Recombinant Enterococcus faecalis 50S ribosomal protein L14 (rplN)

What is the structural composition of Enterococcus faecalis 50S ribosomal protein L14?

E. faecalis ribosomal protein L14 shares structural similarities with the archaeal and eukaryotic L14e protein, which contains an N-terminal region that adopts a classic SH3 fold. Based on structural analysis, the N-terminal portion (approximately 57 residues) forms the core SH3 domain, while the remaining C-terminal portion (about 39 residues) forms a largely extended chain with a short helix . The protein features a tight turn between strands 1 and 2 instead of the typical SH3 RT-loop, suggesting it interacts with neighboring ribosomal proteins through mechanisms distinct from common SH3 binding to proline-rich sequences .

How does E. faecalis L14 protein interact with other ribosomal components?

The L14 protein in E. faecalis interacts with other ribosomal components primarily through its C-terminal domain, which forms a largely extended chain with a short helix that packs onto the surface of the SH3 domain via hydrophobic interactions . This arrangement allows the C-terminal portion to potentially adopt alternative conformations to expose hydrophobic surfaces for protein-protein interactions in the ribosome without disrupting the SH3 fold . These interactions are crucial for the structural integrity of the 50S ribosomal subunit and consequently for proper protein synthesis.

What expression systems are recommended for producing recombinant E. faecalis L14 protein?

For recombinant expression of E. faecalis L14 protein, E. coli expression systems using vectors such as pETBlue-2 in host strains like RosettaBlue(DE3) have been successfully employed . The expression protocol typically involves:

• Cloning the L14 (rplN) gene into an expression vector

• Transforming into an appropriate E. coli strain

• Inducing protein expression with IPTG (1 mM)

• Growing cultures at reduced temperature (27°C) for 8-10 hours post-induction

• Harvesting cells by centrifugation and storing pellets at -80°C

For isotope-labeled protein production necessary for NMR studies, minimal media supplemented with 15NH4Cl and/or 13C-glucose should be used .

What is the most effective purification strategy for recombinant E. faecalis L14 protein?

The most effective purification strategy for recombinant E. faecalis L14 protein involves a combination of thermal precipitation and chromatographic techniques. A recommended protocol includes:

• Cell lysis in buffer containing 10 mM EDTA, 10 mM Tris-HCl (pH 8.0), 0.1% Triton X-100, and 0.5 mM PMSF using sonication

• DNase I treatment (0.5 mg/ml) with brief incubation at 37°C

• Heat treatment (70°C for 40 minutes) to precipitate E. coli proteins while leaving the thermostable L14 protein in solution

• Subsequent purification steps may include ion exchange and size exclusion chromatography

This approach exploits the thermostability of many ribosomal proteins and results in high-purity preparations suitable for structural and functional studies.

What analytical techniques are essential for characterizing recombinant E. faecalis L14 protein?

Comprehensive characterization of recombinant E. faecalis L14 protein requires multiple analytical techniques:

For high-resolution structural studies, NMR spectroscopy using 15N,13C-double-labeled protein samples is particularly valuable, employing experiments such as HCC-TOCSY-NNH and CCC-TOCSY-NNH for side chain assignments .

How can CRISPRi technology be utilized to study the role of L14 protein in E. faecalis pathogenicity?

CRISPRi technology provides a powerful approach to investigate the role of L14 protein in E. faecalis pathogenicity. A scalable dual-vector nisin-inducible CRISPRi system has been developed specifically for E. faecalis that allows for rapid and efficient silencing of target genes . For studying L14 protein:

• Design guide RNAs (sgRNAs) targeting the rplN gene encoding L14 protein

• Clone sgRNAs into the CRISPRi vector system

• Transform the constructs into E. faecalis strains

• Induce gene silencing with nisin

• Assess phenotypic changes in:

  • Growth kinetics

  • Biofilm formation capacity

  • Antibiotic susceptibility profiles

  • Virulence in infection models

This approach allows for conditional knockdown of L14 expression without complete gene deletion, which may be lethal given the essential nature of ribosomal proteins.

What are the challenges in studying ribosomal protein mutations in E. faecalis and how can they be overcome?

Studying ribosomal protein mutations in E. faecalis presents several challenges:

• Essential gene barrier: As ribosomal proteins are often essential, direct knockout approaches may be lethal.

  • Solution: Employ conditional expression systems or CRISPRi for partial knockdown .

• Genetic redundancy: Some ribosomal functions may have backup mechanisms.

  • Solution: Perform double or triple gene silencing using multiplexed CRISPRi approaches .

• Pleiotropy: Changes in ribosomal proteins can affect multiple cellular processes.

  • Solution: Use comprehensive phenotypic profiling and systems biology approaches to deconvolute complex effects.

• Technical difficulties in protein isolation: Ribosomal proteins often co-purify with RNA.

  • Solution: Implement specialized purification protocols with high-salt washes and RNase treatment steps .

• Structural analysis challenges: Obtaining structural information in the context of the intact ribosome.

  • Solution: Combine cryo-EM of ribosomes with high-resolution NMR studies of isolated proteins .

How does the L14 protein contribute to antibiotic resistance mechanisms in E. faecalis?

The L14 protein contributes to antibiotic resistance in E. faecalis through several mechanisms:

Research using CRISPRi systems has enabled the investigation of how alterations in ribosomal proteins like L14 contribute to the development of antibiotic resistance in E. faecalis . The highly recombinogenic nature of E. faecalis further contributes to genetic plasticity that can accelerate resistance development .

What is the relationship between L14 protein structure and E. faecalis biofilm formation?

The relationship between L14 protein and E. faecalis biofilm formation represents an emerging area of research. While direct evidence is still being established, several mechanisms have been proposed:

• Translational regulation: L14 may influence the translation of biofilm-related proteins, affecting matrix production and cell-cell communication.

• Stress response signaling: Changes in ribosomal proteins can trigger stress responses that promote biofilm formation as a protective mechanism.

• Metabolic adaptation: L14 mutations might alter translational efficiency of metabolic enzymes crucial for biofilm matrix synthesis.

Recent studies utilizing CRISPRi technology to silence ribosomal genes have started to uncover the connections between ribosomal proteins and biofilm development in E. faecalis . These studies suggest that ribosomal proteins may have moonlighting functions beyond their canonical roles in protein synthesis.

What experimental approaches are most effective for studying the impact of L14 mutations on ribosome function?

Several experimental approaches have proven effective for studying L14 mutations' impact on ribosome function:

For mutations affecting cysteine residues (positions 10 and 27), site-directed mutagenesis approaches have been successfully employed to convert cysteines to alanines using standard mutagenesis kits .

How can researchers effectively isolate and study E. faecalis ribosomes containing recombinant L14 protein?

To isolate and study E. faecalis ribosomes containing recombinant L14 protein, researchers can implement the following protocol:

• Ribosome isolation:

  • Grow E. faecalis cultures to mid-log phase

  • Harvest cells and lyse in buffer containing 20 mM HEPES-KOH pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, and 6 mM β-mercaptoethanol

  • Clarify lysate by centrifugation (30,000 × g, 30 min)

  • Layer supernatant onto sucrose cushion and ultracentrifuge (100,000 × g, 16 h)

  • Resuspend ribosomal pellet in storage buffer

• Incorporation of recombinant L14:

  • Perform in vitro reconstitution using purified 50S subunits and recombinant L14

  • Alternatively, express tagged recombinant L14 in E. faecalis and isolate ribosomes containing the tagged protein

• Functional and structural analysis:

  • Assess translation activity using in vitro translation systems

  • Perform structural analysis using cryo-EM

  • Analyze L14 dynamics within assembled ribosomes using fluorescence-based approaches if fluorescent tags are incorporated

This approach allows for direct comparison between ribosomes containing native versus recombinant or mutant L14 proteins.

How does E. faecalis L14 differ from homologous proteins in other clinically relevant bacteria?

E. faecalis L14 shows both conserved and distinct features compared to homologous proteins in other clinically relevant bacteria:

These differences can be exploited in developing species-specific antibiotics targeting ribosomal function. The SH3 fold of the N-terminal domain is a conserved feature, though the precise arrangement of the hydrophobic residues and the conformation of the C-terminal extension vary across species .

What insights can structural comparison between archaea, eukaryotic, and E. faecalis L14 proteins provide for evolutionary biology?

Structural comparison between archaeal, eukaryotic, and E. faecalis L14 proteins offers valuable insights into evolutionary biology:

• Conservation of SH3 fold: The presence of an SH3 fold in the N-terminal region across domains of life suggests an ancient origin for this structural feature in ribosomal proteins .

• Divergence in C-terminal regions: While the core SH3 domain is conserved, the C-terminal extensions show greater variability, reflecting domain-specific adaptations in ribosomal assembly.

• Functional constraints: Highly conserved residues across domains likely indicate functionally critical positions under strong selective pressure.

• Horizontal gene transfer assessment: Comparative analysis can help identify potential horizontal gene transfer events in the evolution of ribosomal proteins.

• Co-evolution patterns: Correlation between changes in L14 and interacting ribosomal components can reveal co-evolutionary relationships.

This evolutionary perspective not only contributes to our understanding of bacterial evolution but also informs the development of narrow-spectrum antibiotics targeting domain-specific features of bacterial ribosomal proteins.