Recombinant Enterococcus faecalis 50S ribosomal protein L25 (rplY)

What is the function of L25 ribosomal protein in Enterococcus faecalis?

L25 ribosomal protein in E. faecalis, like its counterpart in E. coli, functions as a component of the 50S ribosomal subunit. Its primary role involves binding to 5S rRNA to form a stable complex. This interaction is critical for the formation of a separate domain within the bacterial ribosome . The protein belongs to a specific set of three ribosomal proteins (L25, L18, and L5) that interact with 5S rRNA in eubacteria .

Unlike some other ribosomal proteins, studies in E. coli have shown that L25 is not essential for cell survival, suggesting functional redundancy or compensatory mechanisms in ribosomes lacking this protein . This non-essential nature may have implications for understanding ribosome assembly and function in E. faecalis as well.

The specific binding of L25 to the loop E domain of 5S rRNA represents a key molecular interaction that contributes to ribosomal architecture and may influence translation efficiency .

How is the rplY gene expression regulated in bacterial systems?

The expression of the rplY gene encoding L25 is subject to two primary regulatory mechanisms as demonstrated in E. coli studies:

• Transcriptional regulation: The rplY transcription is under negative stringent control, which coordinates ribosomal protein synthesis with cellular growth conditions .

• Translational regulation: At the translation level, rplY expression is subject to autogenous regulation, where L25 protein can bind to its own mRNA and repress further translation .

Experiments have shown that when L25 is overexpressed from a plasmid (pL25) in E. coli, the translation yield of an rplY'-'lacZ reporter decreases by approximately threefold, demonstrating the protein's ability to specifically down-regulate its own gene expression .

The autogenous regulation mechanism represents a common control strategy among ribosomal proteins in E. coli and related bacteria, ensuring balanced synthesis of ribosomal components .

What structural elements of the rplY mRNA are important for expression and regulation?

Several key structural elements in the rplY mRNA have been identified as critical for expression and regulation:

• Atypical Shine-Dalgarno sequence: Unlike most bacterial mRNAs, the rplY transcript lacks a canonical Shine-Dalgarno sequence, which is normally required for efficient translation initiation .

• Hairpin structures: The rplY mRNA leaders include:

  • A 5'-proximal imperfect hairpin (HI) that shows significant sequence divergence between species

  • A central imperfect hairpin (HII) that is highly conserved, particularly in its upper part

• AU-rich sequence: An extended AU-rich sequence follows the hairpin structures .

Experimental studies using truncation and mutation analysis have demonstrated that while the 5' proximal stem-loop (HI) contributes to translation efficiency, it is not essential for autogenous regulation. In contrast, the central hairpin (HII) is crucial for L25-mediated regulation, as disrupting this structure eliminates autogenous control while potentially restoring translation efficiency .

These structural features are conserved across multiple γ-proteobacterial families, suggesting their evolutionary importance in regulating L25 synthesis .

What methods are recommended for recombinant expression and purification of E. faecalis L25 protein?

For recombinant expression and purification of E. faecalis L25 protein, researchers should consider implementing a multi-stage methodology:

Expression System Selection:

• E. coli BL21(DE3): This strain is recommended for L25 expression due to its reduced protease activity and controllable induction via IPTG.

• Vector considerations: Vectors containing T7 promoters (pET series) with appropriate affinity tags (His6, GST) facilitate expression and subsequent purification.

Optimization Protocol:

• Temperature modulation: Express at lower temperatures (16-25°C) after induction to enhance proper folding.

• Induction parameters: Optimize IPTG concentration (0.1-1.0 mM) and induction time (3-16 hours).

• Media supplementation: Consider supplementing with specific ions that might be required for proper folding of RNA-binding proteins.

Purification Strategy:

• Affinity chromatography: Initial purification using Ni-NTA for His-tagged protein.

• Ion exchange chromatography: Secondary purification step using appropriate resin based on L25's theoretical pI.

• Size exclusion chromatography: Final polishing step to obtain homogeneous protein preparation.

Activity Verification:

• RNA binding assays using 5S rRNA fragments, particularly the loop E region, to confirm biological activity.

• Thermal shift assays to assess protein stability and proper folding.

Expected Yield and Purity Parameters:

When designing expression constructs, researchers should consider that while L25 in E. coli is non-essential for cell survival , the recombinant protein must retain proper folding to maintain RNA-binding activity.

How do researchers address contradictions in published data regarding L25-5S rRNA interactions?

Addressing contradictions in published data regarding L25-5S rRNA interactions requires a systematic approach:

Methodological Reconciliation:

• Experimental context analysis: Examine whether contradictory results stem from differences in:

  • Organism source (E. coli vs. E. faecalis L25)

  • Experimental conditions (buffer composition, temperature, pH)

  • Protein constructs (full-length vs. truncated versions)

  • RNA constructs (complete 5S rRNA vs. isolated loop E domain)

• Statistical reanalysis: Perform meta-analysis of published binding constants or interaction parameters, accounting for methodological variations.

• Orthogonal validation: Employ multiple independent techniques to verify interactions:

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

  • Electrophoretic mobility shift assays (EMSA)

  • Nuclear magnetic resonance (NMR) spectroscopy

Contradiction Detection Framework:
When evaluating conflicting claims in the literature, utilize systematic contradiction detection approaches :

• Identify whether contradictions arise from semantic inconsistencies

• Determine if numerical data conflicts exist across publications

• Assess whether contradictions represent true biological variation or technical artifacts

Resolving Contradictions Through New Experiments:

• Design experiments that specifically address variables that differ between contradictory studies

• Implement controlled comparative studies using standardized materials and methods

• Consider strain-specific or species-specific variations that might explain apparent contradictions

By approaching contradictions through this framework, researchers can distinguish between technical inconsistencies and genuine biological phenomena, advancing our understanding of L25-5S rRNA interactions.

What is the significance of L25's interactions with other ribosomal components in E. faecalis?

The interactions of L25 with other ribosomal components in E. faecalis have significant implications for ribosome assembly, structure, and function:

Known Interaction Network:
L25 has been demonstrated to interact with:

• 5S ribosomal RNA (forming a stable complex)

• 50S ribosomal protein L16

• L18 and L5 (collectively forming a separate domain of the bacterial ribosome)

Structural Significance:
These interactions contribute to the formation of a distinct structural domain within the 50S ribosomal subunit. In particular, the central protuberance of the ribosome depends on proper assembly of the 5S rRNA with its binding partners L25, L18, and L5 .

Functional Implications:

Experimental Approaches for Studying Interactions:

• Cryo-EM: High-resolution structural studies of E. faecalis ribosomes to map the precise location and interactions of L25.

• Cross-linking coupled with mass spectrometry: To identify direct protein-protein interactions within the ribosome.

• In vivo proximity labeling: To capture transient or dynamic interactions during ribosome assembly.

Understanding these interactions provides insights into ribosome assembly pathways and potential vulnerability points for therapeutic intervention in pathogenic E. faecalis.

How can mutagenesis studies of the rplY gene inform our understanding of E. faecalis antimicrobial resistance?

Mutagenesis studies of the rplY gene can provide valuable insights into E. faecalis antimicrobial resistance through several research avenues:

Targeted Mutagenesis Approaches:

• Site-directed mutagenesis: Creating specific amino acid substitutions in conserved regions of L25 that interact with 5S rRNA.

• Domain swapping: Exchanging functional domains between L25 from susceptible and resistant strains.

• Promoter modifications: Altering the expression levels of L25 to assess the impact on ribosome assembly and function.

Correlation With Antimicrobial Susceptibility:
The relationship between rplY mutations and antimicrobial resistance is particularly relevant for understanding E. faecalis, which exhibits variable resistance patterns:

• ASPR (ampicillin-susceptible but penicillin-resistant) E. faecalis strains have shown high mortality rates (43.8%) when treated with piperacillin-tazobactam, compared to glycopeptide-containing regimens (28.6%) .

• Mutations in ribosomal proteins can affect the binding of antibiotics that target the ribosome, potentially contributing to resistance mechanisms.

Experimental Framework:

Clinical Relevance:
By correlating specific rplY mutations with antimicrobial susceptibility patterns in clinical isolates, researchers can:

• Identify potential molecular markers for predicting treatment outcomes

• Design more effective combination therapies targeting specific resistance mechanisms

• Develop novel antimicrobials that maintain efficacy against resistant strains

This research direction is particularly important given that E. faecalis strains with mutations in other genes like pbp4 have already shown alterations in antibiotic susceptibility profiles .