Recombinant Lactobacillus johnsonii 30S ribosomal protein S5 (rpsE)

What is Lactobacillus johnsonii 30S ribosomal protein S5 (rpsE) and what are its key functions?

The 30S ribosomal protein S5 (rpsE) from Lactobacillus johnsonii is a critical component of the bacterial small ribosomal subunit involved in protein synthesis. This protein belongs to the 30S ribosomal subunit, which has an approximate molecular weight of 0.9 MDa and consists of a 16S rRNA molecule (1542 nucleotides) and 21 ribosomal proteins designated S1-S21 . The S5 protein plays essential roles in:

• Maintaining ribosomal structural integrity

• Facilitating mRNA binding during translation initiation

• Contributing to the decoding center functionality

• Participating in the aminoacyl-tRNA selection process

The protein functions within the context of L. johnsonii, a probiotic bacterium found naturally in the human gastrointestinal tract that has been studied for its potential therapeutic effects in various diseases .

What are the recommended protocols for expression and purification of recombinant L. johnsonii rpsE?

Based on established protocols for similar recombinant ribosomal proteins, the following methodological approach is recommended:

Expression System Selection:

• Heterologous expression in E. coli (BL21 DE3 or similar strains) is commonly used for ribosomal proteins

• Yeast expression systems may provide appropriate post-translational modifications and have been successfully used for similar proteins

Purification Strategy:

• Initial cell lysis using sonication or mechanical disruption in an appropriate buffer (typically containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol)

• Affinity chromatography using a suitable tag (His-tag is common for ribosomal proteins)

• Size exclusion chromatography to separate monomeric protein from aggregates

• Ion exchange chromatography for final polishing

Quality Control:

• SDS-PAGE analysis to confirm purity (target >85%)

• Western blot to verify identity

• Mass spectrometry to confirm molecular weight and sequence integrity

What are the optimal storage conditions for maintaining stability of recombinant L. johnsonii rpsE?

For optimal stability and preservation of biological activity, recombinant L. johnsonii rpsE should be stored under the following conditions:

• Long-term storage: -20°C or preferably -80°C in appropriate buffer with 50% glycerol

• Short-term storage: Working aliquots can be maintained at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and activity loss

• For lyophilized preparations, store at -20°C with expected shelf life of approximately 12 months

• For liquid preparations, store at -20°C/-80°C with expected shelf life of approximately 6 months

Reconstitution Protocol:

• Centrifuge vial briefly before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage

• Aliquot to minimize freeze-thaw cycles

How can researchers effectively study the role of rpsE in L. johnsonii ribosomal assembly and function?

To investigate the role of rpsE in L. johnsonii ribosomal assembly and function, researchers should consider these methodological approaches:

In vitro Ribosomal Assembly Studies:

• Reconstitution experiments with purified L. johnsonii 30S ribosomal components

• Sequential addition of components to determine assembly hierarchy

• Binding assays with fluorescently labeled rpsE to monitor incorporation into ribosomal particles

Functional Translation Assays:

• Cell-free translation systems incorporating wild-type or mutant rpsE protein

• Measuring translation efficiency using reporter constructs

• Polysome profiling to assess ribosomal subunit association

Structural Studies:

• Cryo-electron microscopy to visualize rpsE positioning within the ribosomal complex

• Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

• Cross-linking mass spectrometry to map protein-protein and protein-RNA interactions

Genetic Approaches:

• Site-directed mutagenesis of key residues identified by sequence comparison

• Complementation studies in strains with depleted endogenous rpsE

• CRISPR-Cas9 mediated editing to create conditional mutants

What potential roles might L. johnsonii rpsE play in the probiotic properties of this bacterium?

While ribosomal proteins are primarily associated with protein synthesis, emerging research suggests they may contribute to the probiotic properties of bacteria like L. johnsonii in several ways:

Immunomodulatory Effects:
L. johnsonii has demonstrated significant immunomodulatory capabilities, including altering macrophage, T-cell, and Th2 cytokine levels . Ribosomal proteins, when released from bacteria during stress or lysis, can interact with host immune cells. The rpsE protein could potentially:

• Act as a microbe-associated molecular pattern (MAMP) recognized by specific pattern recognition receptors

• Contribute to dendritic cell regulation observed with L. johnsonii

• Participate in cross-talk with host epithelial cells

Bacterial Adaptation to Host Environment:
The translational machinery, including rpsE, may enable L. johnsonii to adapt to the gastrointestinal environment through:

• Selective translation of stress-response genes

• Controlling expression of colonization factors

• Mediating responses to host-derived signals

Antimicrobial Resistance Considerations:
L. johnsonii strains contain various antibiotic resistance genes, and ribosomal proteins are common targets for antibiotics . The specific structure of rpsE might contribute to natural resistance profiles that allow L. johnsonii to survive in competitive gut environments while affecting microbial community composition.

How might post-translational modifications of L. johnsonii rpsE influence its function in different physiological conditions?

Ribosomal proteins, including rpsE, undergo various post-translational modifications (PTMs) that can significantly impact their function under different conditions. For L. johnsonii rpsE, researchers should consider:

Potential PTMs to Investigate:

• Phosphorylation: Often regulates protein-protein and protein-RNA interactions

• Methylation: Can affect RNA binding properties

• Acetylation: May influence protein stability and interactions

• Glycosylation: Potentially affecting protein folding and recognition

Analytical Approaches:

• Mass spectrometry-based PTM mapping under various growth conditions

• Site-directed mutagenesis of modified residues to determine functional significance

• In vitro modification assays to study enzymatic processes

• Comparison of PTM profiles between planktonic culture and gastrointestinal colonization states

Physiological Relevance:
PTM profiles may change during:

• Adaptation to acid stress in the stomach environment

• Response to bile exposure in the intestine

• Biofilm formation versus planktonic growth

• Interaction with host immune cells

These modifications could serve as molecular switches that allow L. johnsonii to rapidly adapt its translational machinery to changing environmental conditions without requiring new protein synthesis.

What insights can comparative genomic and proteomic analyses of rpsE across Lactobacillus species provide for understanding strain-specific probiotic properties?

Comparative analysis of rpsE across Lactobacillus species can provide valuable insights into evolutionary adaptations and potential functional specializations:

Genomic Approach:

• Whole genome sequencing of multiple L. johnsonii strains to identify rpsE variants

• Analysis of selection pressure on different domains of the protein

• Identification of strain-specific genetic context surrounding the rpsE gene

• Correlation of sequence variations with known probiotic phenotypes

Proteomic Analysis:

• Comparison of rpsE expression levels across strains under standardized conditions

• Identification of strain-specific interacting partners using pull-down assays coupled with mass spectrometry

• Analysis of differential post-translational modification patterns between strains

Structural Biology Perspective:

• Modeling of strain-specific rpsE structures to identify potential functional differences

• Docking studies with various ligands including rRNAs and other ribosomal proteins

• Molecular dynamics simulations to predict flexibility and interaction differences

Evolutionary Considerations:
L. johnsonii has co-evolved with different animals at the species or strain level , which might be reflected in ribosomes adapted to specific host environments. These adaptations could influence translation efficiency of genes related to host interaction and provide clues to strain-specific probiotic efficacy.

What are the recommended approaches for studying rpsE interactions with host cells during L. johnsonii colonization?

To investigate the potential interactions of L. johnsonii rpsE with host cells during colonization, researchers should consider these methodological approaches:

Ex Vivo Interaction Studies:

• Intestinal organoid co-culture systems with labeled recombinant rpsE

• Tissue explant models to maintain host tissue architecture

• Transwell systems to study epithelial translocation of rpsE

In Vivo Tracking:

• Generation of fluorescently tagged rpsE expressed in L. johnsonii

• Conditional expression systems to control rpsE variant production

• Intravital microscopy to visualize interactions in real-time

Molecular Interaction Analysis:

• Surface plasmon resonance to identify host receptors for rpsE

• Immunoprecipitation of rpsE from colonized tissue to identify bound host proteins

• Yeast two-hybrid or BioID proximity labeling to map the interactome

Functional Outcome Assessment:

• Analysis of epithelial transcriptome changes following exposure to wild-type versus mutant rpsE

• Measurement of barrier function parameters (TEER, permeability markers)

• Cytokine profiling to assess immunomodulatory effects

Table 1: Recommended Host Cell Models for L. johnsonii rpsE Interaction Studies

What techniques are most effective for studying the role of rpsE in L. johnsonii stress responses relevant to gastrointestinal survival?

Understanding how rpsE contributes to L. johnsonii stress responses is crucial for deciphering its survival in the gastrointestinal tract. The following methodological approaches are recommended:

Stress Exposure Models:

• Acid stress (pH gradient challenges)

• Bile salt exposure at physiologically relevant concentrations

• Oxidative stress using hydrogen peroxide or other oxidizing agents

• Nutrient limitation to mimic competitive gut environments

• Temperature fluctuations reflecting host fever responses

Analytical Techniques:

• Transcriptomic Analysis:

  • RNA-seq to identify stress-responsive gene networks

  • ribosome profiling to assess translational changes during stress

  • RT-qPCR for targeted gene expression analysis

• Proteomic Approaches:

  • Quantitative proteomics to measure rpsE abundance under different stresses

  • Phosphoproteomics to detect stress-induced modifications

  • Protein turnover studies using pulse-chase labeling

• Structural Studies:

  • Hydrogen-deuterium exchange mass spectrometry to monitor conformational changes

  • Limited proteolysis to identify regions with altered accessibility

  • FRET-based assays to detect intramolecular rearrangements

• Genetic Manipulation:

  • Creation of rpsE variants with modifications at stress-responsive sites

  • Complementation of rpsE knockdown strains with variant proteins

  • Overexpression studies to assess protective effects

Validation in Complex Models:

• Survival assays in simulated gastric and intestinal fluids

• Competition assays against gut commensals

• In vivo tracking using bioluminescent reporters

By employing these methodologies, researchers can gain insights into how rpsE contributes to the remarkable stress tolerance of L. johnsonii, which underlies its probiotic potential and therapeutic applications in various diseases .