Recombinant Pseudomonas syringae pv. syringae 50S ribosomal protein L34 (rpmH)

What is the biological significance of 50S ribosomal protein L34 in Pseudomonas syringae?

The 50S ribosomal protein L34 (rpmH) is a critical component of the bacterial large ribosomal subunit that contributes to protein synthesis machinery. In Pseudomonas syringae, a Gram-negative pathogenic bacterium causing widespread plant diseases, ribosomal proteins like L34 play essential roles in translation processes . Beyond its core role in translation, ribosomal proteins in bacterial pathogens can have moonlighting functions that influence virulence. While specific research on P. syringae rpmH is limited, studies on related Pseudomonas species indicate that ribosomal proteins can influence pathogenicity through various regulatory mechanisms that affect critical virulence pathways. The protein contributes to bacterial fitness and may influence the expression of pathogenicity factors involved in plant-microbe interactions.

How does the sequence and structure of P. syringae rpmH compare to homologous proteins in other bacterial species?

The sequence of P. syringae rpmH likely shows significant conservation with homologous proteins from related bacteria, though with strain-specific variations. Based on the data available for the related P. aeruginosa 50S ribosomal protein L34, which has the sequence "MKRTFQPSTL KRARVHGFRA RMATKNGRQV LSRRRAKGRK RLTV" (44 amino acids) , we can anticipate similar characteristics in the P. syringae variant.

Comparative sequence analysis typically reveals a pattern of conserved regions responsible for core ribosomal functions, particularly in RNA-binding domains and interaction sites with neighboring ribosomal proteins. Structural analyses using techniques such as X-ray crystallography or cryo-electron microscopy would be needed to definitively map any P. syringae-specific structural features that might contribute to its pathogenic lifestyle.

What expression systems are most effective for producing recombinant P. syringae rpmH?

For experimental production of recombinant P. syringae rpmH, several expression systems have proven effective, with selection depending on research objectives:

• Bacterial expression systems: E. coli remains the workhorse for recombinant protein production, particularly for bacterial proteins like rpmH. BL21(DE3) or Rosetta strains are recommended for ribosomal proteins due to their reduced protease activity and enhanced expression of rare codons .

• Mammalian cell expression: For applications requiring post-translational modifications or when bacterial expression proves problematic, mammalian cell systems provide an alternative. These systems can produce properly folded recombinant proteins with high purity (>85% by SDS-PAGE) .

• Cell-free protein synthesis: For rapid production, especially for functional studies, cell-free systems circumvent growth limitations and can be optimized for ribosomal protein expression.

The choice depends on downstream applications, with structural biology studies often requiring higher purity and yield than functional assays.

How can recombinant P. syringae rpmH be used in studying T3SS functionality and virulence mechanisms?

Recombinant rpmH can serve as a valuable tool in investigating the relationship between translation machinery and virulence mechanisms in P. syringae. The Type III Secretion System (T3SS) is critical for P. syringae pathogenicity, enabling the bacterium to inject effector proteins into plant cells . To study potential regulatory links between ribosomal proteins and T3SS:

• Protein-protein interaction studies: Use purified recombinant rpmH as bait in pull-down assays or yeast two-hybrid screens to identify potential interactions with T3SS components or regulatory proteins.

• Translational regulation analysis: Compare translation efficiency of T3SS mRNAs in wild-type versus rpmH-depleted conditions, using the recombinant protein for complementation studies.

• Structural studies: Elucidate whether rpmH forms part of specialized ribosomes that preferentially translate virulence-associated transcripts.

Researchers should note that T3SS components like HrpB, HrpD, HrpF, and HrpP have been identified as pathway substrates contributing to the hypersensitive response in plants and to the translocation of effector proteins . Investigating potential associations between rpmH and these components could reveal novel regulatory mechanisms.

What approaches are recommended for evaluating interactions between rpmH and other ribosomal components?

Investigating the interactions between rpmH and other ribosomal components requires a multi-faceted approach:

• In vitro reconstitution: Using purified recombinant rpmH and other ribosomal proteins to reconstitute partial or complete ribosomal assemblies under controlled conditions.

• Cryo-electron microscopy: For structural determination of rpmH within the context of the assembled ribosome, potentially revealing P. syringae-specific interaction networks.

• Cross-linking mass spectrometry (XL-MS): To map proximal relationships between rpmH and neighboring proteins or rRNA regions within the ribosome.

• Surface plasmon resonance or isothermal titration calorimetry: For quantitative measurement of binding affinities between rpmH and other ribosomal components.

• Hydrogen-deuterium exchange mass spectrometry: To identify regions of rpmH that undergo conformational changes upon interaction with other ribosomal components.

These methodologies can reveal how rpmH contributes to ribosome assembly, stability, and functional dynamics specifically in P. syringae.

What are the optimal conditions for purification of recombinant P. syringae rpmH?

Successful purification of recombinant P. syringae rpmH typically follows this optimized protocol:

• Initial preparation: After expression, centrifuge bacterial cultures at 5000×g for 15 minutes at 4°C to collect cell pellets.

• Cell lysis: Resuspend pellets in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, and protease inhibitor cocktail). Sonication or mechanical disruption should be performed on ice to prevent protein degradation.

• Affinity chromatography: For His-tagged rpmH, use Ni-NTA or TALON resin with graduated imidazole elution (50-250 mM).

• Ion exchange chromatography: Further purification can be achieved using cation exchange chromatography, as ribosomal proteins typically have high pI values.

• Size exclusion chromatography: Final polishing step to remove aggregates and achieve >85% purity as verified by SDS-PAGE .

• Buffer exchange: The final product should be exchanged into a storage buffer containing 20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, and 5% glycerol.

For optimal results, maintain all purification steps at 4°C and include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation of cysteine residues.

How can researchers troubleshoot poor yield or solubility issues with recombinant rpmH?

When encountering challenges with recombinant rpmH production, consider these evidence-based troubleshooting approaches:

• Expression temperature optimization: Lowering induction temperature to 16-20°C often improves folding and solubility of ribosomal proteins.

• Codon optimization: Adapt the rpmH gene sequence to the codon usage of the expression host to enhance translation efficiency.

• Fusion tags selection: For ribosomal proteins with solubility issues, SUMO or MBP fusion tags often outperform traditional His-tags.

• Expression host selection: Specialized strains like Arctic Express or C41/C43 may better accommodate ribosomal protein expression.

• Lysis buffer optimization: Screen additives including increased salt concentration (300-500 mM), mild detergents (0.1% Triton X-100), or stabilizing agents (5-10% glycerol).

• Co-expression strategies: Co-express rpmH with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to improve folding.

For severe solubility issues, denaturation-refolding approaches using 6M guanidine hydrochloride followed by step-wise dialysis can be effective, though structural validation post-refolding is essential.

What storage conditions ensure maximum stability of recombinant rpmH preparations?

To maintain the stability and activity of purified recombinant rpmH, follow these evidence-based storage recommendations:

• Short-term storage (1-7 days): Store working aliquots at 4°C to avoid freeze-thaw cycles, which can significantly reduce protein activity .

• Medium-term storage (up to 6 months): Store in buffer containing 20-50% glycerol at -20°C or -80°C .

• Long-term storage (6-12+ months): Lyophilization is recommended for extended storage, with reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL prior to use .

• Stability considerations:

  • Include protease inhibitors in storage buffers

  • Avoid repeated freeze-thaw cycles

  • For highest stability, store small aliquots (50-100 μL) to minimize freeze-thaw events

  • Add reducing agents (1-5 mM DTT) to prevent oxidation

Stability testing should be performed periodically using activity assays or structural analyses to ensure protein quality over time, particularly for samples stored longer than 6 months.

How can recombinant rpmH be utilized in structural biology studies of P. syringae ribosomes?

Recombinant rpmH serves as a valuable tool for structural biology investigations of P. syringae ribosomes, with several methodological approaches:

• Cryo-electron microscopy (cryo-EM): Purified recombinant rpmH can be used for in vitro reconstitution of ribosomal subunits or complete ribosomes for high-resolution structural analysis. This approach has become the method of choice for ribosome structural biology, allowing visualization of protein-RNA interfaces and conformational states.

• X-ray crystallography: For atomic-level resolution of specific interactions, crystallization trials using recombinant rpmH in complex with neighboring ribosomal proteins or RNA fragments can reveal critical interaction sites.

• NMR spectroscopy: For studying dynamic aspects of rpmH-RNA interactions, solution NMR with isotopically labeled recombinant protein provides insights into binding interfaces and conformational changes.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique identifies solvent-accessible regions and conformational changes in rpmH upon binding to other ribosomal components.

• Single-particle reconstruction: Using tagged recombinant rpmH for image alignment and classification in cryo-EM studies can help resolve challenging regions of the ribosome structure.

These approaches collectively contribute to understanding P. syringae-specific features of the ribosome that may relate to its pathogenic lifestyle or environmental adaptations.

How does rpmH contribute to the regulation of virulence factors in P. syringae?

The potential regulatory role of rpmH in P. syringae virulence involves several interconnected mechanisms:

• Selective translation: Similar to other specialized ribosomal proteins, rpmH may participate in selectively translating specific mRNAs, particularly those encoding virulence factors. Transcription factors like HrpL, which activates T3SS genes by binding to the motif 5′-GGAAC-N16-17-CCACNNA-3′ in the hrp promoters, could be subject to such regulation .

• Integration with stress responses: Ribosomal proteins often serve as sensors of cellular stress, potentially linking environmental cues to virulence gene expression. In P. syringae, this could involve cross-regulation with factors like RhpR, which is known to regulate T3SS genes .

• Direct interaction with regulatory RNAs: rpmH may interact with small regulatory RNAs that modulate virulence gene expression.

To investigate these possibilities, researchers should consider:

• Ribosome profiling comparing wild-type and rpmH-depleted strains

• RNA-immunoprecipitation to identify mRNAs preferentially associated with rpmH-containing ribosomes

• Comparative proteomics to identify proteins whose expression is specifically affected by rpmH mutations

Such studies would position rpmH within the complex regulatory network controlling P. syringae virulence, potentially revealing new targets for disease management.

What are the implications of rpmH structural conservation for developing targeted antimicrobial compounds?

The conservation of ribosomal proteins across bacterial species presents both opportunities and challenges for antimicrobial development:

This research direction represents a promising approach for developing new agricultural antimicrobials with potentially reduced environmental impact compared to broad-spectrum compounds.

What techniques are most effective for studying the role of rpmH in P. syringae translation fidelity?

To investigate how rpmH influences translation fidelity in P. syringae, researchers should consider these methodological approaches:

• In vitro translation assays: Using purified recombinant rpmH in reconstituted translation systems to measure misincorporation rates, frameshifting, and stop codon readthrough frequencies.

• Reporter systems: Employing dual-luciferase reporters with programmed errors to quantify translation fidelity in vivo in wild-type versus rpmH-mutant strains.

• Ribosome profiling: This next-generation sequencing technique provides genome-wide analysis of ribosome positioning and can reveal error-prone sites in rpmH mutants.

• Mass spectrometry analysis: Proteomic approaches to directly identify mistranslation products in bacteria with wild-type versus mutant rpmH.

• Single-molecule FRET studies: Using fluorescently labeled tRNAs and mRNAs to observe real-time translation dynamics in the presence of wild-type or mutant rpmH.

These approaches collectively provide comprehensive insights into how rpmH contributes to translation quality control, which may have downstream effects on protein folding, activity, and ultimately bacterial fitness and virulence.

How can genetic approaches be combined with recombinant protein studies to understand rpmH function?

An integrated approach combining genetics and recombinant protein biochemistry reveals comprehensive insights into rpmH function:

• Conditional knockdown systems: Since complete deletion of essential ribosomal genes may be lethal, conditional expression systems allow for controlled depletion of rpmH in vivo to study phenotypic consequences.

• Complementation studies: Expressing recombinant wild-type or mutant rpmH variants in knockdown strains to determine structure-function relationships.

• Site-directed mutagenesis: Creating targeted mutations in recombinant rpmH followed by in vivo and in vitro functional testing to identify critical residues.

• Domain swapping: Replacing regions of P. syringae rpmH with corresponding regions from non-pathogenic bacteria to identify virulence-associated domains.

• Suppressor screens: Identifying second-site mutations that suppress rpmH mutant phenotypes, revealing functional networks.

• CRISPR interference (CRISPRi): For targeted, tunable repression of rpmH expression without permanently altering the genome.

These approaches create a feedback loop between in vivo observations and in vitro mechanistic studies, accelerating functional characterization of this important ribosomal protein.

What controls and validation steps are essential when performing interaction studies with recombinant rpmH?

When investigating protein-protein or protein-RNA interactions involving recombinant rpmH, these controls and validation steps are critical for reliable results:

• Negative controls:

  • Non-specific proteins of similar size and charge characteristics

  • Heat-denatured rpmH to confirm specificity of structural interactions

  • Scrambled RNA sequences for RNA-binding studies

• Positive controls:

  • Known interaction partners from conserved ribosomal assemblies

  • Synthetic RNA fragments corresponding to established binding sites

• Validation through multiple techniques:

  • Primary screening by pull-down or co-immunoprecipitation

  • Secondary validation by biophysical methods (ITC, SPR, MST)

  • Tertiary confirmation through functional assays

• Structural validation:

  • Circular dichroism to confirm proper folding of recombinant rpmH

  • Limited proteolysis to verify domain integrity

  • Dynamic light scattering to assess aggregation state

• Concentration dependence:

  • Testing interactions across physiologically relevant concentration ranges

  • Determining binding constants to distinguish specific from non-specific interactions

• In vivo confirmation:

  • Bacterial two-hybrid or split-GFP systems to verify interactions in a cellular context

  • Co-localization studies using fluorescently tagged proteins

Rigorous application of these controls ensures that interactions identified with recombinant rpmH reflect genuine biological phenomena rather than experimental artifacts.