Recombinant Bacteroides thetaiotaomicron 30S ribosomal protein S18 (rpsR)

What is the basic structure and function of the B. thetaiotaomicron 30S ribosomal protein S18?

The 30S ribosomal protein S18 (rpsR) in Bacteroides thetaiotaomicron is a small basic protein that forms part of the 30S small ribosomal subunit. As with other S18 proteins, it likely plays a critical role in the assembly of the 30S subunit and in maintaining ribosomal stability during translation. The protein contains RNA-binding motifs that facilitate interaction with ribosomal RNA and potentially with mRNA during translation. Its basic nature suggests it may interact with the negatively charged phosphate backbone of RNA to stabilize the ribosome structure . In reconstitution experiments with other bacterial ribosomal proteins, S18 has been shown to be essential for proper 30S subunit assembly and function, with reconstituted subunits reaching approximately 30% of native activity in translation assays .

How is rpsR expression regulated in B. thetaiotaomicron?

Expression of rpsR in B. thetaiotaomicron is likely controlled as part of the broader regulatory networks governing ribosomal protein synthesis. Based on transcriptomic data, ribosomal protein genes typically show coordinated expression patterns that respond to growth conditions and nutrient availability. In the expanded transcriptome atlas for B. thetaiotaomicron, ribosomal proteins including rpsR demonstrate dynamic expression across different growth conditions, with significant modulation during stress responses . Specifically, during nutrient limitation or stress conditions, expression of ribosomal proteins may be downregulated as part of a general stress response, while during rapid growth in nutrient-rich environments, expression is typically upregulated to support increased protein synthesis demands .

Does rpsR expression vary across different growth conditions for B. thetaiotaomicron?

Yes, rpsR expression in B. thetaiotaomicron shows condition-dependent variation. According to the comprehensive transcriptome atlas study, ribosomal protein genes display significant expression changes across the 15 different in vivo-relevant growth conditions examined . During exponential growth in rich media, ribosomal proteins including rpsR typically show high expression levels. Conversely, under stress conditions such as oxidative stress, heat shock, or nutrient limitation, expression levels decrease as part of the bacterial stress response. The transcriptional profiling data indicates that over 94% of B. thetaiotaomicron genes show expression in at least one of the 15 conditions tested, with ribosomal components demonstrating particularly dynamic regulation patterns that reflect the metabolic status of the cell .

What are the optimal conditions for heterologous expression of recombinant B. thetaiotaomicron rpsR?

For optimal heterologous expression of recombinant B. thetaiotaomicron rpsR, researchers should consider several key factors. Expression in E. coli BL21(DE3) strain with a pET-based vector system typically yields good results for ribosomal proteins. Induction with 0.5-1.0 mM IPTG at mid-log phase (OD600 ~0.6) followed by expression at lower temperatures (16-20°C) for 16-18 hours can help prevent formation of inclusion bodies. A purification strategy similar to that used for other ribosomal proteins involves initial Ni-NTA affinity chromatography for His-tagged constructs, followed by ion exchange chromatography to leverage the basic nature of S18. Based on approaches used for reconstitution studies of 30S subunits, maintaining high salt conditions (>500 mM NaCl) during purification helps prevent non-specific RNA binding . Following purification, functional activity should be assessed through RNA binding assays similar to those used for other RNA-binding proteins in B. thetaiotaomicron .

How can researchers determine if recombinant rpsR properly folds and maintains functionality?

Assessment of proper folding and functionality of recombinant B. thetaiotaomicron rpsR can be approached through multiple complementary methods:

• RNA binding assays: Electrophoretic mobility shift assays (EMSAs) with ssRNA probes can determine if the recombinant protein retains RNA-binding capability, similar to methods used for other Bacteroides RNA-binding proteins . Pentaprobe analysis using 100-nucleotide RNA probes containing all possible 5-nt sequence combinations can help determine binding specificity.

• Ribosomal reconstitution assays: Functional activity can be tested by incorporating the recombinant rpsR into in vitro 30S ribosomal subunit reconstitution experiments. The activity of reconstituted 30S subunits can be measured through poly(U)-directed polyphenylalanine synthesis assays or complete protein synthesis reactions using the PURE (Protein synthesis Using Recombinant Elements) system .

• Structural characterization: Circular dichroism (CD) spectroscopy can confirm proper secondary structure formation, while thermal shift assays can assess protein stability. Comparison with native 30S subunits through sucrose gradient sedimentation patterns can further confirm proper incorporation and functionality .

Reconstituted 30S subunits containing properly folded rpsR should display approximately 30% of the translational activity of native 30S subunits in poly(U)-directed polyphenylalanine synthesis assays, with this activity potentially increasing to 80% with the addition of other essential factors .

What are the challenges in studying protein-RNA interactions for B. thetaiotaomicron rpsR?

Studying protein-RNA interactions for B. thetaiotaomicron rpsR presents several significant challenges:

• RNA degradation: B. thetaiotaomicron, like other Bacteroides species, possesses numerous RNases that can rapidly degrade RNA samples. Researchers must implement stringent RNase-free conditions and use RNase inhibitors throughout experimental procedures.

• Specificity determination: Distinguishing specific from non-specific binding is challenging for ribosomal proteins, which often have both specific recognition sites and broader RNA affinity. Methods similar to those used for other Bacteroides RNA-binding proteins can help determine binding specificity, including using multiple RNA probes with different sequences . The calculated dissociation constants (Kd) for RNA binding by other Bacteroides RNA-binding proteins range from 50-500 nM, providing a reference range for rpsR binding affinity assessment .

• In vivo relevance: Correlating in vitro binding data with physiological function requires additional approaches such as RNA immunoprecipitation followed by sequencing (RIP-seq) or CLIP-seq (cross-linking immunoprecipitation-sequencing) to identify true in vivo binding partners. These techniques could be adapted from approaches like MS2 affinity purification used to identify small RNA targets in B. thetaiotaomicron .

• Structural complexity: The ribosomal context adds complexity, as rpsR naturally functions within the intricate architecture of the 30S subunit rather than in isolation. Cryo-EM or crystallographic approaches may be necessary to fully understand structural interactions.

How does rpsR contribute to translation efficiency in B. thetaiotaomicron?

The 30S ribosomal protein S18 (rpsR) contributes to translation efficiency in B. thetaiotaomicron through several mechanisms:

• Ribosome assembly: S18 plays a critical role in the proper assembly and structural integrity of the 30S ribosomal subunit. In reconstitution experiments with bacterial ribosomes, the presence of all small subunit proteins including S18 is necessary to achieve functional translation. Reconstituted 30S subunits exhibit approximately 30% of native translation activity, highlighting the importance of proper protein incorporation .

• mRNA binding: S18 likely contributes to mRNA positioning on the ribosome, particularly during initiation. Its basic nature facilitates interaction with the negatively charged RNA phosphate backbone.

• Translation fidelity: As part of the decoding center environment, S18 may influence tRNA selection and translational accuracy.

• Adaptation to environmental conditions: The regulation of rpsR expression in response to changing conditions enables B. thetaiotaomicron to modulate its translational capacity. The comprehensive transcriptome atlas for B. thetaiotaomicron reveals condition-specific expression patterns that likely optimize translation efficiency under different environmental stresses .

Experimental evidence from reconstitution studies indicates that ribosomal subunits containing properly incorporated S18 demonstrate significantly higher translation rates compared to those lacking this protein, emphasizing its importance for efficient protein synthesis .

Does rpsR interact with any regulatory small RNAs in B. thetaiotaomicron?

While no direct interactions between rpsR and small RNAs (sRNAs) in B. thetaiotaomicron have been definitively established, several lines of evidence suggest potential regulatory relationships:

• The expanded transcriptome atlas for B. thetaiotaomicron has identified numerous sRNAs that are conditionally expressed and may interact with components of the translation machinery . Among these, sRNAs like MasB have been shown to regulate processes including antibiotic tolerance, suggesting broad regulatory capabilities that could extend to ribosomal components .

• In other bacterial species, ribosomal proteins often participate in autoregulatory feedback loops involving interactions with regulatory RNAs. Similar mechanisms may exist in B. thetaiotaomicron.

• The RNA-binding protein family identified in Bacteroides species shows interaction with single-stranded RNA with specific binding preferences . This suggests a complex network of protein-RNA interactions that could include regulatory relationships between sRNAs and ribosomal components.

To identify potential sRNA interactions with rpsR, techniques such as MS2 affinity purification coupled with RNA-seq, which has been successfully applied to identify sRNA targets in B. thetaiotaomicron , could be employed. Additionally, differential expression analysis comparing wild-type and rpsR mutant strains could reveal sRNAs whose expression is dependent on or influenced by this ribosomal protein.

What role might rpsR play in stress responses of B. thetaiotaomicron?

The rpsR protein likely plays significant roles in B. thetaiotaomicron stress responses through multiple mechanisms:

• Translational reprogramming: During stress conditions, B. thetaiotaomicron exhibits distinct transcriptional signatures, as revealed in the comprehensive transcriptome analysis across 15 different conditions . Modulation of ribosomal protein expression, including rpsR, would allow the bacterium to adjust its translational capacity to prioritize synthesis of stress-response proteins.

• Selective translation: Under stress conditions, changes in ribosome composition or modification could potentially alter mRNA selectivity, enabling preferential translation of specific transcripts needed for stress adaptation.

• Regulatory functions beyond translation: Some ribosomal proteins in bacteria have been shown to perform extraribosomal functions, particularly during stress conditions. The regulatory RNA-binding proteins in Bacteroides have been demonstrated to coordinate expression of carbohydrate utilization genes , suggesting potential regulatory roles for ribosomal RNA-binding proteins like rpsR.

Transcriptomic data from B. thetaiotaomicron under various stress conditions shows coordinated regulation of ribosomal components as part of the bacterial stress response . Gene set enrichment analysis of these stress-specific transcriptional changes reveals patterns that suggest ribosomal proteins, including rpsR, are integral to the adaptive response to environmental challenges faced by this gut commensal bacterium.

What purification strategies yield the highest activity for recombinant B. thetaiotaomicron rpsR?

Based on approaches used for other ribosomal proteins and RNA-binding proteins in Bacteroides, the following purification strategy is recommended for obtaining high-activity recombinant B. thetaiotaomicron rpsR:

Optimized Purification Protocol:

• Lysis buffer composition: 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 1 mM DTT, protease inhibitor cocktail. The high salt concentration helps prevent non-specific RNA binding .

• Initial capture: Ni-NTA affinity chromatography for His-tagged constructs with step gradient elution (50, 100, 250, and 500 mM imidazole).

• Intermediate purification: Heparin affinity chromatography, which leverages the RNA-binding properties of rpsR and removes contaminating nucleic acids.

• Polishing step: Size exclusion chromatography using a Superdex 75 column in a buffer containing 25 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, and 1 mM DTT.

• Quality control: SDS-PAGE should show >95% purity, with activity assessment through RNA binding assays similar to those used for B. thetaiotaomicron RbpB protein, which binds ssRNA with Kd values in the 50-500 nM range .

This protocol typically yields 5-10 mg of purified protein per liter of bacterial culture with retained RNA-binding activity. The use of E. coli Rosetta strain can improve expression of proteins with codon usage differing from E. coli, which is relevant for Bacteroides-derived proteins.

How can researchers effectively incorporate recombinant rpsR into functional 30S ribosomal subunits?

Researchers can effectively incorporate recombinant B. thetaiotaomicron rpsR into functional 30S ribosomal subunits through a modified reconstitution protocol based on established methods:

Step-wise Reconstitution Protocol:

• Preparation of 16S rRNA: Purify 16S rRNA from B. thetaiotaomicron or produce it through in vitro transcription. Heat the RNA at 42°C for 20 minutes in reconstitution buffer (20 mM Tris-HCl pH 7.5, 20 mM MgCl₂, 400 mM NH₄Cl, 0.2 mM EDTA, 5 mM β-mercaptoethanol) to ensure proper folding.

• Sequential protein addition: Add ribosomal proteins in a specific order to mimic natural assembly. Based on E. coli reconstitution studies, this typically involves adding primary binding proteins first, followed by secondary binding proteins including rpsR .

• Two-step incubation: Incubate the mixture first at 4°C for 20 minutes, then at 42°C for 20 minutes, as this temperature shift enhances incorporation efficiency.

• Purification of reconstituted subunits: Separate successfully reconstituted 30S subunits using sucrose gradient ultracentrifugation (10-30% sucrose).

• Activity assessment: Evaluate the functionality of reconstituted subunits through poly(U)-directed polyphenylalanine synthesis or DHFR synthesis in the PURE system .

Reconstituted 30S subunits typically show approximately 30% of the activity of native subunits in translation assays . Addition of specific factors like protein S1 can increase activity to approximately 80% of native levels, as demonstrated in other reconstitution systems . The sedimentation pattern of successfully reconstituted particles should be similar to that of native 30S subunits when analyzed by sucrose gradient ultracentrifugation.

What techniques are most effective for studying rpsR interactions with RNA in vitro?

Several complementary techniques are particularly effective for studying B. thetaiotaomicron rpsR interactions with RNA in vitro:

• Electrophoretic Mobility Shift Assays (EMSAs): This approach has been successfully used for other Bacteroides RNA-binding proteins to determine binding affinities and specificities . For rpsR, using ssRNA pentaprobes (100 nt in length containing all possible 5-nt sequence combinations) can help determine sequence preferences. The calculated Kd values for Bacteroides RNA-binding proteins range from 50-500 nM, providing a reference point for rpsR binding .

• Fluorescence Anisotropy: This technique allows real-time monitoring of protein-RNA interactions in solution. By titrating increasing concentrations of rpsR against fluorescently labeled RNA, binding constants can be determined with greater precision than EMSAs.

• Filter Binding Assays: This method provides quantitative measurement of RNA-protein interactions using radiolabeled RNA. It's particularly useful for comparing binding affinities across multiple RNA sequences or protein variants.

• Surface Plasmon Resonance (SPR): SPR allows kinetic analysis of binding interactions, providing both on- and off-rates in addition to equilibrium binding constants.

• RNA Footprinting: Chemical or enzymatic footprinting techniques can identify specific nucleotides or regions of RNA protected by rpsR binding, providing structural insights into the interaction.

For identifying in vivo RNA targets, crosslinking immunoprecipitation (CLIP) approaches similar to those used to identify targets of other regulatory RNAs in B. thetaiotaomicron would be most appropriate . These methodologies, combined with next-generation sequencing, can generate comprehensive maps of protein-RNA interactions within the cell.

How can recombinant rpsR be used to study B. thetaiotaomicron adaptation to the gut environment?

Recombinant B. thetaiotaomicron rpsR can serve as a valuable tool for investigating this organism's adaptation to the gut environment through several research approaches:

• Ribosome profiling experiments: Using recombinant rpsR in conjunction with ribosome profiling techniques can reveal condition-specific translation patterns. Comparing ribosome occupancy profiles between different gut conditions (inflammation, nutrient availability, presence of competitors) would provide insights into translational adaptation mechanisms.

• Structure-function studies: Mutations in rpsR that alter RNA binding or ribosome assembly can be introduced to examine how ribosome composition affects B. thetaiotaomicron survival and function under gut-relevant conditions. Research on B. thetaiotaomicron has demonstrated its importance in inflammatory bowel disease models, where it provides protective effects against weight loss and histopathological damage . Understanding how ribosomal function contributes to these protective effects could provide new therapeutic insights.

• Interaction studies with host factors: Recombinant rpsR can be used to identify potential interactions with host-derived molecules that might influence bacterial translation in the gut environment. Such interactions could represent important adaptation mechanisms.

• Stress response analysis: Given the dynamic transcriptional changes observed in B. thetaiotaomicron under different conditions , studying how rpsR contributes to stress adaptation in gut-relevant challenges (oxidative stress, pH fluctuations, bile exposure) would provide functional insights.

The transcriptome atlas showing condition-specific gene expression in B. thetaiotaomicron across 15 different growth conditions provides an excellent foundation for designing targeted experiments examining rpsR's role in environmental adaptation .

What insights can rpsR studies provide about evolution of translation machinery in gut bacteria?

Studies on B. thetaiotaomicron rpsR can offer significant evolutionary insights about translation machinery in gut bacteria:

• Comparative analysis: Sequence and structural comparisons of rpsR across diverse gut bacteria can reveal evolutionary conservation patterns and lineage-specific adaptations. The clustering analysis of RNA-binding proteins in Bacteroidetes demonstrates a complicated history of divergence and duplication , suggesting similar evolutionary patterns may exist for ribosomal components.

• Functional conservation: Functional complementation experiments using rpsR from different bacterial species can determine the degree of functional conservation across evolutionary distance. Such experiments could involve expressing recombinant rpsR variants in ribosome reconstitution systems to assess compatibility.

• Horizontal gene transfer assessment: Analysis of rpsR sequences can provide evidence of horizontal gene transfer events that might have influenced ribosomal evolution in gut bacteria. The clustering of RNA-binding protein genes in Bacteroidetes revealed evidence of duplication or horizontal gene transfer among particular lineages , suggesting similar dynamics might apply to ribosomal components.

• Host-microbe co-evolution: Studying how rpsR and other ribosomal components have evolved in gut specialists like B. thetaiotaomicron compared to generalists can provide insights into host-microbe co-evolutionary processes.

The complex evolutionary history observed for RNA-binding proteins in Bacteroidetes, with evidence of duplication events and horizontal gene transfer , suggests that ribosomal components like rpsR may have similarly complex evolutionary trajectories that reflect adaptation to specific ecological niches within the gut environment.

How might rpsR function contribute to B. thetaiotaomicron's therapeutic potential in inflammatory bowel disease?

The function of rpsR in B. thetaiotaomicron may contribute to its therapeutic potential in inflammatory bowel disease (IBD) through several mechanisms:

• Translational adaptation during inflammation: As a component of the translation machinery, rpsR likely plays a role in the adaptive response that enables B. thetaiotaomicron to thrive during inflammation. B. thetaiotaomicron has demonstrated protective effects in preclinical models of IBD, significantly ameliorating weight loss, colon shortening, and histopathological damage .

• Production of anti-inflammatory factors: Efficient translation machinery is essential for the production of bacterial factors that mediate anti-inflammatory effects. B. thetaiotaomicron has been shown to reduce pro-inflammatory NF-κB signaling in intestinal epithelial cells through proteins like the pirin-like protein (PLP) . Proper ribosomal function, dependent on components like rpsR, is necessary for the expression of such protective factors.

• Stress response during IBD conditions: The gut environment during IBD is characterized by increased oxidative stress, altered nutrient availability, and immune activation. Ribosomal adaptation through components like rpsR may enable B. thetaiotaomicron to maintain essential functions under these challenging conditions.

• Regulation of carbohydrate metabolism: B. thetaiotaomicron's beneficial effects in the gut are linked to its extensive carbohydrate metabolic capabilities. RNA-binding proteins in Bacteroides regulate polysaccharide utilization and capsular polysaccharide loci . If rpsR has regulatory functions beyond its ribosomal role, it may contribute to the metabolic flexibility that underlies B. thetaiotaomicron's therapeutic potential.

Research has demonstrated that B. thetaiotaomicron displays strong efficacy in preclinical models of IBD . Understanding the molecular mechanisms behind this efficacy, including the role of fundamental cellular processes like translation, could lead to new approaches for treating inflammatory bowel diseases.

What are the most promising future research directions for B. thetaiotaomicron rpsR studies?

Several promising research directions for B. thetaiotaomicron rpsR studies include:

• Structural biology approaches: Cryo-EM or crystallographic studies of B. thetaiotaomicron ribosomes would provide detailed insights into the structural role of rpsR and potentially reveal species-specific features that contribute to this organism's ecological success.

• Translatomics: Ribosome profiling studies comparing wild-type and rpsR-modified strains under gut-relevant conditions would reveal how this protein influences the translation landscape in response to environmental challenges.

• Extraribosomal functions: Investigation of potential regulatory roles for rpsR beyond its canonical function in the ribosome, potentially involving interactions with regulatory RNAs or other cellular components.

• Therapeutic applications: Exploring how modulation of rpsR function might enhance B. thetaiotaomicron's protective effects in inflammatory bowel disease models , potentially leading to new probiotic approaches.

• Synthetic biology applications: Using knowledge of rpsR structure and function to engineer ribosomes with enhanced properties for biotechnological applications, such as improved translation of specific mRNAs or incorporation of non-standard amino acids.

The comprehensive transcriptome atlas for B. thetaiotaomicron provides an excellent foundation for these studies by establishing baseline expression patterns across diverse conditions . Integration of this data with targeted functional studies of rpsR will yield valuable insights into the molecular mechanisms underlying B. thetaiotaomicron's remarkable adaptability and therapeutic potential.

What are the current technical limitations in studying B. thetaiotaomicron ribosomal proteins?

Current technical limitations in studying B. thetaiotaomicron ribosomal proteins include:

• Genetic manipulation challenges: While genetic tools for Bacteroides have improved, they remain more limited than those available for model organisms like E. coli. Creating precise mutations or deletions in essential genes like rpsR requires sophisticated approaches such as conditional knockouts or depletion systems.

• Expression systems: Heterologous expression of Bacteroides proteins can be challenging due to codon usage differences and potential toxicity. Current reconstitution systems achieve only approximately 30% of native activity , indicating room for improvement in recapitulating authentic ribosomal assembly and function.

• Structural analysis limitations: High-resolution structures of Bacteroides ribosomes are not yet available, limiting our understanding of species-specific features. Existing ribosome purification protocols often yield heterogeneous preparations that are challenging for structural studies.

• In vivo functional assessment: Methods for studying translation dynamics in vivo within the complex gut environment remain limited. Current approaches often rely on in vitro systems that may not fully recapitulate the conditions experienced by B. thetaiotaomicron in its natural habitat.

• RNA stability issues: Working with RNA in Bacteroides is complicated by the prevalence of RNases. RNA preparation protocols need further optimization to maintain RNA integrity for ribosome studies.