Recombinant Pseudomonas syringae pv. tomato 50S ribosomal protein L3 (rplC)

What is the role of 50S ribosomal protein L3 (rplC) in Pseudomonas syringae pv. tomato?

The 50S ribosomal protein L3 (rplC) is an essential component of the bacterial ribosome, specifically the large 50S subunit, where it plays a critical role in ribosome assembly and protein synthesis functions. In Pseudomonas syringae pv. tomato, as in other bacteria, L3 is positioned near the peptidyl transferase center (PTC), making it crucial for proper ribosomal function and susceptibility to certain antibiotics. Mutations in L3 can affect the binding of PTC-targeting antibiotics and potentially alter bacterial fitness and virulence properties .

Methodological approach for investigation: To study L3 function, researchers typically employ knockout complementation systems, where the chromosomal rplC gene is deleted and replaced with plasmid-carried wild-type or mutated versions. This approach allows for the examination of L3 variants without interference from wild-type protein, as demonstrated in similar studies with E. coli .

How does the structure of P. syringae pv. tomato L3 compare to other bacterial L3 proteins?

The L3 protein structure in P. syringae pv. tomato shares significant homology with other bacterial L3 proteins, particularly in the loops near the peptidyl transferase center that influence antibiotic interactions. Comparative analysis reveals that conserved regions, especially around residues such as G144, G147, and Q148, are critical for ribosomal function across different bacterial species .

Research methodology: Structural comparison typically involves computational modeling based on homology with solved crystal structures from model organisms such as E. coli. This approach allows researchers to predict key functional domains and potential impact of mutations, even when direct crystallographic data for P. syringae L3 is not available .

What are the optimal expression systems for producing recombinant P. syringae pv. tomato L3 protein?

The optimal expression system for recombinant P. syringae pv. tomato L3 protein is typically E. coli, due to its established genetic tools and high protein yields. Expression vectors such as pET3a or pBR322 derivatives under the control of constitutive or inducible promoters (like the T7 promoter system) have proven effective for ribosomal proteins. For functional studies, the protein can be expressed with or without fusion tags depending on the intended application .

Experimental methodology: A typical protocol involves:

• Cloning the rplC gene from P. syringae pv. tomato into an appropriate expression vector

• Transforming into an E. coli expression strain (BL21(DE3) or similar)

• Inducing expression with IPTG (for T7-based systems)

• Harvesting cells and lysing under conditions that maintain protein solubility

• Purification using appropriate chromatography techniques

What purification strategies are most effective for isolating recombinant L3 protein while maintaining its native structure?

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been demonstrated as a highly effective one-step purification method for recombinant proteins from E. coli cell lysates, with excellent recovery rates and purity levels. For the 50S ribosomal protein L3, a strategy employing a C8 column with a shallow acetonitrile gradient (0.1%/min) in the presence of 0.1% trifluoroacetic acid (TFA) can achieve >94% purity with >94% recovery .

Alternative methodologies include:

• Affinity chromatography using His-tags or other fusion tags

• Ion-exchange chromatography

• Size exclusion chromatography

• A combination of these methods in a multi-step purification process

Table 1: Comparison of purification methods for recombinant ribosomal proteins

How can recombineering techniques be applied to create site-specific mutations in the P. syringae pv. tomato rplC gene?

Recombineering techniques using RecTE from Pseudomonas syringae provide an efficient method for introducing site-specific mutations into the rplC gene. This approach enables targeted gene modifications without traditional cloning constraints and has been successfully demonstrated in P. syringae pv. tomato DC3000 .

Protocol for recombineering in P. syringae:

• Identify RecT and RecE homologs in P. syringae pv. syringae B728a that promote homologous recombination

• Express these recombination proteins in the target strain (P. syringae pv. tomato)

• Design linear DNA substrates with the desired mutations flanked by homology regions to the rplC gene

• Introduce these substrates directly into cells by electroporation

• Screen transformants for successful integration using appropriate selection markers or screening methods

• Verify mutations by sequencing

This method is particularly valuable for creating specific point mutations in rplC to study structure-function relationships and antibiotic resistance mechanisms.

What are the key considerations when designing mutations in L3 to study antibiotic resistance mechanisms?

When designing L3 mutations to study antibiotic resistance, researchers should focus on regions near the peptidyl transferase center that interact with antibiotics. Key considerations include:

• Target conserved residues in the loops of L3 near the PTC (such as positions G144D, G147R, Q148F, N149S/R, Q150L, and T151P)

• Consider mutations observed in antibiotic-resistant clinical isolates

• Design mutations that alter interactions with specific antibiotics without completely disrupting ribosome assembly

• Include controls to assess growth fitness costs associated with mutations

• Use computational modeling to predict structural changes and potential impacts on antibiotic binding before experimental validation

Comprehensive mutational analysis should employ both single amino acid substitutions and more complex mutations to fully understand structure-function relationships. For example, in studies of E. coli L3, mutations including Q148F, N149R, 136SQF138, and 136SHL138 affected susceptibility to antibiotics like linezolid and tiamulin .

What techniques are most effective for analyzing the impact of L3 mutations on ribosome function and antibiotic resistance?

Multiple complementary techniques are necessary to comprehensively analyze the impact of L3 mutations:

• Growth rate analysis: Comparing growth curves of strains expressing wild-type versus mutated L3 proteins to assess fitness costs of mutations.

• Antibiotic susceptibility testing: Determining minimum inhibitory concentrations (MICs) of various antibiotics for mutant strains compared to wild-type.

• In vitro translation assays: Measuring the efficiency of protein synthesis using purified ribosomes containing mutant L3.

• Computational modeling: Assessing structural changes in the 50S ribosomal subunit and predicting effects on antibiotic binding using molecular dynamics simulations.

• Western blotting and mass spectrometry: Verifying expression and stability of mutated L3 proteins .

Table 2: Sample data from antibiotic susceptibility testing of L3 mutants

Note: Values in this table are representative of typical experimental results based on similar studies and should be validated for specific P. syringae strains.

How can computational modeling be used to predict the effects of L3 mutations on antibiotic binding?

Computational modeling provides valuable insights into the structural consequences of L3 mutations and their effects on antibiotic binding. A recommended methodology includes:

• Generate a structural model of the P. syringae 50S ribosomal subunit based on homology with solved structures (e.g., from E. coli)

• Create an atom sphere (approximately 85 Å) centered at the antibiotic binding site

• Prepare the model using protein preparation tools (e.g., Schrödinger Suite) to assign atom types, add hydrogen atoms, and optimize the structure

• Introduce the desired L3 mutations into the model

• Use molecular docking (e.g., XP Glide methodology) to quantify the binding mode and energy of antibiotic binding

• Calculate glide scores to reflect binding strength of antibiotics to the wild-type versus mutant structures

• Perform molecular dynamics simulations to account for protein flexibility

This approach enables researchers to predict whether specific L3 mutations will affect antibiotic binding before experimental validation, streamlining the research process.

How does the study of L3 mutations contribute to understanding the evolution of antibiotic resistance in plant pathogens like P. syringae?

Studying L3 mutations in P. syringae provides critical insights into the evolutionary mechanisms of antibiotic resistance in plant pathogens. Unlike clinical pathogens, plant pathogens like P. syringae are exposed to different selective pressures, including agricultural antibiotics and naturally occurring antimicrobial compounds produced by plants and competing microorganisms.

Research approach:

• Compare L3 sequences across different P. syringae pathovars and isolates from diverse environments

• Identify natural polymorphisms in L3 and correlate them with habitat and antibiotic exposure

• Use experimental evolution under antibiotic selection to identify emerging L3 mutations

• Assess the fitness costs of resistance mutations in plant colonization and virulence

• Examine horizontal gene transfer patterns of rplC variants between environmental and pathogenic strains

This research helps predict the potential for resistance development in agricultural settings and informs strategies for antibiotic stewardship in plant disease management.

What is the relationship between L3 mutations and virulence in P. syringae pv. tomato?

The relationship between L3 mutations and virulence in P. syringae pv. tomato is complex and multifaceted. While primarily functioning in protein synthesis, alterations in ribosomal proteins like L3 can have pleiotropic effects on bacterial physiology that influence virulence:

• Growth rate effects: Mutations that reduce translation efficiency may decrease growth rates, affecting the bacteria's ability to multiply within host tissues and cause disease .

• Stress response modulation: L3 mutations can alter how bacteria respond to environmental stresses encountered during plant infection, potentially affecting survival within the host.

• Virulence factor expression: Changes in translational efficiency or accuracy can differentially affect the expression of virulence factors such as effector proteins secreted through the type III secretion system .

• Antibiotic tolerance: L3 mutations conferring resistance to antibiotics might provide cross-protection against plant antimicrobial peptides or other host defense molecules.

Experimental approaches to investigate this relationship include:

• Comparing virulence of P. syringae strains carrying different L3 variants in plant infection assays

• Measuring expression levels of key virulence genes in L3 mutant strains

• Assessing bacterial survival under conditions mimicking the plant apoplast

• Examining potential interactions between L3 and known virulence regulators like HrpL or TvrR

How can reversed-phase liquid chromatography (RPLC) be optimized for purification and analysis of recombinant L3 protein?

Optimizing RPLC for recombinant L3 protein requires careful consideration of the protein's physicochemical properties. A data-driven approach to RPLC method development ensures optimal separation and purification efficiency .

Recommended optimization protocol:

• Stationary phase selection: For ribosomal proteins like L3, wide-pore SPP (superficially porous particle) columns with phenyl bonding minimize on-column adsorption and improve selectivity.

• Mobile phase optimization: Design experiments to determine optimal:

  • Organic modifier (acetonitrile typically performs better than methanol)

  • Ion-pairing agent (0.1% TFA or 0.1% formic acid)

  • pH (typically 2-3 for maximum protein solubility)

• Gradient optimization: Employ shallow gradients (0.1-0.5% acetonitrile/min) starting at 5-10% acetonitrile to ensure optimal separation.

• Temperature control: Elevated column temperatures (40-60°C) can improve peak shape and resolution.

• Method robustness testing: Use Design of Experiment (DoE) approaches to challenge method parameters and establish a Method Operable Design Region (MODR) .

This approach has shown excellent results for ribosomal protein purification, yielding >94% purity in a single step with minimal sample preparation .

What are the key considerations for designing a recombineering system to modify the P. syringae pv. tomato rplC gene in vivo?

Designing an effective recombineering system for modifying the P. syringae pv. tomato rplC gene requires careful planning of multiple factors:

• Selection of recombineering proteins:

  • P. syringae RecT is sufficient for single-stranded DNA recombination

  • Both RecT and RecE are required for efficient double-stranded DNA recombination

  • Expression levels must be optimized to minimize toxicity while maintaining recombination efficiency

• Design of DNA substrates:

  • For point mutations, single-stranded oligonucleotides (60-90 nucleotides) targeting the lagging strand of DNA replication are most effective

  • For larger modifications, double-stranded DNA with homology arms (≥500 bp) is recommended

  • Include selection markers for identifying successful recombinants

• Genomic context considerations:

  • The rplC gene is part of the essential S10 ribosomal protein operon

  • Modifications must maintain the reading frame and expression of downstream genes

  • A complementation strategy is necessary since rplC is essential

• Delivery optimization:

  • Electroporation conditions must be optimized for P. syringae (typically 2.5 kV, 25 μF, 200 Ω)

  • Cell preparation is critical (growth phase, washing to remove salts)

  • Recovery media and conditions affect recombination efficiency

• Verification strategy:

  • Sequencing to confirm the intended modification

  • Phenotypic testing to ensure rplC function

  • Ribosome profiling to verify incorporation into mature ribosomes

This system allows for precise genomic modifications without the limitations of traditional cloning methods, enabling detailed structure-function studies of the L3 protein in its native context.

How do L3 mutations in P. syringae pv. tomato compare to those in E. coli regarding their effects on antibiotic resistance?

Comparative analysis reveals both similarities and differences between L3 mutations in P. syringae pv. tomato and E. coli:

Similarities:

• In both species, mutations in the loops of L3 near the peptidyl transferase center can confer resistance to certain antibiotics

• Key conserved residues (G144, G147, Q148, N149) influence antibiotic binding in similar ways

• Mutations often come with fitness costs affecting growth rates in both organisms

Differences:

• P. syringae may show distinct patterns of resistance due to its different ecological niche and exposure to plant-derived antimicrobials

• The broader phylogenetic diversity of P. syringae (with 13 phylogroups and 23 clades) suggests greater potential variation in L3 structure and function compared to E. coli

• The relationship between L3 mutations and virulence factors is potentially more complex in P. syringae due to its plant pathogen lifestyle

Research methodology: A comprehensive comparison requires:

• Analyzing sequence conservation of L3 between P. syringae and E. coli

• Creating equivalent mutations in both species

• Testing antibiotic susceptibility profiles

• Comparing growth characteristics

• Conducting computational modeling of ribosomal structures from both species

This comparative approach provides insights into the evolutionary conservation of resistance mechanisms and helps identify species-specific features that might be targeted for antimicrobial development.

What techniques are most effective for analyzing the phylogenetic relationships of the rplC gene across different P. syringae pathovars?

Effective phylogenetic analysis of the rplC gene across P. syringae pathovars requires a multi-faceted approach combining several techniques:

• Multi-Locus Sequence Typing (MLST): While full MLST typically uses multiple housekeeping genes, the citrate synthase (cts) gene has been shown to accurately predict phylogenetic affiliation for >97% of P. syringae strains. This can be complemented with rplC sequencing to create a more robust phylogeny .

• Core genome phylogeny: Analysis of the rplC gene in the context of the core genome provides a more comprehensive evolutionary perspective. This approach has confirmed the robustness of MLST-derived phylogroups in P. syringae .

• Sequence analysis parameters:

  • Alignment methods should account for codon structure in coding sequences

  • Models of nucleotide substitution should be selected based on likelihood ratio tests

  • Both nucleotide and amino acid sequences should be analyzed to identify synonymous vs. non-synonymous changes

• Analytical software recommendations:

  • MEGA X for basic phylogenetic tree construction

  • RAxML or MrBayes for maximum likelihood or Bayesian inference

  • PAML for detecting selection signatures on specific codons

• Visualization and classification:

  • Interactive tree visualization tools (e.g., iTOL)

  • Integration with metadata about strain origin, host range, and phenotypic traits

The analysis should incorporate strains representing the full breadth of P. syringae diversity, including the 13 established phylogroups and 23 clades, to ensure comprehensive evolutionary coverage .

How might recombinant L3 protein be used to investigate interactions between P. syringae pv. tomato and host immune responses?

Recombinant L3 protein can serve as a valuable tool for investigating host-pathogen interactions through several innovative approaches:

• Immunological detection: Purified recombinant L3 can be used to generate specific antibodies for tracking bacterial colonization and localization within plant tissues.

• Protein-protein interaction studies: L3 variants can be used in pull-down assays or yeast two-hybrid screens to identify potential interactions with host proteins during infection.

• MAMP/PAMP analysis: Investigating whether L3 fragments act as microbe-associated molecular patterns (MAMPs) that trigger plant immune responses.

• Structural immunology: Comparing L3 structures from virulent and avirulent strains to identify features that might influence recognition by plant immune receptors.

• Ribosome engineering: Creating P. syringae strains with modified L3 proteins to alter translation of virulence factors and study their impact on host interactions .

Methodological considerations include purification of L3 under conditions that maintain native conformation, validation of functional activity, and careful controls to distinguish direct L3 effects from secondary consequences of altered bacterial physiology.

What is the relationship between ribosomal protein mutations and the expression of virulence factors in P. syringae pv. tomato?

The relationship between ribosomal protein mutations and virulence factor expression involves complex regulatory networks affecting bacterial pathogenicity:

Experimental approach: To investigate these relationships, researchers should employ:

• Transcriptomics and proteomics to compare virulence factor expression profiles

• Reporter gene assays to monitor expression of key virulence genes

• Plant infection assays to correlate molecular changes with virulence phenotypes

• Comparative analysis across multiple P. syringae strains and L3 variants

Understanding these relationships provides insight into potential evolutionary trade-offs between antibiotic resistance and virulence in plant pathogens.