Recombinant Enterococcus faecalis 50S ribosomal protein L18 (rplR)

What is the structural composition of Enterococcus faecalis 50S ribosomal protein L18?

Enterococcus faecalis 50S ribosomal protein L18 (rplR) is a full-length protein comprising 118 amino acids. Its complete amino acid sequence is: MITKPDKNKTRQKRHRRVRN KISGTAECPRLNIFRSNKNIYAQVIDDVAGVTLASASALDKEISGGTKETAAAVGKLVAERAAEKGIKKVVFDRGGYLYHGRVQALAEAARENGLEF . The protein contains structural domains that allow it to bind to 5S rRNA via its C-terminal region, while its N-terminal region mediates interactions between 5S rRNA and 23S rRNA . These structural characteristics are essential for its role in ribosome assembly.

What expression systems are available for producing recombinant E. faecalis rplR protein?

Multiple expression systems are available for producing recombinant E. faecalis rplR protein, each with distinct advantages for different research applications:

The choice of expression system should be guided by the specific requirements of your research . For instance, the E. coli system with Avi-tag biotinylation utilizes BirA technology, where BirA catalyzes an amide linkage between biotin and a specific lysine in the AviTag peptide, enabling precise targeting for streptavidin-based applications .

How does the function of L18 in E. faecalis compare to its homologs in other organisms?

While bacterial L18 proteins like the one in E. faecalis are required for cell viability, as demonstrated by the difficulty in obtaining viable knockout strains of rplR genes , the plant homologs such as AtPRPL18 play specialized roles in processes like chloroplast development and plant embryo development . This evolutionary divergence highlights the adaptation of ribosomal proteins to specialized cellular compartments in eukaryotes while maintaining core functions in ribosome assembly.

What methodological approaches can be used to study the interactions between E. faecalis L18 and rRNA components?

To study interactions between E. faecalis L18 and rRNA components, researchers can employ multiple complementary approaches:

• RNA binding assays: Electrophoretic mobility shift assays (EMSA) with purified recombinant L18 protein and in vitro transcribed 5S rRNA can quantify binding affinity. This approach can be enhanced by using the biotinylated version (CSB-EP774379ELW-B) for streptavidin-based detection systems .

• Structural biology techniques: X-ray crystallography or cryo-electron microscopy of L18-rRNA complexes can reveal detailed binding interfaces. Based on prokaryotic studies, focus should be placed on the C-terminal domain for 5S rRNA binding and the N-terminal domain for mediating 5S-23S rRNA interactions .

• Site-directed mutagenesis: Systematic mutation of conserved residues in the L18 protein, particularly in the C-terminal and N-terminal regions, followed by functional assays can identify critical amino acids for rRNA binding and ribosome assembly .

• In vivo analysis: Conditional depletion systems can be employed since complete knockout of rplR is challenging due to its essential nature, as evidenced by the low recombination efficiency (~10 colonies per 10^8 viable cells) observed in gene replacement experiments .

• RNA-protein crosslinking: UV-induced or chemical crosslinking followed by mass spectrometry can identify specific contact points between L18 and rRNA molecules in intact ribosomes.

How do mutations in E. faecalis L18 affect ribosome assembly and bacterial viability?

Mutations in E. faecalis L18 can have profound effects on ribosome assembly and bacterial viability, though the specific impacts vary based on the mutation type and location. Studies on ribosomal protein gene knockouts in E. coli provide valuable insights applicable to E. faecalis:

Complete knockout of the rplR gene (encoding L18) yields extremely low recombination efficiency (~10 colonies per 10^8 viable cells) , indicating the essential nature of this protein. Successful recombinants typically retain an intact copy of the rplR gene alongside the knockout construct (rplR<>cat/rplR+), suggesting that partial diploidy is necessary for viability when this gene is targeted .

This contrasts with the knockout of rplY (encoding L25), which shows much higher recombination efficiency (~10^4 colonies per 10^8 viable cells) and can be completely replaced without retaining an intact copy, indicating its non-essential nature . The table below summarizes these differential effects:

These findings indicate that L18's role in incorporating 5S rRNA into the 50S ribosomal subunit is critical for proper ribosome assembly and function . Point mutations that disrupt the protein's interaction with 5S rRNA would likely affect growth rates, antibiotic susceptibility, and translation fidelity.

What are the implications of L18 structure-function relationships for developing antimicrobial agents targeting E. faecalis?

The essential nature of L18 for bacterial viability makes it a promising target for novel antimicrobial development against E. faecalis, particularly given the increasing role of this bacterium in nosocomial infections . Several aspects of L18 structure-function can be exploited:

• Ribosome assembly inhibition: Compounds that specifically interfere with L18's ability to bind 5S rRNA or facilitate its incorporation into the 50S subunit would prevent ribosome assembly. The difficulty in obtaining viable L18 knockouts demonstrates that disrupting this function would be lethal to the bacteria .

• Species-specific targeting: While L18's core function is conserved across bacteria, sequence variations between species could be exploited to develop compounds with specificity for E. faecalis over beneficial gut bacteria or human ribosomes.

• Combination therapy approaches: Targeting L18 in conjunction with other ribosomal components could create synergistic effects. For instance, combined targeting of the three 5S rRNA-binding proteins (L5, L18, and L25) might overcome the partial redundancy in their functions .

• Resistance development considerations: The essential nature and functional constraints of L18 suggest that resistance mutations might come with significant fitness costs to the bacteria, potentially reducing the rate of resistance development.

These approaches would be particularly valuable against strains of E. faecalis that have acquired virulence traits and antibiotic resistance, which are increasingly common in clinical settings .

What are the optimal conditions for reconstitution and storage of recombinant E. faecalis L18 protein?

For optimal reconstitution and storage of recombinant E. faecalis L18 protein, follow these evidence-based protocols:

Reconstitution Protocol:

• Briefly centrifuge the vial containing lyophilized protein before opening to bring contents to the bottom.

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (50% is recommended for maximum stability).

• Aliquot the reconstituted protein to minimize freeze-thaw cycles .

Storage Conditions:

• Store reconstituted protein aliquots at -20°C for routine use or -80°C for long-term storage.

• Avoid repeated freeze-thaw cycles as they can compromise protein structure and activity.

• For working stocks, maintain small aliquots at 4°C for up to one week .

Protein integrity can be assessed using SDS-PAGE, with expected purity >85% as specified by manufacturers . For functional assays, verify protein activity through RNA binding assays before proceeding with complex experiments.

How can researchers effectively design experiments to study E. faecalis L18 interactions within the bacterial ribosome?

Designing effective experiments to study E. faecalis L18 interactions within the bacterial ribosome requires a multi-faceted approach:

• Ribosome profiling approaches:

  • Isolate intact ribosomes from E. faecalis under different growth conditions

  • Perform sucrose gradient ultracentrifugation to separate ribosomal subunits and assess L18 incorporation

  • Use mass spectrometry to identify interaction partners of L18 within the ribosome

• Genetic manipulation strategies:

  • Implement conditional expression systems rather than direct knockouts, given the essential nature of L18

  • Create point mutations in key functional residues rather than whole gene deletions

  • Use merodiploid strains expressing both wild-type and mutant L18 to study dominant-negative effects

• Structural biology considerations:

  • For cryo-EM studies, purify intact ribosomes rather than reconstituting from components

  • Consider using recombinant L18 with minimal tags that won't interfere with ribosome assembly

  • Compare structures of ribosomes with wild-type versus modified L18 to identify conformational changes

• Functional assay design:

  • Measure translation rate and fidelity using reporter systems

  • Assess antibiotic susceptibility patterns in strains with L18 modifications

  • Examine polysome formation under various stress conditions

Remember that the low recombination efficiency observed with rplR gene replacements (~10 colonies per 10^8 viable cells) indicates that complete loss of L18 function is likely lethal, so experimental designs should account for this constraint.

What analytical techniques are most appropriate for studying the structural features of recombinant E. faecalis L18?

Multiple analytical techniques can be employed to study the structural features of recombinant E. faecalis L18, each providing complementary information:

When selecting analytical techniques, consider using recombinant L18 without bulky tags that might interfere with native structure, unless those tags (like the Avi-tag) are specifically required for the analytical method.

How can E. faecalis L18 be used to study the evolution of ribosomal proteins across bacterial species?

E. faecalis L18 provides an excellent model for evolutionary studies of ribosomal proteins due to several key features:

• Comparative genomic approaches:

  • Sequence alignment of L18 across diverse bacterial phyla can reveal conserved functional domains versus species-specific adaptations

  • The 118-amino acid sequence of E. faecalis L18 can be compared with homologs from other bacteria to identify selection pressures on different protein regions

  • Special attention should be paid to the C-terminal (5S rRNA binding) and N-terminal (5S-23S rRNA interaction) regions that are functionally critical

• Functional complementation studies:

  • Test whether E. faecalis L18 can functionally replace L18 in distant bacterial species

  • The essential nature of L18 makes complementation studies particularly informative about functional conservation

  • Create chimeric proteins with domains from different species to map functional regions

• Structural conservation analysis:

  • Compare structural features of L18 across species that have adapted to different ecological niches

  • Examine how L18-rRNA interactions are preserved despite sequence divergence

  • Use recombinant proteins from multiple species to perform comparative binding studies

• Horizontal gene transfer investigation:

  • Analyze whether the rplR gene shows evidence of horizontal transfer in enterococci

  • This is particularly relevant given E. faecalis' known genomic plasticity and ability to acquire new genetic traits

These approaches would contribute to understanding how essential ribosomal proteins evolve while maintaining their critical functions in ribosome assembly and protein synthesis.

What role does L18 play in antibiotic resistance mechanisms in E. faecalis?

L18's role in antibiotic resistance in E. faecalis is multifaceted, involving both direct and indirect mechanisms:

This research area is particularly important given the increasing prevalence of E. faecalis in nosocomial infections and its growing antibiotic resistance profile . Studies should include comparative analysis of L18 sequences and expression levels between antibiotic-sensitive and resistant clinical isolates.

How might understanding E. faecalis L18 contribute to developing new approaches for studying and treating bacterial infections?

Understanding E. faecalis L18 can open several novel avenues for studying and treating bacterial infections:

• Targeted antimicrobial development:

  • The essential nature of L18 makes it a promising target for new antibiotics

  • Compounds that specifically disrupt L18-rRNA interactions could inhibit ribosome assembly

  • High-throughput screening assays using recombinant L18 could identify potential inhibitors of its function

• Diagnostic applications:

  • Species-specific epitopes in L18 could be targeted for rapid identification of E. faecalis in clinical samples

  • The availability of recombinant protein facilitates antibody development for such diagnostic tools

  • Antibodies against L18 could be used in immunofluorescence microscopy for direct visualization of E. faecalis in tissue samples

• Vaccine development:

  • Conserved, surface-exposed epitopes of L18 could potentially serve as vaccine targets

  • Recombinant L18 could be evaluated as a potential component of multi-subunit vaccines against E. faecalis

  • This approach would be particularly valuable for preventing infections in high-risk hospital settings

• Understanding bacterial physiology:

  • Studying L18's role in different growth conditions could reveal adaptation mechanisms

  • The protein's function in stress responses might explain survival strategies during infection

  • As E. faecalis transitions from commensal to pathogen , changes in ribosome composition including L18 modifications might play a role

• Probiotic strain development:

  • Understanding differences in L18 between pathogenic and probiotic E. faecalis strains could guide development of safer probiotic strains

  • Probiotic strains like DSM 16440 (Symbioflor-1) lack many virulence factors but retain essential genes like rplR

These applications highlight how fundamental research on ribosomal proteins like L18 can translate into clinical tools and therapeutic strategies for addressing E. faecalis infections.