Recombinant Enterococcus faecalis 50S ribosomal protein L32-3 (rpmF3)

Basic Research Questions

What is the structural and functional role of L32-3 (rpmF3) in the E. faecalis ribosome?

L32-3 (rpmF3) is a component of the 50S ribosomal subunit in Enterococcus faecalis. It belongs to the bL32 ribosomal protein family that contributes to the structural integrity of the large ribosomal subunit. In the E. faecalis 70S ribosome, L32-3 interacts with rRNA and neighboring proteins to maintain the conformation of the functional ribosome .

How conserved is the L32 protein across bacterial species, and how does E. faecalis L32-3 compare to homologs in other bacteria?

E. faecalis L32-3 shares significant sequence similarity with L32 proteins from other Gram-positive bacteria, particularly within the Enterococcaceae family. When compared to other bacterial species, E. faecalis L32-3 maintains the core functional regions while exhibiting species-specific variations in non-essential regions .

Interestingly, unlike some ribosomal proteins that are universally conserved, L32 is among the set of ribosomal proteins that can be nonessential in certain bacterial species, suggesting a more specialized or condition-dependent role in ribosome function .

What are the expression patterns and regulation mechanisms for rpmF3 in E. faecalis?

The expression of rpmF3 in E. faecalis, like other ribosomal protein genes, is subject to complex regulatory mechanisms at multiple levels:

• Transcriptional regulation: rpmF3 expression is coordinated with other ribosomal protein genes as part of the translation machinery in response to growth conditions.

• Post-transcriptional regulation: mRNA stability and processing influence the steady-state levels of rpmF3 transcripts.

• Translational regulation: The production of L32-3 protein is tightly regulated through mechanisms including autogenous regulation, where excess ribosomal proteins can bind their own mRNAs to inhibit further translation .

• Response to stress conditions: Under stress conditions such as nutrient limitation or antibiotic exposure, rpmF3 expression patterns can change as part of the bacterial adaptive response .

In E. faecalis V583, co-transcription with neighboring genes has been observed for some ribosomal protein genes, which may include rpmF3, suggesting operon-like regulation patterns .

Expression and Purification Methodologies

What are the optimal expression systems and conditions for producing recombinant E. faecalis L32-3 protein?

For successful expression of recombinant E. faecalis L32-3 protein, researchers should consider the following optimal systems and conditions:

Recommended expression systems:

Optimal expression conditions:

• Vector selection: pET vectors with T7 promoter systems are most commonly used for ribosomal proteins.

• Induction parameters: IPTG concentration of 0.1-0.5 mM typically yields best results, with induction at OD600 of 0.6-0.8.

• Temperature: Lowering temperature to 16-20°C after induction often improves solubility of ribosomal proteins.

• Media supplements: Adding 0.1-0.2 mM ZnCl2 can improve folding if zinc-finger motifs are present in the L32-3 structure .

• Solubility enhancement: Fusion partners such as MBP or SUMO can significantly improve solubility of recombinant L32-3 protein.

For challenging expression cases, testing multiple constructs with varied N- and C-terminal boundaries can help identify the most expressible form of the protein.

What purification strategies yield the highest purity and biological activity for recombinant L32-3 protein?

A systematic multi-step purification approach is recommended to obtain high-purity, biologically active recombinant L32-3 protein:

• Initial capture: Affinity chromatography using His-tag (IMAC) is most efficient, with binding in 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole and elution with an imidazole gradient (50-250 mM).

• Intermediate purification: Ion exchange chromatography (typically cation exchange at pH 6.5) exploits the basic nature of ribosomal proteins.

• Polishing step: Size exclusion chromatography in 20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2 removes aggregates and ensures homogeneity.

• Tag removal considerations: If tag removal is required, TEV protease cleavage followed by reverse IMAC yields tag-free protein.

Critical buffer components for maintaining activity:

• Include 5-10 mM MgCl2 to stabilize structure

• Add 1-2 mM DTT or 0.5 mM TCEP to maintain cysteine residues in reduced state

• Consider 5-10% glycerol to prevent aggregation during concentration steps

Purity assessment should be performed using SDS-PAGE (>95% purity) and mass spectrometry to confirm identity. Western blot using anti-L32 antibodies can confirm immunological activity of the recombinant protein.

How can researchers verify the correct folding and assembly competence of recombinant L32-3?

Verification of proper folding and assembly competence for recombinant L32-3 requires multiple complementary approaches:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Differential scanning fluorimetry (DSF) to determine thermal stability (Tm)

  • Dynamic light scattering (DLS) to verify monodispersity and absence of aggregation

• Functional assays:

  • RNA binding assays using electrophoretic mobility shift assay (EMSA) with specific rRNA fragments

  • In vitro reconstitution assays with E. faecalis 50S ribosomal components

  • Incorporation into E. faecalis ribosome assembly intermediates followed by sucrose gradient analysis

• Structural validation:

  • Limited proteolysis to confirm compact folding (properly folded proteins show resistance to proteolytic degradation)

  • NMR 1H-15N HSQC fingerprinting for structural integrity (if isotopically labeled)

  • Comparison with native L32-3 extracted from E. faecalis ribosomes using comparative proteomics approaches

A properly folded L32-3 protein should demonstrate specific rRNA binding properties, resistance to proteolysis under native conditions, and the ability to incorporate into ribosomal subunits in reconstitution experiments .

Advanced Research Applications

How can L32-3 be used to study antibiotic resistance mechanisms in E. faecalis?

L32-3 provides a valuable tool for investigating antibiotic resistance mechanisms in E. faecalis, particularly for antibiotics targeting the ribosome:

• Binding site analysis: Purified recombinant L32-3 can be used in structural studies to determine whether ribosomal antibiotics (like oxazolidinones) interact with regions near or including L32-3. Techniques such as X-ray crystallography or cryo-EM of drug-bound ribosomes containing labeled L32-3 can map precise interaction sites .

• Mutation studies: Site-directed mutagenesis of conserved residues in L32-3 can identify positions that, when altered, affect antibiotic susceptibility. This approach helps distinguish between direct binding effects and allosteric influences on antibiotic action sites .

• In vivo reporter systems: Creating reporter constructs with modified L32-3 sequences can monitor how mutations influence antibiotic resistance phenotypes in living E. faecalis cells.

• Ribosome heterogeneity assessment: Investigating whether different L32-3 variants affect the composition of specialized ribosomes that might preferentially translate resistance factors.

Experimental approaches often involve complementing L32-3 deletion strains with mutant variants and testing antibiotic susceptibility profiles. For E. faecalis specifically, researchers should consider combinations of common therapeutics such as ampicillin plus ceftriaxone, which have shown efficacy against resistant strains .

What role does L32-3 play in E. faecalis virulence and infection models?

The role of L32-3 in E. faecalis virulence is multifaceted and can be investigated through several approaches:

• Virulence gene expression regulation: L32-3 may participate in specialized ribosomes that preferentially translate virulence factors. Analysis of translational profiles in wildtype versus L32-3 mutant strains can reveal differential expression of virulence genes like cylA, gelE, esp, and ace .

• Stress response during infection: L32-3 may contribute to adaptation during host invasion, as ribosomal proteins often serve dual functions under stress conditions:

• In vivo studies: Using animal infection models (particularly endocarditis or UTI models) with L32-3 mutant strains can determine if altered L32-3 function affects:

  • Bacterial load in tissues

  • Persistence during antibiotic treatment

  • Ability to establish chronic infection

  • Host inflammatory response

• Surface exposure potential: Some ribosomal proteins can be exposed on the bacterial surface and interact with host factors. Proteomic "shaving" experiments can determine if L32-3 has surface accessibility during infection .

Research findings indicate that E. faecalis translation machinery adaptations, potentially including L32-3 modifications, may contribute to its ability to cause persistent infections in challenging environments such as the urinary tract and endocardium .

How does gene loss or mutation of rpmF3 affect E. faecalis fitness and ribosome function?

The impact of rpmF3 gene loss or mutation on E. faecalis fitness reveals important insights about ribosome adaptability:

• Essentiality status: Unlike some core ribosomal proteins, L32 falls into the category of potentially nonessential ribosomal proteins in some bacteria. In E. faecalis, experimental determination of whether rpmF3 can be deleted without lethal consequences provides fundamental information about its role in ribosome function .

• Fitness consequences: Even if not strictly essential, loss or mutation of rpmF3 likely imposes fitness costs that can be measured through:

  • Growth rate analysis in different media conditions

  • Competition assays between wildtype and mutant strains

  • Ribosome assembly kinetics measurements

  • Translation fidelity and rate assessments

• Compensatory mechanisms: E. faecalis may employ several strategies to compensate for L32-3 dysfunction:

• Structural implications: Cryo-EM analysis of ribosomes from rpmF3 mutants can reveal structural adaptations and conformational changes that accommodate the absence or alteration of L32-3 .

Research findings suggest that while some bacteria can tolerate loss of certain ribosomal proteins, the consequences often include decreased translational efficiency and reduced fitness in stress conditions. The specific impact on E. faecalis would depend on whether functional paralogs exist or if the ribosome structure can adapt to the absence of L32-3 .

Methods for Studying L32-3 Interactions

What techniques are most effective for studying L32-3 interactions with rRNA and other ribosomal proteins?

Multiple complementary techniques provide comprehensive insights into L32-3 interactions with rRNA and other ribosomal components:

• Structural biology approaches:

  • Cryo-electron microscopy of E. faecalis 70S ribosomes provides high-resolution (2-3Å) visualization of L32-3 positioning within the ribosomal architecture

  • X-ray crystallography of reconstituted substructures containing L32-3 can reveal specific interaction interfaces

  • NMR spectroscopy of isotopically labeled L32-3 with RNA fragments identifies binding sites at atomic resolution

• Biochemical interaction mapping:

  • UV crosslinking followed by mass spectrometry (CXMS) identifies points of contact between L32-3 and rRNA or neighboring proteins

  • Hydroxyl radical footprinting maps RNA regions protected by L32-3 binding

  • Filter binding assays quantify binding affinities between purified L32-3 and rRNA fragments

• In vivo interaction validation:

  • CLASH (crosslinking, ligation, and sequencing of hybrids) captures physiologically relevant RNA-protein interactions

  • Bacterial two-hybrid assays detect protein-protein interactions involving L32-3

  • Co-immunoprecipitation with tagged L32-3 followed by mass spectrometry identifies interaction partners

• Computational approaches:

  • Molecular dynamics simulations predict dynamic interactions not captured in static structures

  • Coevolution analysis identifies residues likely to form interaction interfaces

The 70S ribosome structure from E. faecalis shows that L32-3 is positioned at a key interface region in the 50S subunit, with both rRNA and protein interaction partners that can be systematically mapped using these techniques .

How can researchers investigate the impact of L32-3 variants on ribosome assembly and function in E. faecalis?

Investigating the impact of L32-3 variants requires systematic approaches that combine genetics, biochemistry, and functional assays:

• Genetic system development:

  • Construction of an E. faecalis strain with chromosomal rpmF3 under control of an inducible promoter

  • Complementation system with plasmid-expressed L32-3 variants in a conditional depletion background

  • CRISPR-Cas9 editing for precise chromosomal mutation introduction

• Ribosome assembly analysis:

  • Sucrose gradient profiling to quantify 30S, 50S, 70S and polysome levels in strains expressing L32-3 variants

  • Pulse-chase experiments with 35S-methionine to track assembly kinetics

  • Quantitative mass spectrometry to determine stoichiometry of ribosomal components

• Functional assessment:

  • In vitro translation assays using purified ribosomes containing L32-3 variants

  • Measurement of misincorporation rates using reporter systems

  • Translational fidelity assessment with dual luciferase reporters

  • Antibiotic susceptibility testing of strains with altered L32-3

• Structure-function correlations:

  • Cryo-EM analysis of ribosomes containing L32-3 variants to detect structural perturbations

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered dynamics

Examples of informative L32-3 variants include mutations of conserved cysteine residues involved in potential zinc coordination, alterations to positively charged residues that interact with rRNA, and truncations that probe domain contributions to assembly and function .

What methodological approaches can identify potential moonlighting functions of L32-3 outside the ribosome?

Ribosomal proteins, including L32-3, may have secondary "moonlighting" functions beyond their roles in protein synthesis. To identify such functions for E. faecalis L32-3:

• Extracellular and surface localization:

  • Cell surface "shaving" proteomics to determine if L32-3 is exposed on the bacterial surface

  • Subcellular fractionation followed by immunoblotting to identify non-ribosomal pools of L32-3

  • Immunofluorescence microscopy with anti-L32-3 antibodies to visualize cellular distribution

• Interaction partner identification:

  • Pull-down assays with tagged L32-3 followed by mass spectrometry

  • Bacterial two-hybrid screening against E. faecalis genomic libraries

  • Protein microarray probing with purified L32-3 to identify non-ribosomal binding partners

• Functional screening:

  • Phenotypic analysis of L32-3 overexpression strains

  • Conditional depletion combined with stress challenge panels

  • Biofilm formation assays with L32-3 variants

• Host interaction studies:

  • Binding assays between L32-3 and host extracellular matrix proteins

  • Assessment of L32-3 immunogenicity and potential as pathogen-associated molecular pattern

  • Testing for interference with host immune signaling pathways

Previous research has identified surface-exposed ribosomal proteins in E. faecalis, suggesting the possibility that L32-3 might also have non-canonical functions, potentially in adhesion, immune evasion, or stress response regulation .

Advanced Structural and Functional Characterization

How do post-translational modifications of L32-3 influence ribosome function in E. faecalis?

Post-translational modifications (PTMs) of ribosomal proteins represent an emerging area of research that impacts ribosome heterogeneity and specialized functions:

• Identification of L32-3 PTMs:

  • High-resolution mass spectrometry analysis of purified native L32-3 from E. faecalis grown under various conditions

  • Site-specific detection using targeted proteomics approaches (MRM/PRM)

  • Antibodies against specific modifications (e.g., phosphorylation, methylation)

• PTM mapping relative to structure:

  • Spatial analysis of modification sites relative to functional regions using the E. faecalis ribosome structure

  • Correlation of modification positions with interaction interfaces

• Functional impact assessment:

  • In vitro reconstitution with modified vs. unmodified L32-3

  • Creation of modification-mimicking or modification-preventing mutations

  • Ribosome activity assays under varying environmental conditions

• Regulation of PTMs:

  • Identification of enzymes responsible for L32-3 modifications

  • Analysis of modification patterns during stress responses and infection

Potential PTMs for E. faecalis L32-3 may include phosphorylation, methylation, or acetylation, with patterns potentially changing during antibiotic exposure or host colonization. The zinc-binding motifs in L32 proteins can also be affected by oxidation state in response to cellular redox conditions .

How does L32-3 contribute to specialized ribosome populations in E. faecalis under different stress conditions?

The concept of specialized ribosomes - heterogeneous ribosome populations with distinct compositions and functions - is increasingly recognized as important for bacterial adaptation:

• Detecting L32-3 variability:

  • Quantitative proteomics to measure L32-3 stoichiometry in ribosomes under different conditions

  • RNA-seq analysis of rpmF3 expression relative to other ribosomal genes

  • Polysome profiling coupled with selective ribosome profiling

• Stress-specific responses:

  • Analysis of L32-3 levels during:

    • Antibiotic exposure (particularly cell-wall targeting antibiotics)

    • Nutrient limitation

    • Host-relevant stresses (pH shifts, bile exposure)

    • Biofilm formation

• Translational impact assessment:

  • Ribosome profiling (Ribo-seq) in wildtype vs L32-3 variant strains to identify differentially translated mRNAs

  • Correlation of translation patterns with stress response and virulence gene expression

  • In vitro translation assays comparing activity of ribosomes with different L32-3 status

• Heterogeneity visualization:

  • Single-molecule imaging of fluorescently tagged L32-3 variants

  • Cryo-EM classification to identify structural heterogeneity in ribosome populations

In E. faecalis, L32-3 variation might be particularly relevant during antibiotic exposure, as the organism is known for developing resistance to multiple antibiotics including vancomycin, tetracycline, and erythromycin .

How can computational approaches enhance our understanding of L32-3 evolution and function?

Computational and bioinformatic approaches provide powerful insights into L32-3 evolution and function:

• Evolutionary analysis:

  • Phylogenetic profiling of L32 across bacterial species reveals evolutionary patterns

  • Analysis of sequence conservation identifies critical functional residues

  • Coevolution analysis detects coordinated changes with interaction partners

  • Identification of horizontal gene transfer events affecting rpmF3

• Structural prediction and modeling:

  • Molecular dynamics simulations predict L32-3 behavior within the ribosome

  • AlphaFold2 or RoseTTAFold predictions for variant structures

  • Docking simulations with rRNA and protein partners

  • Computational prediction of PTM sites and their effects on structure

• Systems biology approaches:

  • Integration of transcriptomic and proteomic data to model L32-3 in regulatory networks

  • Genome-scale models incorporating ribosome heterogeneity

  • Prediction of synthetic lethal interactions involving rpmF3

• Comparative genomics:

  • Analysis of rpmF3 presence/absence patterns across bacterial genomes

  • Correlation of L32 variants with lifestyle and pathogenicity traits

  • Identification of compensatory mechanisms in species lacking L32

Computational analysis reveals that while many ribosomal proteins are strictly conserved, L32 belongs to a set that can be nonessential in some bacterial species. This pattern suggests functional redundancy or compensatory mechanisms that can be further investigated in E. faecalis .