Recombinant Enterococcus faecalis 50S ribosomal protein L16 (rplP)

Basic Research Questions

• What is the structure and function of the L16 ribosomal protein in Enterococcus faecalis?

The L16 ribosomal protein in Enterococcus faecalis is a critical component of the 50S ribosomal subunit, involved in peptidyltransferase activity and antibiotic binding. Research indicates that specific amino acid positions (especially positions 51, 52, and 56) are particularly significant for ribosomal function and antibiotic interactions.

Methodologically, structural characterization typically involves:

• X-ray crystallography of the ribosomal subunit

• Cryo-electron microscopy for high-resolution structural analysis

• Sequence alignment with homologous proteins from related species

• Computational modeling of protein-rRNA interactions

In E. faecalis, the rplP gene sequence can be compared with reference sequences such as those used in comparative studies (e.g., GenBank accession no. AF291861 for E. faecium, which is often used as reference for detection of mutations across Enterococcus species) .

• How do mutations in the rplP gene correlate with antibiotic resistance in E. faecalis?

Mutations in the rplP gene encoding the L16 ribosomal protein have been strongly associated with resistance to oligosaccharide antibiotics like evernimicin (EVN). Research has identified specific amino acid substitutions that correlate with increased minimum inhibitory concentrations (MICs).

Key findings from experimental studies include:

• Arg51His substitution in L16 protein has been associated with high-level resistance (MIC of 8 μg/ml) in E. faecalis strain Z-123

• Arg56His and Ile52Thr changes have been found in strains with MICs in the range of 1-3 μg/ml

• Multiple silent mutations may occur alongside amino acid substitutions

This data table from published research illustrates the relationship between specific mutations and antibiotic resistance:

Methodological approach: To investigate these correlations, researchers typically sequence the rplP gene from clinical isolates with varying levels of antibiotic resistance, then perform comparative analysis against susceptible control strains .

• What are the most effective methods for expressing recombinant E. faecalis L16 protein?

Expressing functional recombinant L16 protein requires careful consideration of several factors:

Step-by-step methodological approach:

• Gene optimization: Codon optimization for the expression system (typically E. coli)

• Vector selection: pET expression systems are commonly used due to their tight regulation

• Expression conditions:

  • Use of BL21(DE3) or Rosetta strains for expression

  • Induction at lower temperatures (16-25°C) to enhance folding

  • IPTG concentration optimization (typically 0.1-0.5 mM)

• Purification strategy:

  • His-tag affinity chromatography followed by size exclusion

  • Ion exchange chromatography for higher purity

  • Inclusion body recovery protocols if expression results in insoluble protein

Critical considerations:

• Testing multiple fusion tags (His, GST, MBP) to improve solubility

• Addressing potential toxicity issues by using tightly regulated promoters

• Verifying protein functionality through ribosome binding assays

• Including RNase-free conditions when studying RNA-protein interactions

• How can researchers detect mutations in the rplP gene from clinical isolates?

Detection of mutations in the rplP gene requires a systematic approach:

Methodological workflow:

• Sample preparation:

  • Culture clinical isolates and extract genomic DNA

  • Determine antibiotic susceptibility using standardized methods (e.g., E-test)

• PCR amplification:

  • Design primers targeting a ~414 bp fragment of the rplP gene

  • Optimize PCR conditions for high specificity and yield

• Sequencing:

  • Purify PCR products and sequence in both forward and reverse directions

  • Use established reference sequences for comparison (e.g., GenBank accession AF291861)

• Analysis:

  • Align sequences using tools like MUSCLE or CLUSTAL

  • Identify nucleotide changes and predict resulting amino acid substitutions

  • Correlate identified mutations with antibiotic susceptibility data

This approach has successfully identified various mutations in clinical isolates, including silent mutations and those resulting in amino acid substitutions at key positions like 51, 52, and 56 .

• What controls should be included when studying L16 protein mutations?

Proper experimental controls are essential for reliable interpretation of results:

Recommended controls:

• Susceptible wild-type strains: Include strains with low MICs (e.g., ≤0.25 μg/ml) as negative controls

• Known resistant mutants: Include previously characterized resistant strains as positive controls

• Reference sequences: Use validated sequences (e.g., GenBank accessions) for comparative analysis

• Multiple species controls: Include strains from related Enterococcus species to account for species-specific variations

• Biological replicates: Test multiple colonies from the same strain

For example, in one comprehensive study, researchers included three control strains with MICs in the range of 0.023 to 0.19 μg/ml alongside 19 strains with increased EVN MICs (0.75 to 16 μg/ml) .

Advanced Research Questions

• How can researchers investigate the interaction between L16 mutations and 23S rRNA in antibiotic resistance mechanisms?

Investigating the complex interplay between L16 protein mutations and 23S rRNA requires sophisticated methodological approaches:

Advanced experimental strategies:

• Co-selection analysis:

  • Examine clinical isolates for concurrent mutations in both L16 and 23S rRNA

  • Quantify the frequency of co-occurring mutations

  • Example finding: While some E. faecalis strains (like Z-123) exhibit both L16 mutations (Arg51His) and 23S rRNA mutations (G2581A), others show only one type of mutation

• Structural biology approaches:

  • Cryo-EM analysis of ribosomes containing mutated components

  • Molecular dynamics simulations to predict structural changes

  • Surface plasmon resonance to measure altered antibiotic binding kinetics

• Gene editing techniques:

  • CRISPR-Cas9 modification to introduce specific mutations

  • Allelic exchange to compare effects of individual vs. combined mutations

  • Complementation studies to confirm causality

• Functional assays:

  • In vitro translation assays with reconstituted ribosomes

  • Antibiotic binding studies using fluorescently labeled compounds

  • Ribosome profiling to assess translational effects of mutations

This multifaceted approach can help resolve the mechanistic contribution of each component to antibiotic resistance.

• What methodological challenges exist in studying L16 protein from different Enterococcus species?

Studying L16 protein across different Enterococcus species presents several significant challenges:

Key methodological challenges and solutions:

• Sequence variation challenges:

  • Challenge: Significant sequence diversity across species makes primer design difficult

  • Solution: Design degenerate primers targeting conserved regions or use species-specific primer sets

• Reference sequence limitations:

  • Challenge: Some species lack published reference sequences (e.g., E. hirae and E. durans)

  • Solution: Use well-characterized references from related species and document all variations

• Ribosomal operon multiplicity:

  • Challenge: Enterococci contain 5-6 ribosomal RNA operons (rrn) , potentially with sequence heterogeneity

  • Solution: Clone and sequence multiple PCR products or use next-generation sequencing for comprehensive analysis

• Strain-specific adaptations:

  • Challenge: Some mutations may be strain adaptations rather than resistance mechanisms

  • Solution: Include appropriate control strains and perform statistical analyses on larger sample sets

• Functional equivalence assessment:

  • Challenge: Determining if L16 proteins from different species are functionally equivalent

  • Solution: Conduct complementation studies and heterologous expression experiments

For example, researchers studying E. hirae found that all five strains examined (including a susceptible control) showed a Pro110Ser change in L16 protein compared to E. faecium reference, suggesting this is a species-specific variation rather than a resistance mechanism .

• How can researchers address contradictory findings regarding the role of specific L16 mutations in antibiotic resistance?

Contradictory findings are common in complex biological systems. A structured approach to resolve these contradictions includes:

Systematic resolution framework:

• Meta-analysis of published data:

  • Compile results from multiple studies into standardized formats

  • Analyze discrepancies in experimental conditions, strains, and methodologies

  • Identify patterns that might explain contradictions

• Standardized resistance phenotyping:

  • Use consistent methods for MIC determination (e.g., E-test, broth microdilution)

  • Include quality control strains recommended by CLSI or EUCAST

  • Report MICs as distributions rather than single values

• Genetic background considerations:

  • Investigate the influence of genetic background on mutation effects

  • Use isogenic strains with introduced mutations to control for background effects

  • Example finding: The effect of Arg56His mutation might differ between E. faecium and E. hirae backgrounds

• Multivariate analysis:

  • Consider multiple factors simultaneously (mutation type, species, other resistance genes)

  • Use statistical models that account for interactions between variables

  • Develop predictive models for resistance based on multiple genetic factors

• Independent validation:

  • Reproduce key findings in independent laboratories

  • Use different methodological approaches to test the same hypothesis

  • Conduct blinded analyses to reduce confirmation bias

• What experimental design is optimal for investigating the effects of L16 mutations on ribosome assembly and function?

Investigating L16 mutations requires careful experimental design:

Optimal design parameters:

• Factorial experimental design:

  • Systematically test multiple variables: mutation type, antibiotic class, growth conditions

  • Include appropriate controls for each factor level

  • Analyze interactions between factors using ANOVA or regression models

• Time-course analysis:

  • Monitor ribosome assembly at different time points

  • Track protein synthesis rates before and after antibiotic exposure

  • Analyze adaptation responses over multiple generations

• In vitro to in vivo validation pipeline:

  • Start with in vitro binding and assembly assays

  • Progress to cell-free translation systems

  • Validate findings in cellular models

  • Test in animal infection models when applicable

• Quantitative measurements:

  • Use quantitative RT-PCR for gene expression analysis

  • Apply ribosome profiling for translational efficiency

  • Employ quantitative proteomics for global effects analysis

• Visual confirmation techniques:

  • Apply fluorescence microscopy with labeled ribosomal components

  • Use FRET to measure dynamic interactions

  • Implement super-resolution techniques for detailed structural analysis

• How can Grad-seq analysis contribute to understanding L16 protein interactions in Enterococcus faecalis?

Gradient profiling by sequencing (Grad-seq) is a powerful approach for global RNA-protein complex analysis:

Methodological implementation:

• Sample preparation and gradient separation:

  • Prepare cell lysates under conditions that preserve native complexes

  • Separate complexes by glycerol gradient ultracentrifugation

  • Fractionate gradient into multiple samples (typically 20-25 fractions)

• Molecular analysis:

  • Extract RNA and protein from each fraction

  • Perform RNA-seq for transcriptome profiling

  • Conduct mass spectrometry for proteome analysis

• Data integration:

  • Generate sedimentation profiles for all RNA and protein components

  • Identify co-migrating molecules as potential interaction partners

  • Cluster components with similar profiles

• L16-specific applications:

  • Track L16 protein distribution across gradient fractions

  • Identify RNAs co-sedimenting with L16 (expected: 23S rRNA)

  • Discover unexpected interaction partners

• Validation experiments:

  • Confirm direct interactions through RNA-protein crosslinking

  • Verify functional relevance through mutational analysis

  • Test the effect of antibiotics on complex formation

Grad-seq analysis of Enterococcus has revealed that L16 primarily co-sediments with other large subunit ribosomal proteins and 23S/5S rRNAs in high-molecular-weight fractions, confirming its integration into the ribosomal complex .