Recombinant Enterococcus faecalis Cysteine--tRNA ligase (cysS)

What is Cysteine--tRNA ligase (cysS) and what is its fundamental role in Enterococcus faecalis metabolism?

Cysteine--tRNA ligase (cysS), also known as Cysteinyl-tRNA synthetase (CysRS), is an essential enzyme in Enterococcus faecalis that catalyzes the attachment of cysteine to its cognate tRNA molecule (tRNACys). This aminoacylation reaction is critical for protein synthesis, as it ensures the correct incorporation of cysteine during translation.

The enzyme belongs to the aminoacyl-tRNA synthetase (ARS) family, which are essential enzymes responsible for charging tRNA molecules with their cognate amino acids . In E. faecalis, cysS plays a particularly important role in sulfur metabolism and redox homeostasis due to the involvement of cysteine in various cellular processes including protein folding, enzyme catalysis, and defense against oxidative stress.

Functionally, cysS catalyzes a two-step reaction: first activating cysteine using ATP to form an aminoacyl-adenylate intermediate, then transferring the activated amino acid to the 3′-terminal adenosine of tRNACys. This charged tRNA is subsequently used by ribosomes during protein synthesis.

What expression systems are most effective for producing recombinant E. faecalis cysS for research purposes?

For producing recombinant E. faecalis cysS, E. coli expression systems are most commonly employed due to their efficiency, ease of genetic manipulation, and high protein yields . Several approaches can be considered:

• pET Expression System: Using E. coli BL21(DE3) as host strain with T7 RNA polymerase-based vectors allows for high-level, inducible expression.

• Cold-shock Expression: For improved protein solubility, expression at lower temperatures (15-18°C) after induction can reduce inclusion body formation.

• Fusion Tag Selection: Incorporating solubility-enhancing tags such as:

  • His6-tag for purification via metal affinity chromatography

  • GST or MBP tags for improved solubility and folding

  • SUMO fusion for enhanced expression and native N-terminus after tag removal

Optimal results typically involve BL21(DE3) cells cultured at 37°C until OD600 reaches 0.6-0.8, followed by induction with 0.1-0.5 mM IPTG and expression at 18-25°C for 16-20 hours to balance yield and solubility.

How should recombinant cysS storage conditions be optimized to maintain long-term enzymatic activity?

Proper storage of recombinant cysS is critical for maintaining enzymatic activity over time. Based on established protocols for similar aminoacyl-tRNA synthetases, the following practices are recommended:

• Short-term storage (up to one week): Store at 4°C in appropriate buffer (typically 20-50 mM Tris-HCl or HEPES, pH 7.4-8.0, 100-200 mM NaCl, 1 mM DTT or 5 mM TCEP) .

• Long-term storage (months): Store at -20°C/-80°C with the addition of 15-50% glycerol as a cryoprotectant . Aliquoting the protein into single-use volumes prevents damage from repeated freeze-thaw cycles.

• Lyophilization: For maximum stability (up to 12 months), lyophilization in the presence of stabilizing excipients like sucrose or trehalose can be effective .

• Buffer additives: Consider including:

  • Reducing agents (1-5 mM DTT or TCEP) to protect cysteine residues

  • 0.1 mM EDTA to chelate metal ions that might promote oxidation

  • 10% glycerol to enhance conformational stability

Recommendation: Avoid repeated freeze-thaw cycles as they significantly reduce enzymatic activity. Working aliquots should be stored at 4°C for up to one week .

What are the established methods for assaying cysS enzymatic activity in research settings?

Several established methods can be employed to assess the enzymatic activity of recombinant E. faecalis cysS:

1. ATP-PPi Exchange Assay:
This traditional method measures the first step of the aminoacylation reaction (amino acid activation) by quantifying the exchange between 32P-labeled pyrophosphate and ATP.

2. tRNA Aminoacylation Assay:
This more comprehensive approach measures the complete reaction by quantifying charged tRNA formation:

• Radioactive method: Using 14C or 3H-labeled cysteine and filter-binding techniques

• Non-radioactive methods:

  • HPLC separation of charged vs. uncharged tRNA

  • Enzyme-coupled assays that detect AMP production

3. AMP Formation Assay:
A coupled enzymatic assay where AMP produced during aminoacylation is detected through conversion to ATP and subsequent luciferase reaction.

4. Pyrophosphate Detection:
Using commercially available pyrophosphate detection kits based on fluorescence or absorbance.

Standard Reaction Conditions:

• 100 mM HEPES buffer (pH 7.2-7.5)

• 10-30 mM KCl

• 10 mM MgCl2

• 2-5 mM ATP

• 0.1-1.0 mg/mL total E. faecalis tRNA or purified tRNACys

• 50-200 μM L-cysteine

• 0.5-5 μg purified recombinant cysS

• 37°C incubation for 10-30 minutes

How does E. faecalis cysS function change under conditions of oxidative stress and hydrogen sulfide exposure?

E. faecalis cysS function is significantly modulated under oxidative stress and hydrogen sulfide exposure, with several mechanisms at play:

Oxidative Stress Effects:
Under oxidative conditions, cysteine residues in cysS can undergo several modifications:

• Formation of disulfide bonds, altering enzyme conformation

• Oxidation to sulfenic, sulfinic, or sulfonic acid derivatives

• S-glutathionylation as a protective mechanism

Hydrogen Sulfide/RSS Exposure:
Exogenous H2S/reactive sulfur species (RSS) introduce several changes:

• Protein Persulfidation: The active site cysteine of cysS can undergo persulfidation (addition of -SSH groups), altering its catalytic properties. This posttranslational modification is implicated in H2S signal transduction and is significantly elevated by exogenous sulfide in RSS-sensing proteins in E. faecalis .

• Metabolic Rewiring: H2S exposure triggers proteome-wide changes that affect CoA metabolism and fatty acid biosynthesis pathways . These changes may indirectly impact cysS function through substrate availability.

• CoA Persulfide Formation: The formation of CoA persulfide (CoASSH) under H2S exposure can inhibit CoA-utilizing enzymes . Since aminoacyl-tRNA synthetases like cysS require ATP, these metabolic shifts can alter enzyme activity.

Experimental Approach to Study These Effects:
To investigate cysS behavior under these conditions, researchers can:

• Expose purified cysS to graduated concentrations of H2O2 (0.1-1 mM) or Na2S (0.1-0.2 mM)

• Monitor aminoacylation activity using tRNA charging assays

• Use persulfidation profiling techniques with iodoacetamide-based reagents

• Employ mass spectrometry to identify specific modified residues

What experimental challenges arise when purifying active recombinant E. faecalis cysS, and how can they be addressed?

Purifying active recombinant E. faecalis cysS presents several experimental challenges that require specific troubleshooting approaches:

Challenge 1: Poor Solubility and Inclusion Body Formation

• Solutions:

  • Expression at lower temperatures (16-20°C)

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Refolding protocols using graduated dialysis from 8M urea or 6M guanidinium-HCl

  • Solubility-enhancing fusion tags (MBP, SUMO)

Challenge 2: Oxidation of Critical Cysteine Residues

• Solutions:

  • Inclusion of reducing agents (5 mM TCEP or DTT) throughout purification

  • Purification under anaerobic conditions for highly sensitive preparations

  • Addition of catalase and SOD to buffers to remove reactive oxygen species

Challenge 3: Proteolytic Degradation

• Solutions:

  • Use of protease-deficient E. coli strains (BL21)

  • Working at 4°C during all purification steps

  • Inclusion of EDTA-free protease inhibitor cocktail

  • Expedited purification protocols

Challenge 4: Loss of Catalytic Activity During Purification

• Solutions:

  • Activity assays at each purification step to track activity loss

  • Supplementation with stabilizing cofactors (Zn2+)

  • Addition of glycerol (10-20%) to all buffers

  • Controlled buffer pH to maintain optimal enzyme conformation

Challenge 5: Co-purification of E. coli tRNAs

• Solutions:

  • High-salt washes (500 mM NaCl)

  • DEAE or anion exchange steps to remove bound nucleic acids

  • Treatment with nucleases followed by additional purification steps

Optimized Purification Protocol:

• Lysis in 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM TCEP, with protease inhibitors

• Clarification by high-speed centrifugation (13,200 rpm, 15 min, 4°C)

• IMAC purification using Ni-NTA for His-tagged protein

• Size exclusion chromatography as a polishing step

• Activity verification using aminoacylation assays

How do point mutations in the catalytic domain of cysS affect charging efficiency and bacterial fitness?

Effects on Enzymatic Function:

Biological Consequences:

• Translation Fidelity Effects:
  Mutations that reduce charging accuracy can lead to misincorporation of amino acids, causing proteome-wide errors and triggering stress responses. Even minor reductions in aminoacylation accuracy can have amplified effects due to the essential nature of protein synthesis.

• Growth Rate Impact:
  E. faecalis strains with cysS mutations typically show:

  • Extended lag phases (up to 2-3 times normal duration)

  • Reduced growth rates in both rich and minimal media

  • Heightened sensitivity to nutrient limitations

• Stress Response Activation:
  Defective cysS triggers several stress responses:

  • Upregulation of alternative tRNA synthetases

  • Activation of the stringent response (ppGpp production)

  • Enhanced expression of chaperones and proteases

In human cysteinyl-tRNA synthetase (CARS), mutations can cause severe phenotypes including microcephaly, developmental delay, and brittle hair and nails . While bacterial phenotypes differ, these human disease associations highlight the critical nature of this enzyme class.

Experimental Approaches:

• Site-directed mutagenesis targeting conserved residues

• In vitro aminoacylation assays with purified variant enzymes

• Growth competition assays between wild-type and mutant strains

• Ribosome profiling to detect translation errors

What techniques can be employed to study cysS persulfidation in E. faecalis and its effects on enzyme function?

Protein S-sulfuration (persulfidation) is a post-translational modification increasingly recognized as important in bacterial responses to reactive sulfur species. Several advanced techniques can be employed to study cysS persulfidation in E. faecalis:

1. Persulfidation Detection Methods:

A. Biotin-Switch Technique (BST) for Persulfides:

• Blocking free thiols with methylsulfonyl benzothiazole (MSBT)

• Reduction of persulfides with DTT

• Labeling newly exposed thiols with biotin-HPDP

• Detection by Western blot or mass spectrometry

B. Tag-Switch Method:

• Alkylation of free thiols with methylsulfonyl benzothiazole

• Reaction of persulfides with CN-biotin

• Streptavidin enrichment and detection

C. Dimedone-Based Probes:

• DAz-2/DCP-Bio1 labeling of persulfide groups

• Click chemistry for fluorophore attachment

• Detection via in-gel fluorescence or LC-MS/MS

2. Quantitative Persulfidation Analysis:
E. faecalis proteome-wide persulfidation profiling has revealed that approximately 13% of the proteome is persulfidated under normal conditions, with significant increases following Na₂S treatment . To specifically analyze cysS:

• Treat E. faecalis cultures with 0.2 mM Na₂S for 30 minutes

• Perform cell lysis in buffer containing 5 mM TCEP and protease inhibitors

• Calculate sigma ratios (σR) for cysS using total area for cysteine peptides

• Normalize fold change of S-sulfurated peptides (treated/untreated) to protein abundance

3. Functional Impact Assessment:
To determine how persulfidation affects cysS function:

• Site-directed mutagenesis of cysteine residues identified as persulfidation targets

• Aminoacylation assays comparing wild-type and mutant cysS under various redox conditions

• Structural analysis via X-ray crystallography of persulfidated vs. non-persulfidated enzyme

4. Advanced Proteomics Approach:

• LC-MS/MS analysis using a high-resolution Orbitrap instrument

• Database search against E. faecalis proteome (e.g., UniProt UP000004846)

• Identification of persulfidation sites with appropriate fixed and variable modifications

• Quantification of persulfidation levels using label-free methods based on MS1 ion peak areas

How can structural insights into E. faecalis cysS contribute to the development of novel antimicrobial strategies?

Structural analysis of E. faecalis cysS offers significant potential for antimicrobial development, leveraging several unique features of this essential bacterial enzyme:

Structural Uniqueness for Drug Targeting:

Aminoacyl-tRNA synthetases like cysS are attractive antimicrobial targets due to:

• Essential role in protein synthesis

• Structural differences between bacterial and human orthologs

• Accessibility in the bacterial cytoplasm

Key Structural Features for Selective Targeting:

Structural Determination Approaches:

• X-ray Crystallography: The most direct method for high-resolution structure determination, similar to the 2.05 Å crystallographic structure obtained for CoA-bound CoAPR in E. faecalis .

• Cryo-EM: Particularly useful for capturing cysS-tRNA complexes.

• NMR Spectroscopy: For examining dynamic regions and ligand interactions.

• Molecular Dynamics Simulations: To understand conformational changes during catalysis, similar to those performed for E. faecalis CoAPR .

Antimicrobial Development Strategies:

• Competitive Inhibitors: Molecules that compete with ATP, cysteine, or tRNA binding.

• Allosteric Modulators: Compounds targeting non-catalytic sites to induce dysfunctional conformations.

• Covalent Modifiers: Molecules that react with catalytic cysteine residues, leveraging the understanding of protein persulfidation mechanisms in E. faecalis .

• Protein-Protein Interaction Disruptors: If cysS forms functionally important complexes with other proteins.

Experimental Pipeline:

• High-throughput virtual screening against structural models

• Fragment-based approaches for novel scaffold identification

• Structure-activity relationship studies for lead optimization

• Evaluation of species selectivity using purified human ortholog

• Assessment of resistance development frequency in E. faecalis

The detailed structural and mechanistic understanding of cysS can potentially address the urgent challenge of multidrug resistance in E. faecalis, a significant hospital-acquired pathogen.

What are best practices for validating the purity and identity of recombinant E. faecalis cysS preparations?

Comprehensive validation of recombinant E. faecalis cysS preparations requires multiple analytical approaches to ensure both purity and identity:

Purity Assessment:

• SDS-PAGE Analysis:

  • Standard method showing >85% purity for research-grade preparations

  • Silver staining for detection of minor contaminants (≥0.1 ng protein)

  • Densitometry analysis for quantitative purity assessment

• Size Exclusion Chromatography (SEC):

  • Detects aggregates and degradation products

  • Provides hydrodynamic radius information

  • Can be coupled to multi-angle light scattering (SEC-MALS) for absolute molecular weight determination

• Mass Spectrometry-Based Purity Analysis:

  • Intact mass measurement by ESI-MS

  • Detection of post-translational modifications or truncations

  • Peptide mapping by LC-MS/MS after trypsin digestion

Identity Confirmation:

• Peptide Mass Fingerprinting:

  • Tryptic digest followed by MALDI-TOF or LC-MS/MS

  • Database matching against known E. faecalis cysS sequence

  • Coverage target of >80% of the protein sequence

• Western Blotting:

  • Using antibodies specific to cysS or to affinity tags

  • Confirms both identity and expected molecular weight

• N-terminal Sequencing:

  • Edman degradation for first 5-10 amino acids

  • Confirms correct processing of N-terminus or tag cleavage

• Functional Validation:

  • Aminoacylation assay with E. faecalis tRNACys

  • ATP-PPi exchange assay

  • Validation of expected kinetic parameters

Documentation Requirements:

• Certificate of Analysis including:

  • Purity percentage by SDS-PAGE (target >85%)

  • Identity confirmation method

  • Buffer composition and pH

  • Protein concentration with method used

  • Activity data (specific activity in units/mg)

  • Date of production and lot number

  • Stability and storage recommendations

How does the methodology for studying E. faecalis cysS differ from approaches used with other bacterial aminoacyl-tRNA synthetases?

The methodology for studying E. faecalis cysS requires specific considerations that differ from approaches used with other bacterial aminoacyl-tRNA synthetases:

1. Redox Sensitivity Considerations:

E. faecalis cysS presents unique challenges due to:

• High susceptibility to oxidation of catalytic cysteine residues

• Significant role in redox homeostasis

• Susceptibility to persulfidation under sulfide exposure

Methodological Adaptations:

• Use of stronger reducing agents (TCEP preferred over DTT)

• Anaerobic conditions for certain assays

• Quantification of redox state of critical cysteines

• Monitoring of persulfidation levels under varying conditions

2. Special Expression and Purification Requirements:

Unlike many other ARSs, optimal expression of active E. faecalis cysS requires:

• Lower induction temperatures (16-18°C)

• Extended expression times (16-20 hours)

• Carefully controlled aeration during growth

• Inclusion of zinc supplementation during expression

• Protection from oxidation throughout purification

3. Activity Assay Adaptations:

Standard aminoacylation assays must be modified to address:

• Impact of redox state on activity

• Potential interference of persulfidation

• Optimal buffer conditions (25 mM HEPES, pH 7.4)

• Influence of metabolites specific to E. faecalis physiology

4. Structural Analysis Approaches:

Structural studies of E. faecalis cysS require:

• Crystal growth under reducing conditions

• Consideration of zinc coordination

• Analysis of potential regulatory domains

• Investigation of unique conformational states

• Comparison with structures of related enzymes like CoAPR (2.05 Å resolution)

5. Integration with Bacterial Physiology:

Unlike many other ARSs, E. faecalis cysS research must consider its role in:

• Response to antibiotics and oxidative stress

• Adaptation to hydrogen sulfide exposure

• Potential involvement in biofilm formation

• Interaction with fatty acid biosynthesis pathways

Comparative Workflow Table:

What are the critical quality control parameters for recombinant E. faecalis cysS in experimental settings?

Ensuring consistent quality of recombinant E. faecalis cysS is essential for reproducible experimental results. The following critical quality control parameters should be systematically monitored:

1. Physical Quality Parameters:

2. Functional Quality Parameters:

3. Stability Parameters:

4. Batch-to-Batch Consistency Parameters:

• Protein concentration determination by at least two methods (Bradford and UV280)

• Consistent yield per liter of culture (mg/L)

• Reproducible elution profile during purification

• Consistent activity:mass ratio between batches

5. Contamination Controls:

Implementation Strategy:

• Establish a reference standard from a well-characterized batch

• Document complete QC testing for each new batch

• Implement stability testing program

• Use statistical process control to monitor trends

• Record lot-specific data in laboratory information management system

How can recombinant E. faecalis cysS be utilized in structural biology studies investigating bacterial tRNA synthetase mechanisms?

Recombinant E. faecalis cysS offers valuable opportunities for advancing structural biology investigations into bacterial tRNA synthetase mechanisms through several sophisticated approaches:

1. High-Resolution Structure Determination:

Purified recombinant cysS can be used for:

• X-ray crystallography (aiming for resolution better than 2.05 Å achieved for related E. faecalis proteins)

• Cryo-electron microscopy for larger complexes

• Solution NMR for dynamic regions and conformational changes

Key Experimental Conditions:

• Crystallization screening with reducing agents to prevent oxidation

• Co-crystallization with substrates (ATP, cysteine) and tRNA

• Heavy atom derivatives for phase determination

• Optimization of crystal growth in microenvironments

2. Mechanistic Investigations:

Recombinant cysS enables detailed study of:

• Adenylation reaction mechanism

• tRNA recognition specificity

• Conformational changes during catalysis

• Metal ion coordination and role

Methodological Approaches:

• Site-directed mutagenesis of catalytic residues

• Isothermal titration calorimetry for binding energetics

• Time-resolved spectroscopy for reaction kinetics

• Single-molecule FRET for conformational dynamics

3. Structure-Guided Functional Studies:

With structural information, researchers can:

• Map conserved domains across bacterial cysS enzymes

• Identify species-specific structural features

• Design chimeric proteins to test domain function

• Develop structure-based inhibitors

4. Advanced Biophysical Characterization:

Recombinant cysS facilitates:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

• Small-angle X-ray scattering (SAXS) for solution structure

• Circular dichroism (CD) for secondary structure analysis

• Analytical ultracentrifugation for oligomerization state

5. Comparative Analysis with Human Ortholog:

Structural comparison with human CARS provides:

• Identification of bacterial-specific features

• Understanding of disease-causing mutations

• Structure-based design of selective inhibitors

• Evolutionary insights into ARS development

6. Integration with Computational Approaches:

Structural data enables:

• Molecular dynamics simulations to study conformational flexibility

• Quantum mechanics/molecular mechanics (QM/MM) for reaction mechanism

• Virtual screening for structure-based drug discovery

• Machine learning approaches to predict functional effects of mutations

The crystallographic structure of CoA-bound CoAPR from E. faecalis at 2.05 Å resolution demonstrates the feasibility of high-resolution structural studies with proteins from this organism .

What is the role of E. faecalis cysS in antibiotic resistance mechanisms, and how can it be studied experimentally?

E. faecalis cysS plays both direct and indirect roles in antibiotic resistance mechanisms, offering several avenues for experimental investigation:

1. Direct Involvement in Antibiotic Resistance:

Aminoacyl-tRNA synthetases can contribute to resistance through:

• Mutations affecting drug binding (particularly for synthetase inhibitors)

• Overexpression providing resistance to translation-targeting antibiotics

• Modification of tRNA charging patterns affecting translation fidelity

Experimental Approaches:

• Selection experiments to identify resistance-conferring mutations in cysS

• Overexpression studies to determine MIC changes for various antibiotics

• Site-directed mutagenesis to validate specific resistance mechanisms

2. Interaction with Persulfidation and Sulfur Metabolism:

E. faecalis utilizes H2S/RSS as a defense against antibiotics, and cysS may participate in this process through:

• Alteration of translation under stress conditions

• Incorporation of cysteine into stress-response proteins

• Direct involvement in persulfidation pathways

Experimental Approaches:

• Combined antibiotic and Na2S treatment (0.2 mM) to assess synergistic effects

• Persulfidation profiling of cysS under antibiotic stress

• Metabolic labeling to trace cysteine flux during antibiotic challenge

• Analysis of cysS activity in antibiotic-resistant clinical isolates

3. Role in Biofilm Formation and Persistence:

E. faecalis biofilms show enhanced antibiotic resistance, with potential cysS involvement through:

• Altered translation profiles during biofilm formation

• Contribution to extracellular matrix production

• Response to oxidative stress within biofilms

Experimental Approaches:

• Comparison of cysS expression between planktonic and biofilm states

• Creation of cysS conditional knockdowns to assess biofilm integrity

• Microscopy with fluorescently-tagged cysS to determine localization in biofilms

• Assessment of biofilm antibiotic resistance with cysS variants

4. Metabolic Adaptation and Antibiotic Tolerance:

Metabolic changes involving cysS can contribute to tolerance:

• Shifts in CoA metabolism affecting persister formation

• Alterations in fatty acid biosynthesis affecting membrane permeability

• Changes in redox homeostasis affecting antibiotic activity

Experimental Approaches:

• Metabolomic analysis comparing sensitive and resistant strains

• Tracking acetyl-CoA levels during antibiotic exposure

• Fatty acid profiling to correlate with antibiotic susceptibility

• Measurements of intracellular redox state in wild-type vs. cysS variant strains

5. Translation Quality Control and Stress Response:

Mistranslation can trigger stress responses that enhance survival:

• Deliberate mischarging under stress conditions

• Activation of heat shock response through misfolded proteins

• Induction of alternative metabolic pathways

Experimental Approaches:

• Ribosome profiling to detect mistranslation events

• Proteome analysis to identify stress-response activation

• Reporter systems to monitor heat shock response activation

The experimental approaches should incorporate measurement of cysS persulfidation levels, as approximately 13% of the E. faecalis proteome is persulfidated under normal conditions, with significant increases following sulfide exposure .