Recombinant Pseudomonas syringae pv. tomato 30S ribosomal protein S11 (rpsK)

What recombineering approaches are most effective for manipulating the rpsK gene in P. syringae pv. tomato?

For targeted manipulation of the rpsK gene in P. syringae pv. tomato, recombineering using the RecTE system offers a highly effective approach. The RecTE system identified from P. syringae pv. syringae B728a has been demonstrated to work efficiently in P. syringae pv. tomato DC3000 . For modifications of rpsK, the following methodology has proven most successful:

• For single nucleotide changes or small modifications: The P. syringae RecT homolog alone is sufficient to promote recombination using single-stranded DNA oligonucleotides .

• For larger modifications including gene disruptions or replacements: Both RecT and RecE homologs should be expressed together for efficient recombination of double-stranded DNA .

A quantitative experimental approach using the following protocol has been validated:

• Express the RecT (for ssDNA) or RecTE (for dsDNA) from P. syringae using a plasmid like pUCP24/47

• Design DNA substrates with 50-80 bp homology arms flanking the desired modification

• Introduce the DNA by electroporation into cells expressing the recombineering proteins

• Select for recombinants using appropriate markers

This system shows significantly improved efficiency over attempting to use the E. coli lambda Red system in Pseudomonas, addressing the species-specificity issues often encountered with recombineering technologies .

What expression systems are recommended for producing recombinant P. syringae pv. tomato rpsK protein?

For the production of recombinant P. syringae pv. tomato 30S ribosomal protein S11, a methodological approach based on the molecular techniques evident in the research literature would include:

Table 1: Comparison of Expression Systems for rpsK Protein Production

The methodology should include codon optimization for the expression host, as P. syringae genes may contain codons that are rare in E. coli. For functional studies, expression in a Pseudomonas-based system may preserve native folding and post-translational modifications, though with lower yields than E. coli-based systems. Selection of the appropriate expression system should be guided by the specific experimental requirements, balancing protein yield with structural and functional authenticity.

How can researchers effectively design primers for PCR amplification and cloning of the rpsK gene?

When designing primers for PCR amplification and cloning of the rpsK gene from P. syringae pv. tomato, researchers should follow these methodological guidelines:

• Primer Design Strategy:

  • Use the complete genome sequence of P. syringae pv. tomato DC3000 or similar reference strain to identify the precise location and sequence of the rpsK gene

  • Design forward and reverse primers with 18-25 nucleotides of exact complementarity to the target sequence

  • Add appropriate restriction enzyme sites flanked by 4-6 nucleotides at the 5' end to facilitate subsequent cloning

  • Consider adding a Kozak sequence or ribosome binding site if planning for expression studies

  • For recombineering applications, design primers with 50-80 bp homology arms as demonstrated effective for P. syringae

• PCR Optimization Protocol:

  • Initial denaturation: 98°C for 30 seconds

  • 30 cycles of: denaturation at 98°C for 10 seconds, annealing at Tm-5°C for 30 seconds, extension at 72°C (30 seconds per kb)

  • Final extension: 72°C for 5 minutes

  • Use high-fidelity polymerase (e.g., Phusion or Q5) to minimize errors in this essential gene

• Verification Strategy:

  • Confirm PCR product size by agarose gel electrophoresis

  • Verify sequence integrity through Sanger sequencing prior to further applications

  • For recombineering applications, verify recombination frequency using the quantitative assay approach described for P. syringae RecTE system

How does mutation in the rpsK gene affect P. syringae pv. tomato virulence in tomato plants?

The relationship between rpsK mutations and P. syringae pv. tomato virulence represents a complex research question. While direct studies on rpsK mutations are not extensively documented in the search results, parallels can be drawn from research on other essential genes. Mutations in essential ribosomal proteins typically result in pleiotropic effects that may impact multiple virulence systems. Based on virulence assessment methodologies for other P. syringae mutants, researchers investigating rpsK mutations should:

Research on the PsPto-PscC chemoreceptor mutant demonstrates that different inoculation methods reveal distinct aspects of virulence: spray-inoculation showed significant reductions in plant entry for the mutant, while infiltration showed comparable growth to wild-type once inside the plant tissue . Similar differential analysis would be valuable for rpsK mutants to distinguish between effects on entry versus in planta survival and multiplication.

What approaches can be used to study interactions between rpsK and other components of the translation machinery during infection?

Studying interactions between rpsK and other translation components during infection requires sophisticated methodological approaches. Based on current research techniques:

• In vivo Crosslinking and Co-immunoprecipitation (Co-IP):

  • Express epitope-tagged rpsK in P. syringae pv. tomato

  • Inoculate plants using established methods as described for virulence studies

  • Recover bacteria from plant tissue at different infection stages

  • Perform crosslinking followed by Co-IP to capture interaction partners

  • Identify proteins using mass spectrometry

• Cryo-electron Microscopy of Ribosomes:

  • Purify intact ribosomes from P. syringae grown in plant-mimicking media or recovered from infected plants

  • Perform cryo-EM to visualize structural changes in the ribosome, particularly focusing on the S11 protein position

  • Compare structures between free-living and in planta conditions

• Ribosome Profiling:

  • Extract bacterial RNA from infected plant tissue using differential centrifugation to separate bacterial from plant ribosomes

  • Perform ribosome profiling to identify changes in translation efficiency of specific mRNAs

  • Compare wild-type to rpsK mutants to identify genes whose translation is particularly dependent on proper S11 function

These methodologies enable researchers to move beyond simple gene expression studies to understand the functional role of rpsK in the complex environment of the infected plant tissue.

How can researchers leverage comparative genomics to understand the evolution of rpsK across different P. syringae pathovars?

To leverage comparative genomics for understanding rpsK evolution across P. syringae pathovars, researchers should employ the following methodological framework:

• Sequence Collection and Alignment:

  • Extract rpsK sequences from genome databases for multiple P. syringae pathovars, including the well-characterized DC3000 and T1 strains

  • Perform multiple sequence alignment using MUSCLE or MAFFT algorithms

  • Calculate sequence conservation scores for each amino acid position

• Phylogenetic Analysis:

  • Construct phylogenetic trees using Maximum Likelihood or Bayesian methods

  • Compare rpsK-based phylogeny with whole-genome or other gene-based phylogenies

  • Identify evolutionary patterns correlated with host specificity or geographical distribution

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify sites under positive, negative, or neutral selection

  • Compare substitution patterns with functional domains of the S11 protein

  • Identify potential coevolution with other ribosomal components

• Structural Modeling:

  • Generate structural models of S11 proteins from different pathovars

  • Map sequence variations onto the 3D structure

  • Identify structural implications of sequence differences

This approach allows researchers to connect sequence evolution with functional constraints and potentially identify signatures of host adaptation in this essential protein, similar to the divergence patterns observed in other genomic features between P. syringae strains with different host specificities .

What are common challenges when attempting to express recombinant P. syringae pv. tomato rpsK in heterologous systems?

Researchers frequently encounter several technical challenges when expressing recombinant P. syringae pv. tomato rpsK in heterologous systems. These challenges and their solutions include:

• Protein Solubility Issues:

  • Challenge: Ribosomal proteins often form inclusion bodies when overexpressed

  • Solution: Lower induction temperature (16-18°C), use solubility-enhancing fusion tags (SUMO, MBP), or express as a co-complex with interacting ribosomal components

• Functional Activity Assessment:

  • Challenge: Determining if recombinant rpsK is functionally active

  • Solution: Develop in vitro translation assays using purified components; consider complementation studies in conditional E. coli rpsK mutants

• Codon Usage Disparities:

  • Challenge: Codon bias differences between P. syringae and expression hosts

  • Solution: Optimize codons for the expression host or use specialized E. coli strains with expanded tRNA repertoires

• Protein Toxicity:

  • Challenge: Expression of foreign ribosomal proteins may interfere with host translation

  • Solution: Use tightly controlled inducible promoters, consider cell-free expression systems

• Co-factor Requirements:

  • Challenge: Proper folding may require specific ions or chaperones

  • Solution: Supplement expression medium with relevant ions (Mg²⁺, K⁺), co-express with P. syringae chaperones

These challenges parallel the species-specificity issues observed with recombineering systems, where tools developed for one bacterial species often function poorly in others, even closely related ones . The methodological solutions proposed are based on successfully addressing similar challenges in other bacterial protein expression systems.

How can researchers overcome the challenges of generating targeted mutations in the rpsK gene?

Generating targeted mutations in the essential rpsK gene presents significant technical challenges. Based on recombineering approaches developed for P. syringae , researchers should consider the following methodology:

• Conditional Mutation Strategy:

  • Design an inducible promoter system to control expression of a second copy of rpsK

  • Once established, target the native copy for modification using the RecTE system

  • For the modification itself, use the Pseudomonas RecT for single nucleotide changes or RecTE for larger modifications

• Optimized Recombineering Protocol:

  • Use the P. syringae-derived RecTE system rather than E. coli-derived systems

  • For highest efficiency, use a dual-plasmid approach: one expressing RecTE and another carrying a counter-selectable marker

  • Include positive and negative selection markers flanking the mutation site

  • Optimize electroporation conditions specifically for P. syringae pv. tomato (field strength, buffer composition)

• Verification Strategy:

  • Design PCR primers flanking the modified region

  • Sequence the entire gene to confirm the desired modification and absence of off-target mutations

  • Perform phenotypic assays to confirm the expected functional changes

• Alternative Approaches When Recombineering Fails:

  • Consider CRISPR-Cas9 systems adapted for Pseudomonas

  • For complete gene replacement, use suicide vector integration with counter-selection

This methodological framework builds upon the demonstrated success of the P. syringae RecTE system for chromosomal modifications , adapting it for the specific challenges of modifying an essential gene like rpsK.

What methods are available for studying rpsK expression under different infection conditions?

To accurately measure rpsK expression during P. syringae pv. tomato infection under varying conditions, researchers should employ these methodological approaches:

• Quantitative RT-PCR (RT-qPCR):

  • Extract RNA from bacteria recovered from infected plants at different time points

  • Use gene-specific primers for rpsK and reference genes

  • Normalize expression using multiple reference genes that remain stable during infection

  • Calculate relative expression changes using the 2^(-ΔΔCt) method

• Transcriptional Reporter Fusions:

  • Generate translational fusions of rpsK promoter with fluorescent proteins

  • Introduce these constructs into P. syringae strains

  • Measure fluorescence in bacteria recovered from plants using flow cytometry

  • Use confocal microscopy for spatial expression analysis within the plant tissue

• RNA-Seq Analysis:

  • Perform RNA-seq on bacteria recovered from plants under different conditions

  • Use differential expression analysis to identify changes in rpsK expression

  • Compare with expression patterns of other ribosomal proteins and translation factors

  • Identify potential regulatory elements through promoter analysis

• In situ Hybridization:

  • Design fluorescent probes specific to rpsK mRNA

  • Perform fluorescence in situ hybridization on infected plant tissue

  • Visualize expression patterns within the context of plant-pathogen interaction

These approaches can be modeled after the gene expression studies conducted for GABA catabolism genes in P. syringae, where expression changes were measured in response to relevant stimuli by comparing wild-type and mutant strains .

How might rpsK modifications be used to develop attenuated P. syringae strains for research purposes?

The development of attenuated P. syringae pv. tomato strains through rpsK modifications represents an innovative research direction with significant potential applications. Based on current understanding of bacterial attenuation strategies:

• Strategic Approach:

  • Introduce specific amino acid substitutions in conserved functional domains of rpsK

  • Target residues involved in tRNA binding or mRNA decoding

  • Create temperature-sensitive variants that function normally at permissive temperatures but lose function at restrictive temperatures

• Expected Outcomes:

  • Reduced translation efficiency resulting in slower growth

  • Impaired ability to respond to changing environments within the plant

  • Diminished virulence similar to that observed with mutations in other essential systems

• Validation Methodology:

  • Assess growth rates under various conditions

  • Measure translation efficiency using reporter systems

  • Perform virulence assays using both spray-inoculation and infiltration methods

  • Verify stability of the attenuated phenotype across multiple passages

• Potential Applications:

  • Live bacterial vectors for delivering beneficial proteins to plants

  • Research tools for studying plant immune responses without disease development

  • Platforms for testing complementation with variant genes

This approach builds on the understanding that subtle modifications to essential cellular machinery can create viable but attenuated strains, as demonstrated by the differential impacts of various mutations on P. syringae virulence .

What role might rpsK play in adaptation of P. syringae pv. tomato to different host plants?

The potential role of rpsK in P. syringae pv. tomato host adaptation represents an intriguing research question. While effector proteins are known to be major determinants of host specificity , components of the core cellular machinery like rpsK may also contribute in subtler ways:

This research direction connects to the broader understanding that bacterial adaptation to hosts involves both specialized virulence factors and adjustments to core cellular functions for optimal performance in specific host environments.

How can structural biology approaches advance our understanding of rpsK function in P. syringae pv. tomato?

Advanced structural biology approaches offer powerful tools for elucidating the detailed function of rpsK in P. syringae pv. tomato. A comprehensive methodological framework would include:

• Cryo-EM Structure Determination:

  • Purify intact 30S ribosomal subunits from P. syringae pv. tomato

  • Perform cryo-EM to resolve the structure at near-atomic resolution

  • Focus analysis on the S11 protein and its interactions with rRNA and neighboring proteins

  • Compare structures from bacteria grown in different conditions to identify conformational changes

• X-ray Crystallography of Isolated rpsK:

  • Express and purify recombinant rpsK protein

  • Conduct crystallization trials with various conditions and additives

  • Collect diffraction data and solve the structure

  • Perform co-crystallization with RNA fragments to understand binding interactions

• Nuclear Magnetic Resonance (NMR) Studies:

  • Produce isotopically labeled rpsK for NMR analysis

  • Determine solution structure and dynamic properties

  • Study interactions with ligands and binding partners

  • Investigate conformational changes upon binding to functional partners

• In silico Structure-Function Analysis:

  • Use structural data to perform molecular dynamics simulations

  • Predict effects of mutations on protein stability and function

  • Identify potential binding sites for small molecules

  • Model interactions with antibiotics that target the ribosome

These approaches could reveal unique structural features of P. syringae rpsK that might explain its specific functional properties in this plant pathogen, potentially identifying targets for specific inhibition of bacterial growth without affecting plant ribosomes.