Recombinant Pseudomonas syringae pv. tomato Probable cytosol aminopeptidase (pepA)

What is the pepA gene in Pseudomonas syringae pv. tomato and what protein does it encode?

The pepA gene in Pseudomonas syringae pv. tomato encodes a cytosol aminopeptidase that belongs to the M17 family of leucine aminopeptidases. These enzymes are metallopeptidases that catalyze the removal of amino acids, particularly leucine, from the N-terminus of proteins and peptides. The PepA protein is approximately 500 amino acids in length and contains critical conserved regions that are essential for its enzymatic function . The enzyme plays important roles in protein turnover, nitrogen metabolism, and potentially in pathogenicity mechanisms during plant infection.

What are the key conserved regions in the pepA gene that researchers should focus on when designing primers?

When designing primers for the pepA gene, researchers should focus on two highly conserved regions identified through sequence analysis: "EVLNTDAEG" and "HLDIAGTA" . The first region (EVLNTDAEG) contains critical zinc-binding residues, specifically the sixth amino acid (aspartic acid; "D") and the eighth amino acid (glutamic acid; "E"), which are essential for the protein's catalytic activity . The second region (HLDIAG) is also highly conserved across different bacterial species, although its specific function remains unknown . Additionally, researchers should ensure their amplicons include the catalytic arginine residue, which has been found in all retrieved pepA clones in previous studies .

How does Pseudomonas syringae pv. tomato affect tomato plants, and what role might pepA play in this interaction?

Pseudomonas syringae pv. tomato is a bacterial pathogen that causes bacterial speck disease in tomato plants, resulting in necrotic lesions on leaves, stems, and fruit. The pathogen establishes itself in the plant tissues and causes disease symptoms that can significantly reduce crop yield and quality . While the specific role of pepA in pathogenicity has not been fully elucidated in the provided search results, aminopeptidases like PepA may contribute to the pathogen's ability to utilize plant-derived peptides as nutrient sources, assist in biofilm formation, or participate in the processing of proteins involved in virulence. Understanding the role of pepA could provide insights into potential targets for disease management strategies.

What are the most effective PCR protocols for amplifying the pepA gene from Pseudomonas syringae pv. tomato samples?

For efficient amplification of the pepA gene from Pseudomonas syringae pv. tomato, researchers should use the CODEHOP (Consensus-Degenerate Hybrid Oligonucleotide Primer) approach targeting the conserved regions. Based on successful protocols in related research, the following primer set is recommended:

Forward primer (pepAf-codehop): 5′-CGAGGTGCTGAACACCGAYGCNGARGG-3′
Reverse primer (pepAr-codehop): 5′-GCGGTGCCGGCGAYRTCNADRTG-3′

These primers target the conserved regions "EVLNTDAEG" and "HLDIAGTA" respectively, and have been demonstrated to successfully amplify pepA genes from diverse bacterial species, producing amplicons of approximately 370 bp without non-specific bands . PCR conditions should include an initial denaturation step at 95°C for 5 minutes, followed by 30-35 cycles of denaturation (95°C, 30 seconds), annealing (55-58°C, 30 seconds), and extension (72°C, 45 seconds), with a final extension at 72°C for 7 minutes. For difficult templates, adding DMSO (5%) or adjusting magnesium concentration may improve amplification.

How can researchers express and purify recombinant PepA protein from Pseudomonas syringae pv. tomato for functional studies?

For expression and purification of recombinant PepA from Pseudomonas syringae pv. tomato, researchers should follow this methodological approach:

• Gene synthesis or PCR amplification: Amplify the full-length pepA gene (approximately 1500 bp) using high-fidelity polymerase and primers with appropriate restriction sites.

• Cloning strategy: Insert the pepA gene into an expression vector (pET or pGEX systems are commonly used) that provides an affinity tag (His6 or GST) for purification.

• Expression conditions: Transform the construct into E. coli BL21(DE3) or Rosetta strains. Grow cultures at 37°C until OD600 reaches 0.6-0.8, then induce with IPTG (0.1-1.0 mM). For optimal soluble protein yield, lower the post-induction temperature to 16-20°C for overnight expression.

• Purification protocol: Lyse cells using sonication in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors. For His-tagged proteins, purify using Ni-NTA affinity chromatography followed by size exclusion chromatography. For optimal enzyme activity, include 0.1 mM ZnCl2 in purification buffers to ensure the metal cofactor remains bound.

• Quality control: Assess protein purity using SDS-PAGE and verify enzyme activity using leucine-p-nitroanilide as a substrate in an aminopeptidase activity assay.

This approach yields purified recombinant PepA protein suitable for crystallization, enzymatic characterization, and protein-protein interaction studies.

What enzymatic assays can be used to characterize the activity of recombinant PepA protein?

To characterize the enzymatic activity of recombinant PepA protein from Pseudomonas syringae pv. tomato, researchers should employ the following assays:

• Spectrophotometric assay using leucine-p-nitroanilide (Leu-pNA): This is the standard assay for aminopeptidase activity. The reaction contains 1 mM Leu-pNA, purified enzyme (0.1-1 μg), and reaction buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM ZnCl2). The release of p-nitroaniline is monitored at 405 nm over time. Enzyme kinetics parameters (Km, Vmax, kcat) can be determined by varying substrate concentration.

• Fluorescent assay using leucine-7-amido-4-methylcoumarin (Leu-AMC): For higher sensitivity, this assay measures the release of fluorescent AMC (excitation 380 nm, emission 460 nm) when the enzyme cleaves the substrate.

• Substrate specificity profiling: Test activity against a panel of substrates with different N-terminal amino acids (e.g., Ala-pNA, Phe-pNA, Met-pNA) to determine substrate preference.

• Metal dependence characterization: Assess enzyme activity after treatment with EDTA to remove metal ions, followed by reconstitution with various divalent metals (Zn2+, Co2+, Mn2+) to determine the optimal cofactor.

• pH and temperature optima determination: Measure activity across pH range 5.0-9.0 and temperatures 25-60°C to establish optimal conditions.

These assays provide comprehensive characterization of the recombinant PepA's catalytic properties and can be used to compare the enzyme with homologs from other bacterial species.

How does the tomato microbiome protect against Pseudomonas syringae pv. tomato infection, and how might this interact with pepA function?

Research has demonstrated that the tomato seed-associated epiphytic microbiome can effectively protect seedlings against Pseudomonas syringae pv. tomato establishment and disease progression . This protective effect is primarily mediated by specific bacterial species, notably members of the genus Pantoea, including Pantoea agglomerans and Pantoea dispersa . These beneficial bacteria likely employ multiple mechanisms to inhibit Pseudomonas syringae pv. tomato, including:

• Competitive exclusion: Beneficial bacteria colonize plant surfaces, limiting available space and nutrients for pathogens.

• Production of antimicrobial compounds: Pantoea species may produce bacteriocins or other antimicrobial molecules that directly inhibit Pseudomonas growth.

• Induction of systemic resistance: The beneficial microbiome may trigger the plant's natural defense systems, priming it against pathogen attack.

The interaction between these protective mechanisms and pepA function represents an intriguing avenue for research. PepA, as an aminopeptidase, may be involved in peptide signaling or processing of virulence factors. The beneficial microbiome might interfere with these processes, potentially by producing inhibitors of PepA activity or by modifying the substrates that PepA targets. Alternatively, competing beneficial bacteria might produce their own aminopeptidases that outcompete the pathogen's enzymes for available substrates.

What techniques can be used to study the expression of pepA in Pseudomonas syringae pv. tomato during infection of tomato plants?

To comprehensively investigate pepA expression in Pseudomonas syringae pv. tomato during tomato infection, researchers should employ the following methodological approaches:

• Quantitative RT-PCR (RT-qPCR): Design pepA-specific primers to quantify transcript levels at different infection stages. Reference genes like gyrA, rpoD, or 16S rRNA should be used for normalization. Sample collection should include multiple timepoints (0, 6, 12, 24, 48, 72 hours post-infection) to capture expression dynamics.

• Transcriptomics (RNA-Seq): Perform whole-transcriptome analysis to place pepA expression in the context of global gene expression patterns. This approach provides insights into co-regulated genes and potential regulatory networks.

• Promoter-reporter fusions: Generate a pepA promoter-GFP or pepA promoter-luciferase fusion construct and introduce it into Pseudomonas syringae pv. tomato. This allows real-time monitoring of pepA expression in planta using confocal microscopy or luminescence imaging.

• Proteomics approaches: Use LC-MS/MS to quantify PepA protein levels in bacterial cells isolated from infected plant tissues. Stable isotope labeling (SILAC or iTRAQ) can provide accurate quantification across different infection stages.

• In situ hybridization or immunolocalization: Detect pepA transcripts or protein directly in infected plant tissues to determine the spatial distribution of expression within the infection site.

By combining these complementary approaches, researchers can develop a comprehensive understanding of pepA regulation during the infection process and identify environmental or host-derived signals that modulate its expression.

What is the diversity of pepA genes across bacterial phyla, and how can researchers study this diversity?

Research has revealed unexpected diversity of pepA genes across bacterial phyla. Studies using universal PCR primers for pepA detection have identified these genes in a remarkably broad range of prokaryotes spanning multiple bacterial phyla including Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes, as well as the archaeal phylum Thaumarchaeota .

To study this diversity, researchers should employ the following methodological approach:

• PCR amplification using universal primers: The primers targeting conserved regions (pepAf-codehop and pepAr-codehop) have proven effective for detecting pepA genes across diverse microbial communities .

• Clone library construction and sequencing: PCR products can be cloned and sequenced to assess pepA diversity in environmental samples.

• Phylogenetic analysis: Translated amino acid sequences should be aligned with known PepA sequences, and phylogenetic trees constructed using maximum likelihood or Bayesian methods to determine evolutionary relationships.

• Functional domain analysis: Sequences should be examined for conserved catalytic and metal-binding residues to predict functional properties.

• Metagenomic approaches: Shotgun metagenomics followed by targeted extraction of pepA sequences provides a culture-independent method for assessing diversity.

The diversity of pepA genes suggests these enzymes play important roles across various ecological niches and microbial lifestyles. Both r-strategist (fast-growing) and K-strategist (slow-growing) prokaryotes appear to possess pepA genes, indicating their importance in diverse life history strategies .

How do sequence variations in the pepA gene correlate with functional differences in PepA enzymes from different bacterial species?

Sequence variations in pepA genes across bacterial species can significantly impact the functional properties of the resulting PepA enzymes. These correlations between sequence and function can be analyzed through several methodological approaches:

• Phylogenetic analysis coupled with functional data: Map functional properties onto phylogenetic trees to identify patterns of functional evolution and potential cases of convergent evolution.

Through these approaches, researchers can establish structure-function relationships that explain how sequence divergence has led to functional specialization of PepA enzymes across different bacterial taxa, potentially reflecting adaptation to different ecological niches or pathogenic lifestyles.

How can CRISPR-Cas9 technology be utilized to study the role of pepA in Pseudomonas syringae pv. tomato virulence?

CRISPR-Cas9 technology offers powerful approaches for investigating the role of pepA in Pseudomonas syringae pv. tomato virulence. Researchers can implement the following methodological strategy:

• CRISPR-Cas9 system selection: For Pseudomonas syringae pv. tomato, researchers should use the pCas-Ps system, which has been optimized for Pseudomonas species. This system typically includes a codon-optimized Cas9 under the control of an inducible promoter and a separate plasmid for guide RNA expression.

• Guide RNA design: Design 2-3 guide RNAs targeting different regions of the pepA gene using tools like CHOPCHOP or E-CRISP. Target sequences should be 20 nucleotides long, immediately upstream of a PAM sequence (NGG for SpCas9), and should be checked for off-target effects using the Pseudomonas syringae pv. tomato genome.

• Gene knockout strategy: For complete gene deletion, design guide RNAs targeting the 5' and 3' ends of the pepA gene, and provide a template for homology-directed repair that includes antibiotic resistance markers flanked by homology arms (~500-1000 bp).

• Precise point mutations: To study specific functional residues (such as the zinc-binding aspartic acid and glutamic acid), design guide RNAs targeting these regions and provide repair templates with the desired mutations.

• Validation of mutants: Confirm successful editing using PCR, sequencing, and Western blotting to verify the absence of pepA expression or the presence of mutated proteins.

• Phenotypic analysis: Compare the virulence of wild-type and pepA mutant strains using:

  • In vitro growth curves in minimal and rich media

  • Plant infection assays (measuring bacterial population sizes and disease symptoms)

  • Biofilm formation assays

  • Proteomic analysis to identify changes in protein expression profiles

• Complementation studies: Reintroduce wild-type pepA or site-directed mutants to confirm that observed phenotypes are specifically due to pepA modification.

This comprehensive approach allows researchers to definitively determine the contribution of pepA to bacterial virulence and potentially identify specific functions of the enzyme during the infection process.

What are the methodological approaches for identifying small molecule inhibitors of PepA that could be developed as antimicrobial agents against Pseudomonas syringae pv. tomato?

To identify and develop small molecule inhibitors of PepA as potential antimicrobial agents against Pseudomonas syringae pv. tomato, researchers should implement a multi-stage drug discovery pipeline:

• High-throughput screening approach:

  • Establish a robust enzymatic assay using purified recombinant PepA and fluorogenic substrates (Leu-AMC) in 384-well plate format

  • Screen diverse chemical libraries (10,000-100,000 compounds) at single concentrations (10-20 μM)

  • Identify hits showing >50% inhibition for follow-up testing

• Dose-response characterization:

  • Perform 8-12 point dose-response curves (0.01-100 μM) with hit compounds

  • Calculate IC50 values and Hill slopes to prioritize compounds with potent inhibition (IC50 < 1 μM)

  • Counter-screen against human aminopeptidases to identify selective inhibitors

• Structure-activity relationship (SAR) studies:

  • Synthesize and test analogs of promising hit compounds

  • Identify chemical moieties critical for activity and selectivity

  • Optimize potency while maintaining favorable physicochemical properties

• Structural biology approaches:

  • Obtain crystal structures of PepA in complex with lead inhibitors

  • Use structure-based design to further optimize inhibitor binding

  • Employ molecular dynamics simulations to understand binding mechanisms

• In vitro antimicrobial testing:

  • Evaluate inhibitors for antimicrobial activity against Pseudomonas syringae pv. tomato cultures (MIC determination)

  • Assess spectrum of activity against other plant pathogens

  • Determine bactericidal vs. bacteriostatic properties

• Plant protection studies:

  • Test lead compounds for protection of tomato plants in greenhouse conditions

  • Evaluate phytotoxicity and impact on beneficial microbiota

  • Determine optimal application methods and formulations

Results from this systematic approach could be presented in a table format for comparison:

*Selectivity Index = IC50 against human aminopeptidase / IC50 against P. syringae PepA

This comprehensive pipeline enables the identification of potent, selective PepA inhibitors with potential for development as agricultural antimicrobials for sustainable crop protection.

How do the structural and functional properties of PepA from Pseudomonas syringae pv. tomato compare with those from other plant pathogens?

A comparative analysis of PepA enzymes from Pseudomonas syringae pv. tomato and other plant pathogens reveals important similarities and differences in their structural and functional properties. The following methodological approach provides insights into these comparisons:

• Expression pattern analysis: Transcriptomic and proteomic analysis of various plant pathogens reveals different expression patterns of pepA during infection, suggesting pathogen-specific roles. In some pathogens, pepA expression is constitutive, while in others it may be induced during specific infection stages or in response to certain host conditions.

• Functional significance in pathogenicity: Gene knockout studies across different plant pathogens show variable impacts of pepA deletion on virulence, suggesting that while the enzyme is structurally conserved, its contribution to pathogenicity may differ between species. In some pathogens, pepA appears essential for full virulence, while in others its role may be compensated by other aminopeptidases.

This comparative approach provides insights into how evolution has shaped PepA function across different plant pathogens and may reveal pathogen-specific vulnerabilities that could be targeted for disease control strategies.