Recombinant Enterococcus faecalis 30S ribosomal protein S18 (rpsR)

What is the structure and function of Enterococcus faecalis 30S ribosomal protein S18?

Enterococcus faecalis 30S ribosomal protein S18 (rpsR) is a small ribosomal protein component of the 30S small subunit, essential for proper ribosome assembly and translation. Based on related ribosomal protein research:

• S18 consists of approximately 152 amino acids with a molecular weight of approximately 17.7 kDa (extrapolated from rat S18 data)

• Functions as part of the small ribosomal subunit, contributing to mRNA binding and translation fidelity

• Has structural homology with bacterial S18 proteins across species, with significant conservation between gram-positive bacteria

• Post-translationally modified by N-terminal acetylation via RimI acetyltransferase

Experimental approach for structural characterization:

• Recombinant expression with His-tag in E. coli expression systems

• Purification using affinity chromatography

• Structural analysis using X-ray crystallography or cryo-EM in complex with other ribosomal components

What expression systems are optimal for producing recombinant E. faecalis ribosomal protein S18?

For optimal recombinant expression of E. faecalis S18 protein:

E. coli-based expression systems are most commonly employed, with considerations for:

• Vector selection: pET-series vectors with T7 promoter systems provide high expression levels

• Host strain selection: BL21(DE3) or derivatives optimized for recombinant protein expression

• Inclusion of appropriate affinity tags (His-tag being most common) for purification

• Codon optimization for E. coli expression, especially when gram-positive genes are expressed in gram-negative hosts

Expression methodology:

• Clone the rpsR gene into expression vector with appropriate tags (typically N-terminal His-tag)

• Transform into expression host cells

• Induce protein expression with IPTG (typically 0.5-1.0 mM)

• Culture at lower temperatures (16-25°C) post-induction to enhance solubility

• Harvest cells and extract protein using standardized lysis procedures

Signal peptide considerations:

• Signal peptides can significantly impact secretion efficiency for recombinant proteins

• Bioinformatic tools such as SignalP, Phobius, and PrediSi can be used to predict optimal signal peptides

• For cytoplasmic expression of S18, signal peptides are typically unnecessary

How can post-translational modifications of S18 be analyzed in experimental settings?

Post-translational modifications, particularly N-terminal acetylation by RimI acetyltransferase, are critical for S18 function. Methods for analysis include:

Mass spectrometry approaches:

• MALDI-MS for determination of intact mass differences (acetyl group adds 42 Da)

• LC-MS/MS for peptide-level analysis of modifications

• Sample preparation involving trypsin digestion followed by enrichment of acetylated peptides

Comparative analysis methodology between wild-type and modified S18:

• Express recombinant S18 in both wild-type and RimI-knockout strains

• Extract and purify the protein using affinity chromatography

• Analyze by mass spectrometry to detect mass differences

• Compare functional properties through activity assays

Data from RimI knockout studies demonstrate:

• S18 from RimI knockout strains shows mass reduction precisely equal to an acetyl group

• Complementation with plasmid-encoded RimI restores acetylation

• Growth defects are observed in RimI knockout strains, particularly in minimal media conditions

What role does S18 play in antibiotic resistance mechanisms of E. faecalis?

S18 and other ribosomal proteins contribute to antibiotic resistance in E. faecalis through several mechanisms:

Ribosomal protection mechanisms:

• Structural modifications of the ribosome can affect binding of antibiotics that target the 30S subunit

• Mutations in ribosomal proteins like S18 may alter ribosome conformation and reduce antibiotic binding affinity

Research findings on tetracycline-class antibiotics resistance:

• Omadacycline resistance involves ribosomal proteins and potential mutations in 30S subunit genes

• Heteroresistance can develop without mutations in 30S ribosomal subunit genes but may involve overexpression of ABC transporter proteins

Experimental methodology for investigating S18's role in resistance:

• Generate site-directed mutations in the rpsR gene

• Express in susceptible strains and measure MIC values

• Perform ribosome binding assays with fluorescently-labeled antibiotics

• Use cryo-EM to visualize structural changes in ribosomes containing mutated S18

MIC data from clinical isolates of E. faecalis against tetracycline-class antibiotics:

Data derived from 276 clinical isolates collected in China (2011-2015)

How does N-terminal acetylation by RimI affect S18 function and bacterial fitness?

N-terminal acetylation of S18 by RimI acetyltransferase significantly impacts E. faecalis fitness:

Functional impacts:

• Acetylation affects protein stability, interaction capabilities, and possibly ribosomal assembly

• RimI acetylates both S18 and elongation factor Tu (EF-Tu), suggesting coordinated regulation of translation machinery

Experimental evidence from growth studies:

• RimI knockout strains (ΔrimI) show growth disadvantages compared to wild-type strains

• Growth defects are moderate in rich media (LB) but more pronounced in minimal media

• Competition assays demonstrate that acetylation provides a selective advantage

Methodological approach for investigating acetylation effects:

• Generate RimI knockout strains (ΔrimI)

• Complement with plasmid-encoded RimI (ΔrimI/pIB166::rimI)

• Compare growth rates in different media conditions

• Perform competition assays between wild-type and knockout strains

• Analyze protein synthesis rates and ribosome profiles

What experimental designs are most effective for studying S18 mutations and their impact on ribosome function?

Optimal experimental designs for S18 mutation studies include:

Genetic manipulation approaches:

• Allelic replacement strategies using temperature-sensitive plasmids like pJRS233

• CRISPR-Cas9 systems for precise genome editing

• Complementation studies using shuttle vectors (e.g., pIB166)

Structural biology methods:

• Cryo-EM of mutant ribosomes to visualize structural changes

• Ribosome profiling to assess translation impacts

• Polysome profiling to evaluate ribosome assembly and function

Workflow for comprehensive mutation analysis:

• Generate precise mutations in rpsR using PCR-based site-directed mutagenesis

• Introduce mutations into the chromosome using allelic replacement

• Verify mutations by sequencing

• Assess growth phenotypes under various conditions

• Perform ribosome isolation and functional assays

• Determine antibiotic susceptibility profiles

Important considerations from related research:

• Design controls carefully, including empty vector controls

• Include complementation strains to confirm phenotypes are due to specific mutations

• Certain ribosomal protein mutations may confer growth defects or temperature sensitivity

• Consider effects on both translation accuracy and efficiency

How can quantitative proteomics approaches be used to study S18 expression levels and interactions?

Quantitative proteomics offers powerful tools for analyzing S18 expression and interactions:

Methodological options:

• Tandem Mass Tag (TMT) labeling coupled with nano-LC-MS/MS for comparative quantitation

• Stable Isotope Labeling with Amino acids in Cell culture (SILAC) for metabolic labeling

• Label-free quantification for simpler experimental designs

Protocol for membrane protein isolation and analysis (applicable to ribosomal studies):

• Harvest bacterial cells at mid-exponential phase

• Lyse cells by ultrasonication in appropriate buffer with protease inhibitors

• Perform ultracentrifugation to isolate membrane fractions

• Quantitate using Bradford assay and verify by SDS-PAGE

• Process samples for proteomics analysis using TMT-labeling

• Analyze by nano-LC-MS/MS

Data processing and analysis:

• Apply false discovery rate (FDR) cutoff (typically 1%)

• Use statistical significance threshold (p < 0.05)

• Compare expression levels between experimental conditions

• Validate findings using complementary techniques (e.g., western blotting)

What relationships exist between ClpP protease, S18 abundance, and antimicrobial tolerance in E. faecalis?

ClpP protease significantly influences S18 abundance and impacts antimicrobial tolerance:

Key research findings:

• ClpP deletion (ΔclpP) decreases the abundance of multiple ribosomal proteins, including S18 (rpsR)

• These changes are linked to altered stress tolerance and biofilm formation

• The ΔclpP mutant shows modified antimicrobial susceptibility profiles

Experimental approach to investigate this relationship:

• Generate ClpP deletion mutant using temperature-sensitive plasmid (pJRS233)

• Create complemented strain (ΔclpP/pIB166::clpP)

• Perform quantitative proteomics to measure ribosomal protein levels

• Assess biofilm formation capacity

• Determine antimicrobial susceptibility profiles

• Conduct virulence testing using Galleria mellonella infection model

Virulence data from G. mellonella infection model:

Statistical significance: p < 0.01 between wild-type and ΔclpP mutant (log-rank test)

How can RNA-protein interaction studies be designed to investigate S18's role in RNA binding and ribosome assembly?

S18 plays a critical role in RNA binding within the ribosome. Techniques to study these interactions include:

Grad-seq methodology for comprehensive RNA-protein interaction mapping:

• Prepare bacterial lysates from cultures grown to desired phase

• Fractionate using glycerol gradient ultracentrifugation

• Collect fractions and analyze RNA and protein content

• Perform sequencing and mass spectrometry on fractions

• Create sedimentation profiles of RNAs and proteins

• Identify co-sedimenting components as potential complexes

RNA immunoprecipitation sequencing (RIP-seq) approach:

• Express tagged S18 protein

• Cross-link RNA-protein complexes in vivo

• Lyse cells and perform immunoprecipitation

• Extract and sequence bound RNAs

• Analyze binding motifs and interaction patterns

Data visualization and analysis:

• Create browser resources for interactive searching of sedimentation profiles

• Perform unsupervised clustering of RNA and protein profiles

• Identify clusters representing functional complexes

• Validate key interactions using independent methods

What approaches can be used to study the role of S18 in E. faecalis pathogenicity and virulence?

Investigating S18's role in pathogenicity requires multiple complementary approaches:

Infection models:

• Galleria mellonella larval infection model provides rapid assessment of virulence

• Mouse infection models for systemic infections, endocarditis, or UTI studies

• Cell culture models to assess bacterial adhesion, invasion, and intracellular survival

Experimental design framework:

• Generate S18 mutant strains (point mutations or expression level modifications)

• Assess in vitro phenotypes (growth, biofilm formation, stress tolerance)

• Determine antimicrobial susceptibility profiles

• Evaluate virulence in appropriate infection models

• Measure inflammatory responses and immune evasion capabilities

Dual nature of E. faecalis as commensal and pathogen:

• As a commensal, E. faecalis produces vitamins, metabolizes nutrients, and maintains intestinal pH

• As a pathogen, it can cause serious infections when it spreads beyond the intestine

• S18 and other ribosomal proteins may play roles in adaptation between these states