Recombinant Lactobacillus plantarum 30S ribosomal protein S15 (rpsO)

What is the structural and functional role of ribosomal protein S15 in Lactobacillus plantarum?

S15 is a critical primary RNA-binding protein that serves as a component of the 30S ribosomal subunit in Lactobacillus plantarum. Structurally, bacterial S15 typically consists of four alpha helices, with three forming the core structure and one N-terminal helix protruding outward . The protein plays a dual role in bacterial cells: first, it organizes rRNA during ribosome assembly, and second, in many bacterial species, it can interact with structured portions of its own transcript to negatively regulate gene expression . S15 specifically helps organize the central domain of 16S rRNA and facilitates the assembly of the platform region of the 30S subunit. This protein belongs to a conserved family with homologs across bacterial species, though the specific RNA recognition profiles can vary between homologs .

How does S15 contribute to ribosome assembly in bacteria?

S15 functions as a primary binding protein in the hierarchical assembly of the 30S ribosomal subunit. During assembly, S15 binds to the central domain of 16S rRNA, orchestrating the subsequent binding of secondary and tertiary ribosomal proteins - specifically S6, S11, S18, and S21 - to form the platform of the 30S subunit . In vitro reconstitution experiments have demonstrated that the association of S15 with 16S rRNA is typically required for these other proteins to bind to the emerging complex . Additionally, isothermal titration calorimetry studies have shown that the S6/S18 heterodimer does not bind to 16S rRNA in the absence of S15 under in vitro conditions . S15 also contributes to the formation of an interface bridge between the 30S and 50S subunits in the functional 70S ribosome, suggesting a role in subunit association beyond its assembly function .

What techniques are commonly used to study recombinant S15 protein interactions?

Several experimental approaches are employed to investigate S15 interactions:

These techniques provide complementary information about the binding specificity, affinity, and functional consequences of S15-RNA interactions in experimental settings .

What are the optimal conditions for recombinant expression of L. plantarum S15 protein?

While the search results don't provide specific optimization parameters for L. plantarum S15 expression, established protocols for similar ribosomal proteins can be adapted. Expression typically employs E. coli BL21(DE3) or similar strains with expression vectors containing T7 promoters. The methodology should include:

• Codon optimization of the rpsO gene for the expression host

• Temperature optimization (typically 18-30°C for ribosomal proteins to prevent inclusion body formation)

• Induction parameters testing (IPTG concentration 0.1-1.0 mM)

• Expression duration trials (4-24 hours)

• Media composition assessment (rich media vs. minimal media)

Purification generally involves affinity chromatography (His-tag or GST-tag), followed by ion exchange and size exclusion chromatography. The purified protein should be assessed for proper folding using circular dichroism spectroscopy and functional RNA binding assays .

How can researchers verify the functional activity of recombinant S15 protein?

Functional verification of recombinant S15 should include multiple assays:

• RNA binding assays using electrophoretic mobility shift assays (EMSA) or filter binding assays with 16S rRNA fragments

• In vitro ribosome reconstitution to assess the ability of recombinant S15 to promote assembly of 30S subunits

• Comparative association studies measuring the ability of S15-containing vs. S15-deficient 30S subunits to form 70S ribosomes with natural 50S subunits

• Testing the ability of recombinant S15 to complement growth defects in ΔrpsO strains, particularly under suboptimal conditions like low temperature

Functional S15 should bind specifically to its RNA targets, promote assembly of ribosomal proteins in the S15 assembly branch, and support efficient subunit association . Researchers should pay particular attention to temperature-dependent effects, as S15 deletion strains show cold sensitivity with marked ribosome biogenesis defects at low temperatures .

How can researchers investigate the RNA recognition specificity of S15 from different bacterial species?

The RNA recognition specificity of S15 homologs from different bacterial species can be investigated through a multifaceted approach:

• Heterologous complementation assays: Testing whether S15 from one species can functionally replace S15 in another species, particularly focusing on the regulatory function

• Chimeric protein analysis: Creating fusion proteins between S15 homologs to identify domains responsible for specificity

• Binding assays with diverse RNA structures: Measuring affinity of S15 homologs for the four distinct mRNA structures known to interact with S15

• Structural analysis: Comparing solution structures of S15-RNA complexes from different species using NMR or X-ray crystallography

• Evolutionary analysis: Examining patterns of coevolution between S15 coding sequences and their regulatory RNA structures

Research has shown that despite their shared RNA binding function in the rRNA, S15 homologs exhibit distinct RNA recognition profiles, and even minor changes to amino acid sequences or RNA structural motifs can significantly impact RNA-protein recognition . Researchers should design experiments that can distinguish between binding to regulatory RNA structures versus binding to rRNA.

What are the methodological considerations for studying S15 deletion effects in L. plantarum?

Creating and studying S15 deletion in L. plantarum requires careful experimental design:

• Construction of precise in-frame deletion of rpsO using CRISPR-Cas9 or traditional homologous recombination approaches

• Confirmation of deletion through whole genome sequencing to ensure no compensatory mutations arise

• Growth characterization across various conditions, particularly temperature ranges (20-42°C) and stressful environments

• Ribosome profile analysis using sucrose gradient centrifugation to assess subunit ratios and polysome formation

• In vitro association assays to test 30S-50S subunit joining capacity

• Complementation studies with wild-type and mutant S15 variants

• Proteomic analysis of ribosomes to verify assembly of S6, S11, S18, and S21 in the absence of S15

Research on E. coli has shown that ΔrpsO strains are viable but display growth defects and cold sensitivity, suggesting S15 becomes critical for assembly under suboptimal conditions . Researchers should be particularly attentive to potential species-specific differences, as the plasticity of ribosome assembly may vary between E. coli and L. plantarum.

How can researchers assess the impact of S15 modifications on Lactobacillus plantarum probiotic function?

To evaluate how S15 modifications affect L. plantarum probiotic properties, researchers should implement a comprehensive assessment approach:

• Gastrointestinal transit simulation: Testing tolerance to simulated gastric and intestinal conditions using artificial gut models

• Adhesion assays: Measuring adherence to intestinal epithelial cell lines (e.g., Caco-2, HT-29)

• Anti-inflammatory capacity analysis: Assessing the ability to reduce pro-inflammatory cytokine production in LPS-stimulated cell models

• In vivo colitis models: Evaluating the therapeutic efficacy in DSS or TNBS-induced colitis in mice

• Gut microbiome analysis: Assessing impact on microbiota composition through 16S rRNA sequencing

• Immunomodulatory pathway investigation: Measuring effects on TLR4, MyD88, and NF-κB signaling

Lactobacillus plantarum has demonstrated potential therapeutic applications in ulcerative colitis management, with mechanisms involving anti-inflammatory effects and gut microbiota modulation . Researchers should determine whether S15 modifications alter these beneficial properties, potentially affecting the strain's therapeutic efficacy.

What mechanisms explain the viability of bacterial strains lacking S15 despite its apparent importance in ribosome assembly?

The surprising viability of S15 deletion strains presents a fascinating research question. Several hypothetical mechanisms may explain this observation:

• Functional redundancy: Other ribosomal proteins may compensate for S15's role in organizing 16S rRNA

• Alternative assembly pathways: In vivo assembly may follow different pathways than observed in vitro reconstitution

• Conditional essentiality: S15 may only be essential under specific growth conditions (supported by cold sensitivity)

• Assembly factors: In vivo assembly factors may facilitate ribosome formation in the absence of S15

• Growth-rate adaptation: Slow growth phenotype may allow sufficient time for less efficient assembly

Research in E. coli has shown that despite in vitro studies indicating S15 is required for the binding of S6, S18, S11, and S21, these proteins successfully assemble into ribosomes in vivo in ΔrpsO strains . This reveals "a remarkable level of plasticity and perhaps redundancy in ribosome assembly and function in vivo that has not been observed in vitro" . Future research should focus on identifying the molecular mechanisms enabling this plasticity and determining whether similar adaptability exists in L. plantarum.

How does post-translational modification of S15 affect its dual functionality in ribosome assembly and autoregulation?

Post-translational modifications (PTMs) of S15 may significantly influence its functionality, though this area remains underexplored. Research approaches should include:

• Mass spectrometry analysis to identify and quantify PTMs on S15 across growth phases and stress conditions

• Creation of modification-mimicking mutants to test functional consequences

• In vitro and in vivo binding assays comparing modified and unmodified S15

• Structural studies examining how PTMs affect S15 conformation and interaction surfaces

• Temporal analysis of PTM patterns during ribosome assembly vs. autoregulation

The dual functionality of S15 in ribosome assembly and autoregulation may be differentially affected by PTMs, potentially serving as a regulatory mechanism to balance these functions based on cellular needs. Researchers should pay particular attention to conditions that might trigger a switch between these roles, such as nutrient limitation or translational stress.

What are the implications of S15 strain-specific variations for designing recombinant L. plantarum as therapeutic agents?

Strain-specific variations in S15 structure and function have important implications for the development of recombinant L. plantarum strains as therapeutic agents:

L. plantarum has demonstrated therapeutic potential in conditions like ulcerative colitis, with strain-specific effects . The L15 strain shows particularly promising characteristics including high gastrointestinal transit tolerance, strong adhesion properties, and significant anti-inflammatory abilities . Researchers developing recombinant strains should consider how S15 modifications might influence these beneficial properties and carefully characterize any engineered strains across multiple parameters.

What strategies can address purification difficulties with recombinant S15 protein?

Researchers often encounter challenges when purifying recombinant S15. Effective troubleshooting strategies include:

S15's nucleic acid binding properties can cause co-purification with host RNA, requiring stringent purification protocols. Additionally, its role as part of a multi-protein complex may result in exposure of hydrophobic surfaces, leading to aggregation when expressed alone .

How can researchers accurately assess the in vivo assembly of ribosomes in S15-modified L. plantarum strains?

Accurate assessment of ribosome assembly in S15-modified strains requires multiple complementary approaches:

• Ribosome profiling using sucrose gradient ultracentrifugation to quantify 30S, 50S, 70S, and polysome distributions

• Stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry to measure incorporation of ribosomal proteins

• Pulse-chase experiments with radiolabeled precursors to track assembly kinetics

• Electron microscopy to examine structural integrity of assembled ribosomes

• In vivo RNA structure probing (e.g., DMS-MaPseq) to assess 16S rRNA folding

• Functional translation assays measuring rates of protein synthesis and fidelity

What emerging technologies will advance our understanding of S15 function in L. plantarum?

Several cutting-edge technologies hold promise for deepening our understanding of S15:

• Cryo-electron microscopy for high-resolution structural studies of L. plantarum ribosomes with various S15 modifications

• Single-molecule FRET to directly observe S15-RNA interactions in real-time

• Ribosome profiling to assess translation effects of S15 modifications at nucleotide resolution

• RNA-protein interaction mapping via CLIP-seq to identify all RNA targets of S15 in vivo

• Bacterial cytological profiling to examine cellular consequences of S15 modifications

• Microfluidic techniques to analyze single-cell variability in ribosome assembly

• Mathematical modeling of ribosome assembly pathways incorporating thermodynamic and kinetic parameters

These approaches will allow researchers to move beyond traditional bulk biochemical assays to gain dynamic, high-resolution insights into S15 function in living cells. Integrating these approaches will be particularly valuable for understanding the plasticity of ribosome assembly observed in vivo but not captured in in vitro reconstitution systems .

How might S15 engineering be leveraged to develop improved probiotic or therapeutic L. plantarum strains?

Engineering S15 presents several potential avenues for developing enhanced L. plantarum strains:

• Ribosome engineering to optimize expression of therapeutic proteins

• Fine-tuning translational efficiency for improved stress tolerance and GI tract survival

• Modifying autoregulatory mechanisms to achieve controlled expression profiles

• Creating chimeric S15 proteins with altered RNA specificity for synthetic biology applications

• Developing strains with enhanced anti-inflammatory properties through optimized production of beneficial factors