Recombinant Enterococcus faecalis Serine--tRNA ligase 2 (serS2)

What is the fundamental function of Enterococcus faecalis Serine--tRNA ligase 2 (serS2)?

Enterococcus faecalis Serine--tRNA ligase 2 (serS2) belongs to the aminoacyl-tRNA synthetase family and is responsible for attaching serine to its cognate tRNA molecules. Like other seryl-tRNA synthetases (SerRSs), serS2 catalyzes the esterification of serine to the 3'-terminal adenosine of tRNA^Ser, a critical step in protein synthesis. This reaction proceeds in two steps: first, the activation of serine with ATP to form seryl-adenylate, and second, the transfer of the seryl group to the appropriate tRNA^Ser . While the canonical function involves aminoacylation of tRNA with serine, SerRSs in many organisms additionally aminoacylate selenocysteine-specific tRNA (tRNA^Sec) with serine, which serves as the first step in the metabolic pathway for translational incorporation of selenocysteine into selenoproteins .

How is E. faecalis serS2 typically expressed and purified for research purposes?

Recombinant expression of E. faecalis serS2 typically employs bacterial expression systems similar to those used for other aminoacyl-tRNA synthetases. The gene encoding serS2 can be cloned into expression vectors such as those in the pET series, which utilize the T7 promoter system. Expression is commonly performed in E. coli BL21(DE3) or similar strains, with induction using IPTG (isopropyl-β-d-thiogalactopyranoside) at concentrations ranging from 0.1 to 0.5 mM .

For purification, a general protocol would include:

• Cell lysis using buffers containing Tris-HCl (pH 7.5-8.0), NaCl, and reducing agents like DTT

• Initial purification via affinity chromatography (His-tag or other fusion tags)

• Further purification using ion-exchange chromatography

• Final polishing by size-exclusion chromatography

What assays are used to measure serS2 enzymatic activity?

Several complementary assays can be employed to measure serS2 activity:

What methodologies are most effective for studying tRNA recognition by serS2?

Optimal methods for investigating serS2-tRNA interactions include:

• In vitro binding assays:

  • Electrophoretic mobility shift assays (EMSA)

  • Filter binding assays with purified serS2 and tRNA transcripts

  • Surface plasmon resonance to determine binding kinetics

• Structural approaches:

  • X-ray crystallography of serS2-tRNA complexes

  • Cryo-electron microscopy for larger assemblies

  • NMR studies for dynamic interactions

• Aminoacylation assays:

  • Using native tRNAs vs. in vitro transcripts

  • Comparing wild-type and modified tRNAs

  • Kinetic analysis (Km, kcat) with different tRNA variants

How does oxidative stress affect serS2 activity and expression in E. faecalis?

E. faecalis responds to reactive oxygen species by modifying its RNA epitranscriptome, suggesting a broader stress response system that may include regulation of serS2. When exposed to the superoxide generator menadione, E. faecalis shows significant decreases in specific RNA modifications, particularly N2-methyladenosine (m2A) in both 23S rRNA and tRNA . This response appears to be mediated through ROS-mediated inactivation of iron-sulfur cluster-containing enzymes .

The impact on serS2 may occur through several mechanisms:

• Changes in the modification state of tRNA^Ser affecting recognition by serS2

• Altered expression of serS2 in response to oxidative stress

• Post-translational modifications to serS2 that affect its activity

• Redistribution of serS2 within the cell

Experimental analysis of serS2 under oxidative stress conditions should include measurement of enzymatic activity, protein levels, and localization. Real-time RT-PCR methods similar to those used in studies of E. faecalis gene expression could be employed to monitor changes in serS2 expression .

What is the relationship between antibiotic exposure and serS2 function in E. faecalis?

E. faecalis shows distinct responses to different antibiotics that may impact serS2 function. Studies have demonstrated that exposure to bacteriostatic antibiotics including macrolides (erythromycin and spiramycin) and phenicols (chloramphenicol) results in reduced N2-methyladenosine (m2A) in both 23S rRNA and tRNA, while bactericidal antibiotics like ciprofloxacin, ampicillin, and aminoglycosides do not produce this effect .

The relationship between antibiotic exposure and serS2 function should be investigated through:

• Analysis of serS2 expression levels following exposure to different antibiotics

• Measurement of aminoacylation activity in extracts from antibiotic-treated cells

• Characterization of serS2-tRNA binding under antibiotic stress conditions

• Investigation of potential post-translational modifications to serS2

Notably, the observation that deletion of RNA methyltransferase RlmN in E. faecalis confers a 16-fold increase in chloramphenicol resistance suggests complex interactions between RNA modification systems and antibiotic resistance that may involve serS2.

What structural features distinguish serS2 from other aminoacyl-tRNA synthetases?

Based on knowledge of SerRSs from other organisms, E. faecalis serS2 likely possesses certain distinguishing structural features:

• Domain organization:

  • N-terminal catalytic domain containing the active site for ATP binding and aminoacylation

  • C-terminal anticodon-binding domain for tRNA recognition

  • A connecting peptide linking these domains

• Class-specific motifs:

  • As a Class II aminoacyl-tRNA synthetase, serS2 would feature characteristic motifs including:

    • Motif 1: involved in dimer interface formation

    • Motif 2: containing the signature HIGH sequence for ATP binding

    • Motif 3: containing the KMSKS sequence involved in ATP binding and catalysis

• Serine-specific elements:

  • A serine-binding pocket with specific residues for serine discrimination

  • Structural elements for recognition of the small side chain of serine

How can site-directed mutagenesis be utilized to map functional domains of serS2?

Site-directed mutagenesis provides a powerful approach to mapping functional domains and critical residues in serS2. Based on conserved features of seryl-tRNA synthetases, the following mutagenesis strategy would be informative:

What protein-protein interactions might serS2 participate in beyond its canonical role?

SerRSs in various organisms interact with diverse proteins, enabling them to perform alternative functions beyond translation . Based on known interactions of SerRSs in other organisms, E. faecalis serS2 might interact with:

• Other aminoacyl-tRNA synthetases:

  • Potential formation of multi-synthetase complexes

  • Interaction with arginyl-tRNA synthetase (similar to archaeal SerRS)

• Ribosomal components:

  • Interaction with ribosomal P-stalk proteins (observed in archaeal SerRS)

  • Potential tRNA channeling mechanisms

• RNA modification enzymes:

  • Interaction with tRNA methyltransferases (similar to yeast and human SerRSs)

  • Coordination between tRNA modification and aminoacylation

• Transcriptional regulators:

  • Potential nuclear functions (similar to human cytosolic SerRS)

  • Regulation of gene expression under stress conditions

Methodologies to identify these interactions include co-immunoprecipitation, yeast two-hybrid screening, and proximity labeling approaches.

What evidence exists for non-canonical functions of serS2 in E. faecalis pathogenicity?

While direct evidence for non-canonical functions of serS2 in E. faecalis pathogenicity is not provided in the search results, several possibilities can be inferred from studies of SerRSs in other organisms:

• Potential role in stress response:

  • Human cytosolic SerRS acts as a negative regulator of VEGFA gene expression when translocated to the nucleus

  • SerRS can compete with transcription factors and recruit co-repressors like SIRT2

• Connection to oxidative stress response:

  • E. faecalis modifies its RNA in response to ROS

  • SerS2 might participate in coordinating translation and stress adaptation

• Potential involvement in antibiotic resistance:

  • RNA modification systems in E. faecalis affect antibiotic susceptibility

  • SerS2 might influence translation of resistance factors

To investigate these possibilities, researchers should consider:

• Localization studies of serS2 under different stress conditions

• Protein interaction studies focused on stress response pathways

• Transcriptomic and proteomic analysis of serS2 mutants or overexpression strains

• Phenotypic characterization related to virulence traits and antibiotic resistance

How can high-throughput sequencing technologies enhance understanding of serS2 function?

Modern sequencing technologies offer powerful approaches to investigate serS2 biology comprehensively:

• RNA-Seq approaches:

  • Transcriptome analysis of serS2 mutants or overexpression strains

  • Identification of genes regulated by serS2 under different conditions

  • Comparison of wild-type and mutant responses to stress

• tRNA-Seq:

  • Quantification of tRNA abundance and modification status

  • Identification of changes in tRNA^Ser population under stress

  • Analysis of aminoacylation levels of specific tRNAs

• Ribosome profiling:

  • Assessment of translation efficiency and accuracy

  • Identification of potential pausing at serine codons under stress

  • Correlation between serS2 activity and translation patterns

• CLIP-Seq (Crosslinking Immunoprecipitation-Sequencing):

  • Mapping of serS2 binding sites on tRNAs and potentially other RNAs

  • Identification of non-canonical RNA targets

  • Discovery of potential regulatory roles

What are the most effective approaches for functional genomic analysis of serS2?

Comprehensive functional genomic analysis of serS2 should employ multiple complementary approaches:

• Gene manipulation strategies:

  • Conditional knockdown using inducible antisense RNA

  • CRISPR interference for tunable repression

  • Site-specific mutagenesis of key residues

• Phenotypic characterization:

  • Growth under various stress conditions

  • Antibiotic susceptibility profiling

  • Virulence trait assessment (biofilm formation, adherence)

• Omics approaches:

  • Integration of transcriptomics, proteomics, and metabolomics data

  • Analysis of tRNA modification patterns using liquid chromatography-mass spectrometry

  • Correlation of data across multiple stress conditions

• Comparative analysis:

  • Comparison with other aminoacyl-tRNA synthetases in E. faecalis

  • Evolutionary analysis across bacterial species

  • Structure-function relationships based on homology modeling