Recombinant Enterococcus faecalis 30S ribosomal protein S19 (rpsS)

What is the role of 30S ribosomal protein S9 in Enterococcus faecalis?

The 30S ribosomal protein S9 (encoded by the rpsI gene) is a critical component of the small ribosomal subunit in Enterococcus faecalis. It plays essential roles in translational accuracy and efficiency by stabilizing the interaction between mRNA and the ribosome. S9 is positioned near the decoding center of the ribosome and contributes to maintaining the proper conformation of the 30S subunit during protein synthesis. Additionally, it helps coordinate with other ribosomal proteins to ensure accurate codon-anticodon pairing during translation, which is vital for bacterial protein synthesis and survival .

How does recombinant E. faecalis S9 protein differ from native S9?

Recombinant E. faecalis S9 protein is produced in heterologous expression systems (typically yeast, as noted in commercial products) rather than being isolated from E. faecalis cultures. The recombinant protein contains the same amino acid sequence as the native protein but may include additional elements such as purification tags (e.g., His-tags or SUMO-fusion tags) to facilitate isolation. These tags may be removed post-purification through specific protease cleavage. Studies evaluating reconstituted 30S subunits show that properly prepared recombinant S9 can achieve similar structural integration as native proteins, though the functional activity of reconstituted subunits typically reaches approximately 30% of native subunits .

What methods are available for purifying recombinant E. faecalis ribosomal proteins?

The small ubiquitin-related modifier (SUMO) fusion method has proven effective for ribosomal protein purification. This approach involves:

• Cloning the ribosomal protein gene into a SUMO fusion vector

• Expressing the fusion protein in a suitable host system

• Purifying the fusion protein using affinity chromatography

• Cleaving the SUMO tag using SUMO protease

• Conducting a second purification step to isolate the native protein

This method has been successfully employed to purify all S2-S21 ribosomal proteins with sequences identical to native ribosomal proteins, as demonstrated in reconstitution studies. The SUMO fusion approach helps enhance protein solubility and facilitates proper folding, which is particularly important for ribosomal proteins that are often prone to aggregation .

What approaches should be considered when designing experiments for 30S ribosomal subunit reconstitution using recombinant E. faecalis proteins?

When designing 30S subunit reconstitution experiments with recombinant E. faecalis proteins, researchers should implement a multi-faceted approach:

• Component preparation: Ensure high purity of individual components - recombinant proteins (S2-S21) and 16S rRNA. The SUMO fusion method has proven effective for protein purification, while 16S rRNA can be isolated from native 30S subunits or synthesized in vitro.

• Reconstitution conditions: Implement a two-step reconstitution protocol:

  • Form the reconstitution intermediate (RI) by combining 16S rRNA with proteins under low-temperature conditions (0-15°C)

  • Apply heat activation (42°C) under high-salt conditions to transform the RI into mature 30S subunits

• Quality control: Verify reconstitution success through:

  • Sucrose density gradient (SDG) analysis to confirm proper sedimentation compared to native 30S subunits

  • SDS-PAGE analysis to confirm the presence of all ribosomal proteins

  • Functional assays such as poly(U)-directed polyphenylalanine synthesis

• Optimization considerations: Be prepared to optimize salt concentration, temperature, and incubation times to improve reconstitution efficiency, as reconstituted subunits typically show ~30% activity compared to native subunits .

How can researchers analyze contradictory data in E. faecalis ribosomal protein studies?

When faced with contradictory data in E. faecalis ribosomal protein studies, researchers should apply the following analytical framework:

• Assess experimental conditions: Different reconstitution methodologies (salt concentration, temperature, incubation time) can yield varying results. Standardize conditions across experiments for valid comparisons.

• Evaluate protein quality: Analyze protein folding and post-translational modifications. Recombinant proteins may lack native modifications present in E. faecalis, affecting function.

• Compare activity metrics: Different assays measure distinct aspects of ribosomal function. For example:

  • Poly(U)-directed polyphenylalanine synthesis evaluates basic translation capability

  • Full-length protein synthesis in PURE systems assesses complete translational fidelity

  • Biophysical binding assays measure specific molecular interactions

• Consider strain variations: E. faecalis strains (e.g., V583, OG1RF, ATCC 4200RF) have genetic differences that may influence ribosomal protein function. Document strain origins in all experiments.

• Analyze component stoichiometry: As observed in reconstitution studies where S2 showed weaker band intensity compared to native 30S, protein stoichiometry can vary in reconstituted subunits and affect function .

What is the optimal protocol for in vitro reconstitution of functional 30S subunits using recombinant E. faecalis ribosomal proteins?

The optimal protocol for in vitro reconstitution of functional 30S subunits involves:

Materials required:

• Purified recombinant S2-S21 ribosomal proteins (SUMO fusion method recommended)

• Purified 16S rRNA from native 30S subunits or synthesized in vitro

• Reconstitution buffers (precise composition detailed below)

Protocol:

• Preparation of Reconstitution Buffer:

  • Buffer A: 20 mM HEPES-KOH (pH 7.5), 20 mM MgCl₂, 400 mM NH₄Cl, 4 mM β-mercaptoethanol

  • Buffer B: 20 mM HEPES-KOH (pH 7.5), 4 mM MgCl₂, 400 mM NH₄Cl, 4 mM β-mercaptoethanol

• Formation of Reconstitution Intermediate (RI):

  • Combine 16S rRNA with equimolar amounts of all 20 ribosomal proteins (S2-S21)

  • Incubate in Buffer B at 4°C for 20 minutes

• Heat Activation:

  • Transfer the RI mixture to 42°C

  • Incubate for 20 minutes in Buffer A (containing higher Mg²⁺ concentration)

• Purification:

  • Layer the reconstitution mixture onto a 10-30% sucrose gradient

  • Ultracentrifuge at 35,000 rpm for 16 hours at 4°C

  • Collect the 30S peak fractions

• Validation:

  • Analyze by SDS-PAGE to confirm the presence of all ribosomal proteins

  • Assess functionality using poly(U)-directed polyphenylalanine synthesis assay

This protocol typically yields reconstituted 30S subunits with approximately 30% of the activity of native 30S subunits, which is consistent with previously published results .

How can researchers accurately evaluate the activity of reconstituted 30S subunits containing E. faecalis ribosomal proteins?

To accurately evaluate the activity of reconstituted 30S subunits, researchers should employ multiple complementary approaches:

• Poly(U)-Directed Polyphenylalanine Synthesis Assay:

  • This standard assay measures the capacity of ribosomes to translate poly(U) mRNA into polyphenylalanine

  • Components: reconstituted 30S subunits, native 50S subunits, poly(U) mRNA, charged Phe-tRNAs, translation factors

  • Measurement: incorporation of ¹⁴C-labeled phenylalanine into acid-precipitable material

  • Expected results: Reconstituted 30S subunits typically show ~30% activity compared to native subunits

• PURE System Translation:

  • Evaluates complete protein synthesis capability using a defined in vitro translation system

  • Assesses the ability to translate full-length proteins rather than just homopolypeptides

  • More stringent test of functional fidelity

• Sedimentation Profile Analysis:

  • Sucrose density gradient ultracentrifugation comparing reconstituted vs. native 30S subunits

  • Analyzes structural integrity and complex formation capability

• tRNA Binding Assays:

  • Measures the ability of reconstituted 30S subunits to correctly bind tRNAs at A, P, and E sites

  • Can be quantified using fluorescence-labeled tRNAs

• Ribosomal Assembly Mapping:

  • Uses chemical probing techniques (DMS, SHAPE) to assess the correct folding of 16S rRNA within the reconstituted complex

  • Provides structural validation of proper assembly

These multiple assessment methods provide a comprehensive evaluation of both structural and functional properties of reconstituted ribosomes .

How can recombinant E. faecalis ribosomal proteins be used to study antibiotic resistance mechanisms?

Recombinant E. faecalis ribosomal proteins offer powerful tools for studying antibiotic resistance mechanisms through several approaches:

• Site-Directed Mutagenesis Studies:

  • Generate specific mutations in ribosomal proteins known to confer antibiotic resistance

  • Reconstitute 30S subunits with these mutant proteins

  • Compare antibiotic binding and translation inhibition between wild-type and mutant reconstituted ribosomes

  • This approach allows precise molecular characterization of resistance mechanisms

• Antibiotic Binding Assays:

  • Using reconstituted 30S subunits with fluorescently labeled antibiotics

  • Direct measurement of binding affinity changes in resistant variants

  • Competitive binding assays to characterize novel inhibitors

• Comparative Studies Between Susceptible and Resistant Strains:

  • Reconstitute ribosomes with proteins from both MDR and antibiotic-susceptible E. faecalis strains

  • Analyze structural and functional differences

  • Identify key adaptations in ribosomal proteins that contribute to resistance

• Integration with CRISPR-Cas Systems:

  • As described in the literature, CRISPR-Cas systems interact with horizontal gene transfer mechanisms in E. faecalis

  • By combining ribosomal protein studies with CRISPR-Cas manipulation, researchers can examine how translation machinery adaptations correlate with acquisition of resistance genes

This approach is particularly valuable for studying aminoglycoside resistance, as these antibiotics target the 30S subunit and resistance often involves alterations in ribosomal proteins or rRNA modifications.

What research applications exist for using recombinant E. faecalis ribosomal proteins in studying bacterial evolution?

Recombinant E. faecalis ribosomal proteins provide unique opportunities for evolutionary studies through:

• Ancestral Sequence Reconstruction:

  • Computationally predict ancestral sequences of ribosomal proteins

  • Synthesize and reconstitute these ancestral variants

  • Compare functional properties to understand evolutionary adaptations in translation machinery

• Comparative Ribosome Analysis:

  • Reconstitute hybrid ribosomes containing proteins from different bacterial species or strains

  • Analyze how species-specific ribosomal protein variants contribute to translation efficiency

  • Identify evolutionary adaptations in translation machinery across enterococcal species

• Horizontal Gene Transfer Studies:

  • Examine how changes in ribosomal proteins correlate with genome expansion in MDR E. faecalis strains

  • Research shows MDR strains like V583 possess expanded genomes with large segments of mobile DNA

  • Ribosomal proteins may co-evolve with these genomic changes

• Fitness Cost Analysis:

  • Quantify how mutations in ribosomal proteins affect bacterial fitness

  • As seen in CRISPR-Cas studies, where maintenance of certain genetic elements induces fitness costs

  • Apply similar principles to study evolutionary trade-offs in ribosomal protein adaptations

• Selective Pressure Mapping:

  • Reconstitute ribosomes with systematically altered ribosomal proteins

  • Measure functional impacts under various selective pressures

  • Map evolutionary constraints on ribosomal protein sequence and function

This evolutionary approach provides insights into both natural selection processes and potential intervention strategies against MDR enterococcal strains.

What are the common challenges in expressing and purifying recombinant E. faecalis ribosomal proteins, and how can they be addressed?

Research demonstrates that the SUMO fusion method significantly improves the solubility and stability of ribosomal proteins, enabling successful reconstitution of 30S subunits with all 20 proteins (S2-S21). Careful optimization of expression conditions and purification protocols can address most common challenges in working with these technically demanding proteins .

How can researchers optimize the reconstitution of 30S subunits for specific experimental applications?

Optimization strategies for 30S subunit reconstitution depend on the specific experimental application:

• For Structural Studies (Cryo-EM or X-ray Crystallography):

  • Increase reconstitution homogeneity by implementing stepwise addition of proteins

  • Apply additional purification steps (e.g., MonoQ chromatography) post-reconstitution

  • Use chemical crosslinking to stabilize the complex

  • Optimize buffer conditions to minimize conformational heterogeneity

• For Functional Translation Assays:

  • Adjust Mg²⁺ concentration during heat activation step (typically 20 mM optimal)

  • Fine-tune the ratio of ribosomal proteins to 16S rRNA (slight excess of proteins often improves yield)

  • Include molecular crowding agents (PEG, Ficoll) to enhance assembly efficiency

  • Extend heat activation time to increase the proportion of correctly assembled subunits

• For Antibiotic Binding Studies:

  • Modify reconstitution buffers to match physiological conditions

  • Include trace metal ions that might be required for proper folding

  • Implement gradual cooling after heat activation to optimize binding pocket formation

  • Consider including stabilizing ligands during reconstitution

• For In Vivo Relevance:

  • Include native E. faecalis ribosome biogenesis factors in the reconstitution mixture

  • Perform reconstitution at temperatures relevant to E. faecalis growth (37°C)

  • Use physiological salt concentrations after initial assembly

Research shows that reconstituted 30S subunits typically achieve ~30% of native activity, but optimization can significantly improve this percentage for specific applications .

How might research on E. faecalis ribosomal proteins contribute to developing new antimicrobial strategies?

Research on E. faecalis ribosomal proteins opens several promising avenues for novel antimicrobial strategies:

• Ribosome-Targeted Antimicrobial Design:

  • Structural studies of reconstituted ribosomes with species-specific features

  • Identification of unique binding pockets in E. faecalis ribosomal proteins

  • Structure-based design of selective inhibitors targeting these species-specific features

• Combination with CRISPR-Cas Systems:

  • Research demonstrates that CRISPR-Cas systems can be exploited to selectively deplete antibiotic-resistant E. faecalis strains

  • Target ribosomal protein genes or their regulatory elements using CRISPR-Cas

  • Exploit fitness costs associated with ribosomal protein modifications

  • As demonstrated in research: "We present a novel approach to alter the structure of E. faecalis populations by exploiting CRISPR-Cas, selection, and intraspecies competition"

• Translation Fidelity Modulation:

  • Identify ribosomal protein modifications that reduce translational accuracy

  • Design molecules that induce errors in protein synthesis specifically in E. faecalis

  • Target the ribosomal protein S9 which plays a crucial role in translation accuracy

• Disruption of Ribosome Assembly:

  • Target ribosome assembly pathways rather than mature ribosomes

  • Develop inhibitors of specific protein-rRNA interactions critical for E. faecalis ribosome biogenesis

  • This approach may circumvent existing resistance mechanisms that protect mature ribosomes

The unique properties of E. faecalis ribosomes, especially in MDR strains with expanded genomes, provide opportunities for selective targeting that could lead to next-generation therapeutics for these challenging infections .

What are the current knowledge gaps regarding E. faecalis ribosomal proteins that require further research?

Several critical knowledge gaps remain in our understanding of E. faecalis ribosomal proteins:

• Strain-Specific Variations:

  • Comprehensive comparative analysis of ribosomal protein sequences across clinically relevant strains

  • Correlation between ribosomal protein variations and antibiotic resistance profiles

  • Functional consequences of strain-specific ribosomal protein variants

• Post-Translational Modifications:

  • Characterization of post-translational modifications in native E. faecalis ribosomal proteins

  • Impact of these modifications on ribosome function and antibiotic susceptibility

  • Reconstitution with proteins containing these modifications

• Ribosome Heterogeneity:

  • Presence and function of specialized ribosomes with altered protein composition

  • Potential role in stress response or antibiotic resistance

  • Regulation of ribosomal protein expression under different growth conditions

• Integration with Mobile Genetic Elements:

  • How expanded genomes in MDR strains affect ribosomal protein function

  • Interaction between horizontally acquired genes and the translation machinery

  • Research shows MDR E. faecalis strains "have acquired large segments of mobile DNA in the form of prophage, genomic islands, transposons, and plasmids"

• Biogenesis Factors:

  • Identification of E. faecalis-specific ribosome assembly factors

  • Role of these factors in ribosome assembly and function

  • Potential as antimicrobial targets

• Structural Dynamics:

  • High-resolution structures of E. faecalis ribosomes in different functional states

  • Comparative analysis with model organisms

  • Species-specific motion patterns during translation

Addressing these knowledge gaps would significantly advance our understanding of E. faecalis translation machinery and provide new opportunities for therapeutic intervention against this important pathogen.