Recombinant Lactobacillus plantarum 30S ribosomal protein S16 (rpsP)

Basic Research Questions

• What is the optimal expression system for producing Recombinant L. plantarum 30S ribosomal protein S16?

  While E. coli remains the most widely used expression system for recombinant ribosomal proteins due to its high yield and shorter turnaround times, several expression systems can be used for producing recombinant 30S ribosomal protein S16:

  Expression System	Advantages	Disadvantages
  E. coli	High yield, cost-effective, rapid growth	Limited post-translational modifications
  L. plantarum	GRAS status, surface display capabilities, potential for mucosal delivery	Lower yield compared to E. coli
  Yeast	Post-translational modifications, high yield	Longer production time, more complex media
  Insect/Baculovirus	Advanced post-translational modifications	Complex methodology, lower yield
  Mammalian cells	Complete post-translational modifications	Costly, time-consuming, lowest yield

  For L. plantarum rpsP specifically, the pSIP expression vectors have shown promising results for producing recombinant ribosomal proteins in L. plantarum strains .

• What is the function of 30S ribosomal protein S16 in the bacterial translation process?

  The 30S ribosomal protein S16 serves several critical functions in bacterial translation:

  • Forms part of the 30S ribosomal subunit that binds mRNA and initiator tRNA

  • Specifically participates in binding the anticodon stem-loop of tRNA during elongation

  • Contributes to maintaining the translational reading frame when the A site is unoccupied

  • Interacts directly with 16S rRNA to support proper ribosome function

  Crystal structures of the 30S subunit have confirmed that while S16 makes direct contacts with tRNA, the primary interactions are with 16S rRNA. Notably, experiments have shown that 16S rRNA contacts alone are sufficient to support protein synthesis, indicating the fundamental importance of S16 in ribosomal architecture .

• What promoter systems are effective for expressing recombinant proteins in L. plantarum?

  Several promoter systems have been evaluated for recombinant protein expression in L. plantarum, with varying expression strengths:

  Promoter	Origin	Relative Strength	Characteristics
  P₁₆ₛ ᵣᵣₙₐ	L. plantarum	++++	Synthetic library based on 16S rRNA promoter
  Pₗₚᵈₕ	L. plantarum	+++	Constitutive, metabolically regulated
  Pₜᵤf	L. plantarum	+++	Constitutive, moderately high expression
  Pₗₚ₋₃₀₅₀	L. plantarum	++	Signal peptide promoter
  Pₜₗₚₐ	S. typhimurium	+++++	Orthogonal promoter, very high expression
  Pₓ	S. pneumoniae	++++	Effective in multiple L. plantarum strains
  pSIP inducible	L. sakei	+++++	Inducible by autoinducer peptides

  Notably, the orthogonal promoter Pᵗˡᵖᴬ from Salmonella typhimurium has been shown to surpass the expression levels of native L. plantarum promoters like P₄₈ and P₂₃ by approximately fivefold .

• How can L. plantarum expression systems be optimized for recombinant ribosomal protein production?

  Optimization of L. plantarum expression systems involves several key strategies:

  • Codon optimization: Adapting the coding sequence to L. plantarum's codon usage bias

  • Signal peptide selection: Testing different signal peptides for optimal secretion or cell-surface display (Lp_2145, Lp_0373, and Lp_AmyA have shown notably higher yields )

  • Promoter selection: Using strong constitutive promoters or inducible systems like pSIP vectors

  • Growth conditions: Optimizing temperature, pH, and media composition for maximum protein yield

  • Plasmid stability: Ensuring stable maintenance of expression constructs through multiple generations

  • Reference gene selection: Using appropriate reference genes (gmk, gyrA, and gapB) for RT-qPCR analysis when evaluating expression levels

  For ribosomal proteins specifically, intracellular expression rather than secretion may be preferable due to their natural intracellular localization.

• What methods are used to verify successful expression of recombinant ribosomal proteins in L. plantarum?

  Multiple complementary methods should be employed to verify expression:

  • Western blotting: Using antibodies specific to the target protein or added tags (e.g., His-tag)

  • Immunofluorescence assay (IFA): Confirming protein location (surface vs. intracellular)

  • Flow cytometry: Quantitative assessment of expression levels in bacterial populations

  • RT-qPCR: Measuring mRNA levels to confirm transcription of the target gene

  • Mass spectrometry: Precise identification of the recombinant protein and potential modifications

  • Functional assays: Confirming biological activity of the expressed protein

  For example, successful expression of recombinant proteins in L. plantarum can be confirmed using SDS-PAGE followed by Western blot analysis using specific antibodies, as demonstrated in several studies .

Advanced Research Questions

• How does the secondary structure of recombinant L. plantarum 30S ribosomal protein S16 compare to native protein?

  CD spectroscopy analysis of recombinant ribosomal protein S16 has revealed important structural characteristics that can be compared to native proteins:

  • Approximately 21 ± 4% of amino acid sequence forms α-helices

  • About 24 ± 3% forms β-strands

  • The protein exhibits rapid denaturation at pH above 8.0

  • Increasing urea concentration causes slow unfolding of the protein structure

  These findings indicate that the secondary structure of recombinant S16 largely maintains the expected secondary structure elements. For optimal stability, purification and storage conditions should maintain pH below 8.0 to preserve the native conformation .

  When expressing recombinant ribosomal proteins, it's critical to validate whether the recombinant protein maintains the secondary structure characteristics of the native protein, as this directly impacts functional studies.

• What strategies can overcome inclusion body formation when expressing ribosomal proteins in L. plantarum?

  Ribosomal proteins are prone to inclusion body formation due to their interactions with RNA and other proteins. Several strategies can mitigate this issue:

  Strategy	Methodology	Effectiveness
  Optimized refolding	Stepwise dialysis with decreasing denaturant concentration	High for S16, requires optimization
  Fusion tags	Solubility enhancers (SUMO, MBP, TrxA)	Moderate, may affect structure
  Lower expression temperature	25-30°C instead of 37°C	Variable effectiveness
  Co-expression of chaperones	GroEL/GroES or DnaK systems	Significant improvement possible
  pSIP inducible system	Controlled expression rate	Highly effective for L. plantarum

  A successful method for S16 refolding from inclusion bodies involves solubilization in urea or guanidine hydrochloride followed by stepwise dialysis with decreasing denaturant concentration while maintaining pH below 8.0 to prevent denaturation .

• How can the interaction between recombinant 30S ribosomal protein S16 and 16S rRNA be studied?

  Several methodological approaches can be employed to study the interaction between recombinant S16 and 16S rRNA:

  • RNA footprinting: Identifying RNA regions protected by S16 binding

  • Cross-linking studies: Mapping direct contact points between protein and RNA

  • Electrophoretic mobility shift assays (EMSA): Detecting complex formation

  • Surface plasmon resonance (SPR): Measuring binding kinetics and affinity

  • Isothermal titration calorimetry (ITC): Determining thermodynamic parameters

  • Cryo-electron microscopy: Visualizing structural details of the complex

  • Blue-Native/SDS-PAGE: Identifying protein complexes maintained under native conditions

  Blue-Native/SDS-PAGE has been successfully used for studying protein-protein interactions in L. plantarum, generating interaction maps that reveal both heterodimeric and homodimeric complexes . This approach could be adapted to study S16-RNA interactions by including RNA in the native separation step.

• What are the challenges in studying 30S ribosomal protein S16 function in vivo using recombinant L. plantarum?

  Investigating S16 function in vivo presents several methodological challenges:

  • Essential nature: S16 is essential for ribosome assembly and function, making knockout studies challenging

  • Complementation complexity: Requires precise expression levels to maintain ribosome homeostasis

  • RNA interactions: Proper folding and interactions with 16S rRNA are critical for function

  • Growth effects: Perturbations in ribosomal proteins often affect growth rates, confounding analysis

  • Heterologous expression issues: Recombinant expression may not fully recapitulate native regulation

  • Mutagenesis limitations: Many mutations may be lethal or severely affect fitness

  Approaches to address these challenges include:

  • Using inducible expression systems for controlled complementation

  • Creating partial deletions or point mutations rather than complete knockouts

  • Employing ribosome profiling to assess translational impacts

  • Utilizing reporter systems fused to S16 to monitor localization and incorporation into ribosomes

• How can site-directed mutagenesis be used to study functional domains of L. plantarum 30S ribosomal protein S16?

  Site-directed mutagenesis offers powerful insights into structure-function relationships in S16:

  1. Identify conserved residues: Perform multiple sequence alignment across bacterial species to identify highly conserved amino acids

  2. Target RNA-binding domains: Mutate residues implicated in 16S rRNA binding

  3. Design expression constructs: Use the pSIP system for controlled expression in L. plantarum

  4. Generate mutations: Create alanine substitutions or conservative replacements

  5. Functional assays: Assess:

     • Ribosome assembly efficiency

     • Translation rate and accuracy

     • Growth phenotypes

     • Antibiotic sensitivity profiles (certain antibiotics target the 30S subunit)

  The pSIP expression system has been successfully used for mutational analysis of other membrane proteins in L. plantarum and could be adapted for S16 studies . When investigating essential ribosomal proteins, expressing the mutant variant while maintaining the wild-type copy can prevent lethal phenotypes while still allowing functional assessment.

• What immune responses are triggered by recombinant L. plantarum expressing modified ribosomal proteins?

  Recombinant L. plantarum can elicit multiple immune responses relevant to both basic research and vaccine applications:

  Immune Component	Response to Recombinant L. plantarum	Detection Method
  Serum IgG	Significantly increased levels	ELISA
  IgG subtypes	IgG1 and IgG2a elevations	ELISA
  Mucosal sIgA	Enhanced in intestine, lungs, feces	Immunofluorescence, ELISA
  T cells	Increased CD4+IFN-γ+ and CD8+IFN-γ+ cells	Flow cytometry
  T cell proliferation	Enhanced in spleen and MLNs	CFSE staining
  B cells	Increased B220+IgA+ cells in Peyer's patches	Flow cytometry
  Dendritic cells	Activation in Peyer's patches	Flow cytometry

  These immune responses are particularly valuable when L. plantarum is engineered to express antigenic proteins, as the bacterium serves both as expression host and adjuvant. The immune response profile includes both systemic (serum IgG) and mucosal (sIgA) components, making this system potentially useful for vaccine applications .

• How can RNA-seq be used to evaluate the impact of recombinant ribosomal protein expression on L. plantarum physiology?

  RNA-seq provides comprehensive insights into the global transcriptional response to recombinant protein expression:

  1. Experimental design:

     • Compare wild-type L. plantarum to strains expressing recombinant S16

     • Sample at multiple time points post-induction

     • Include appropriate reference genes (gmk, gyrA, gapB have been validated for L. plantarum)

  2. Key parameters to analyze:

     • Stress response genes (heat shock proteins, chaperones)

     • Translation machinery genes (other ribosomal proteins, translation factors)

     • Energy metabolism pathways

     • Cell wall and membrane stress responses

     • Growth phase-dependent expression patterns

  3. Data analysis approach:

     • Differential expression analysis to identify significantly altered gene expression

     • Pathway enrichment to identify affected biological processes

     • Time-course analysis to track adaptation to recombinant protein expression

  RNA-seq has been used to study L. plantarum gene expression under various conditions, including responses to oxidative stress and carbon source availability, providing a methodological framework applicable to studying the effects of recombinant ribosomal protein expression .

• What is the role of reference genes in accurately quantifying recombinant ribosomal protein expression in L. plantarum?

  Selecting appropriate reference genes is critical for accurate quantification of recombinant protein expression:

  1. Validated reference genes for L. plantarum:

     • gmk (guanylate kinase)

     • gyrA (DNA gyrase subunit A)

     • gapB (glyceraldehyde-3-phosphate dehydrogenase)

  2. Impact of growth conditions:

     • Reference gene stability varies with growth phase

     • Environmental factors (pH, temperature, nutrients) affect expression

     • Recombinant protein expression itself may alter housekeeping gene expression

  3. Methodological recommendations:

     • Use multiple reference genes simultaneously (minimum 3)

     • Validate stability under specific experimental conditions

     • Apply geometric averaging of multiple references

     • Use software like geNorm or NormFinder to select optimal references

  Studies have shown that expression of housekeeping genes in L. plantarum can vary significantly with growth phase and experimental conditions. Using a combination of validated reference genes is essential for accurate RT-qPCR analysis of recombinant protein expression levels .

• How does the gut microbiome respond to recombinant L. plantarum expressing ribosomal proteins?

  The introduction of recombinant L. plantarum into the gut ecosystem produces several measurable effects on the microbiome:

  Microbiome Parameter	Observed Effects	Analysis Method
  Species diversity	Significant increase (Shannon-Wiener index)	16S rRNA sequencing
  Microbial structure	Altered community composition	Beta diversity analysis
  OTU abundance	Changed number of operational taxonomic units	16S rRNA analysis
  Functional pathways	Enhanced metabolism and immune regulation	Functional clustering
  Colonization	Varies by strain and expressed protein	Fluorescence tracking

  Recombinant L. plantarum strains expressing fusion proteins have been shown to enhance species diversity of gut bacteria based on the Shannon-Wiener index. Beta diversity analysis demonstrated that microbial structure is measurably changed by recombinant L. plantarum colonization. Furthermore, functional analysis revealed enrichment in immune system, metabolism, and energy metabolism pathways in the gut microbiota after administration of recombinant L. plantarum .

• What proteomics approaches can identify post-translational modifications of recombinant ribosomal proteins in L. plantarum?

  Several proteomics approaches can be employed to characterize post-translational modifications:

  1. Sample preparation:

     • Subcellular fractionation (cytoplasmic vs. membrane-associated)

     • Enrichment of modified peptides (phosphopeptides, glycopeptides)

     • Protein digestion with multiple proteases for better coverage

  2. Analysis techniques:

     • LC-MS/MS with high-resolution mass analyzers

     • Electron transfer dissociation (ETD) for labile modifications

     • SILAC or TMT labeling for quantitative comparison

     • Data-independent acquisition (DIA) for comprehensive detection

  3. Bioinformatics analysis:

     • Database searching with variable modifications

     • De novo sequencing for unexpected modifications

     • Site localization scoring for precise modification mapping

  The combination of two-dimensional electrophoresis with tandem mass spectrometry has been successfully used to identify hundreds of proteins in L. plantarum, including ribosomal proteins. This approach could be adapted to specifically investigate modifications of recombinant ribosomal proteins by comparing the recombinant protein profile with the native protein .