Recombinant Pseudomonas syringae pv. syringae 30S ribosomal protein S5 (rpsE)

What are the key structural domains of P. syringae S5 protein that contribute to ribosome assembly?

Based on homology with other bacterial S5 proteins, the P. syringae S5 likely contains:

The N-terminal domain is particularly important for interactions with 16S rRNA and neighboring proteins in the 30S subunit assembly pathway. Key conserved residues in this domain are critical for proper folding of 16S rRNA and translational fidelity .

What are the optimal expression systems for producing recombinant P. syringae 30S ribosomal protein S5?

For recombinant expression of P. syringae S5 protein, researchers should consider both heterologous and homologous expression systems:

• E. coli expression system: The pET vector system with BL21(DE3) or Rosetta strains provides high yields for bacterial ribosomal proteins. Include a His-tag for purification and optimize with the following parameters:

  • Induction: 0.5-1.0 mM IPTG

  • Temperature: 18-25°C post-induction (reducing inclusion body formation)

  • Duration: 12-16 hours

  • Media: Auto-induction media or LB with glucose supplement

• Native P. syringae expression: For studies requiring post-translational modifications specific to P. syringae, consider using a shuttle vector system compatible with both E. coli and Pseudomonas, such as pDN18 or pRK415 derivatives .

The choice depends on downstream applications, with the E. coli system offering higher yields but potentially lacking pathogen-specific modifications.

How can S5 mutations be introduced to study translational fidelity in P. syringae?

To introduce specific mutations in the P. syringae rpsE gene:

• Site-directed mutagenesis approach:

  • PCR-based methods using overlapping primers containing desired mutations

  • CRISPR-Cas9 genome editing optimized for P. syringae

  • Allelic exchange using suicide vectors like pK18mobsacB

• Mutation targets based on E. coli studies:

  • G28D mutation: Known to affect translational fidelity and produce spectinomycin resistance and cold sensitivity in E. coli

  • Regions involved in tRNA binding

  • Interfaces with other r-proteins

• Phenotypic assays for translational fidelity measurement:

  • Luciferase reporters with programmed frameshifts

  • Nonsense suppression assays

  • Ribosome profiling to analyze translocation rates

Remember to include appropriate controls, including wild-type rpsE and multiple S5 mutants for comparative analysis.

How does S5 protein contribute to the type III secretion system efficiency in P. syringae?

While the S5 protein primarily functions in translation, its potential indirect effects on the type III secretion system (T3SS) encoded by hrp/hrc genes in P. syringae merit investigation. Ribosomal proteins like S5 may influence pathogenicity through:

• Translational regulation of T3SS components: S5 mutations could alter translational efficiency of specific mRNAs encoding secretion system components or regulatory proteins .

• Stress response integration: Ribosomal function affects bacterial stress responses, which in turn regulate virulence gene expression patterns. The tripartite mosaic structure of the Hrp pathogenicity island may be differentially expressed under conditions where S5 function is altered .

• Experimental approach: Measure expression levels of T3SS components in wild-type versus S5 mutant strains using:

  • RT-qPCR of key hrp/hrc genes

  • Western blotting of secreted effector proteins

  • Secretion assays measuring effector translocation efficiency

Comparative studies between pathovars like P. syringae pv. syringae and P. syringae pv. tomato could reveal pathovar-specific differences in how ribosomal proteins influence virulence mechanisms .

What functional consequences result from S5 mutations in the context of 16S rRNA interactions?

S5 mutations, particularly those affecting conserved residues like glycine-28, can significantly alter 16S rRNA folding and interactions. Based on E. coli studies:

• Ribosome assembly alterations: S5(G28D) mutation results in:

  • Abnormal 30S subunit profiles with broader sedimentation patterns

  • Altered distribution of 30S, 50S, and 70S ribosomes

  • Reduced polysome formation

• 16S rRNA conformational changes:

  • Modified protection patterns in chemical probing experiments

  • Altered accessibility of specific nucleotides involved in decoding

  • Changes in tRNA binding efficiency

• Translational fidelity effects:

  • Increased +1 and -1 frameshifting

  • Enhanced nonsense suppression

  • Potential "ribosome ambiguity" (ram) phenotype, although through a mechanism potentially distinct from other ram mutations

These functional consequences highlight the critical role of S5 in maintaining proper ribosomal architecture and function .

How does P. syringae S5 compare with other bacterial pathogens' S5 proteins, and what are the evolutionary implications?

Comparative analysis of S5 proteins across bacterial pathogens reveals important evolutionary patterns:

Researchers should consider these evolutionary patterns when interpreting S5 mutations in different bacterial contexts or when using S5 as a phylogenetic marker.

What methodological approaches can distinguish the contribution of S5 to pathogenicity versus general fitness in P. syringae?

Separating S5's contribution to pathogenicity from its role in general bacterial fitness requires sophisticated experimental designs:

• Conditional expression systems:

  • Temperature-sensitive S5 mutants that maintain basal function but show altered properties

  • Inducible promoter systems to control S5 expression levels during different infection stages

  • Complementation with homologs from non-pathogenic Pseudomonas species

• In planta versus in vitro comparative studies:

  • Compare growth rates and translational profiles in minimal media versus plant extracts

  • Measure relative fitness of S5 mutants during plant colonization versus laboratory conditions

  • Analyze differential gene expression patterns in wild-type versus S5 mutants during infection

• Specific translational regulation assessment:

  • Ribosome profiling to identify pathogenicity-related mRNAs specifically affected by S5 mutations

  • Polysome analysis to determine if virulence factors are differentially translated

  • RNA-protein crosslinking methods to identify specific interactions between S5 and pathogenicity-related transcripts

This methodological framework allows researchers to distinguish between general growth defects and specific pathogenicity impacts when studying S5 mutations.

What are the optimal approaches for analyzing tRNA binding and translational fidelity in recombinant P. syringae S5 systems?

For comprehensive analysis of tRNA binding and translational fidelity in recombinant P. syringae S5 systems:

• In vitro tRNA binding assays:

  • Filter binding assays using purified 30S subunits containing recombinant S5 variants

  • Fluorescence-based methods with labeled tRNAs to measure binding kinetics

  • Competition assays to determine relative affinities for different tRNA species

• Translational fidelity measurement:

  • Dual luciferase reporters with programmed frameshift sites

  • β-galactosidase readthrough assays for nonsense suppression quantification

  • Cell-free translation systems reconstituted with purified components including recombinant S5

• Structural analysis methods:

  • Chemical probing of 16S rRNA in the presence of wild-type or mutant S5

  • Cryo-EM to visualize structural changes in the 30S subunit

  • SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension) to examine RNA structural changes induced by S5 variants

The combination of these approaches provides complementary data on how S5 variants affect translation at the molecular level.

How can researchers integrate S5 protein studies with pathogenicity island analysis in P. syringae?

To effectively integrate ribosomal protein S5 studies with pathogenicity island analysis:

• Transcriptomic approaches:

  • RNA-seq comparing wild-type and S5 mutant strains, focusing on differential expression of genes in the Hrp pathogenicity island

  • Targeted RT-qPCR of effector genes from both the exchangeable effector locus (EEL) and conserved effector locus (CEL)

  • 5' end mapping to identify transcription start sites affected by S5-mediated translational feedback

• Proteomics integration:

  • Quantitative proteomics comparing effector protein levels in wild-type versus S5 mutant strains

  • Secretome analysis to measure T3SS efficiency with different S5 variants

  • Ribosome profiling to identify translational efficiency changes for specific pathogenicity-related mRNAs

• Functional genomics correlation:

  • Construct a correlation matrix between S5 genetic variants and effector protein repertoires across P. syringae pathovars

  • Analyze co-evolution patterns between ribosomal components and pathogenicity determinants

  • Develop predictive models for translational impact on virulence expression

This integrated approach bridges fundamental ribosomal biology with pathogen-specific virulence mechanisms.

How might S5 protein variants be used to study phage resistance mechanisms in P. syringae?

Ribosomal protein S5 variants offer unique opportunities to study phage resistance mechanisms:

• Translational manipulation of phage resistance:

  • S5 mutations affecting translation fidelity could alter expression of phage receptors

  • Specific S5 variants might influence translation of host restriction-modification systems

  • Study differential susceptibility of S5 mutants to phages like the novel phage identified in P. syringae from Callery pear

• Phage-host co-evolution experimental system:

  • Evolve phages against P. syringae strains with different S5 variants

  • Analyze adaptive mutations in phage genomes responding to host translational machinery changes

  • Map the genetic basis of resistance and counter-resistance mechanisms

• Biocontrol applications:

  • Determine if S5 mutations affect phage therapy efficacy

  • Assess whether phage resistance mechanisms can be predicted based on host S5 status

  • Evaluate how translational variants influence phage host range across P. syringae pathovars

These approaches can inform both fundamental phage-host interaction studies and practical biocontrol applications for P. syringae infections in plants.

What bioinformatic approaches are most effective for analyzing S5 protein conservation across P. syringae pathovars in the context of host specificity?

Effective bioinformatic analysis of S5 conservation requires:

• Sequence-based approaches:

  • Multiple sequence alignment of S5 proteins across pathovars with different host specificities

  • Detection of selection signatures using dN/dS ratio analysis

  • Identification of co-evolving residues between S5 and other ribosomal components

• Structural bioinformatics:

  • Homology modeling of S5 variants from different pathovars

  • Molecular dynamics simulations to predict functional impacts of sequence variations

  • Protein-RNA docking to assess changes in 16S rRNA interactions

• Integrative analysis with pathogenicity determinants:

  • Correlation analysis between S5 sequence variants and effector repertoires

  • Network analysis of translational machinery components and virulence factors

  • Machine learning approaches to predict host specificity based on combined translational and effector features

These complementary approaches provide a comprehensive framework for understanding how translational machinery evolution relates to host specificity in P. syringae pathovars .