related strains or species . This has important implications for understanding the distribution of functional variants across bacterial taxa and their potential contributions to ecological adaptation.

What structural features determine YedY substrate specificity across different Pseudomonas species?

Understanding the structural determinants of YedY substrate specificity across Pseudomonas species requires detailed structural and biochemical analysis. While the search results don't provide specific structural information about YedY in P. syringae, we can outline a research approach to investigate this question:

• Comparative structural analysis:

  • Obtain crystal structures or generate homology models of YedY from different Pseudomonas species

  • Identify variations in the active site and substrate-binding regions

  • Use molecular docking to predict interactions with various methionine sulfoxide-containing substrates

• Structure-guided mutagenesis:

  • Create chimeric proteins by swapping domains between YedY orthologs from different species

  • Perform site-directed mutagenesis of residues predicted to determine specificity

  • Measure activity against a panel of substrates to correlate structural features with specificity

• Substrate profiling experiments:

  • Develop a methionine sulfoxide-containing peptide library

  • Screen YedY orthologs against this library to identify preferred sequence contexts

  • Perform proteomics to identify natural substrates in the periplasm of different Pseudomonas species

The E. coli YedY (MsrP) has the unique ability to reduce both R and S diastereomers of methionine sulfoxide , and determining whether this capability is conserved in P. syringae YedY would provide important insights into its functional evolution.

Potential structural features that might determine specificity include:

• Active site architecture and accessibility

• Surface charge distribution affecting protein-protein interactions

• Conformational flexibility accommodating different substrate orientations

• Binding pockets for specific amino acid sequences surrounding methionine sulfoxide