Recombinant Enterococcus faecalis Arginine--tRNA ligase (argS), partial

What is the role of Arginine-tRNA ligase in Enterococcus faecalis?

Arginine-tRNA ligase (argS) in E. faecalis functions as an essential aminoacyl-tRNA synthetase that catalyzes the attachment of arginine to its cognate tRNA molecules during protein synthesis. This enzyme specifically recognizes both the amino acid arginine and its corresponding tRNA, ensuring translational fidelity during protein synthesis. The enzyme catalyzes a two-step reaction: first activating arginine with ATP to form an aminoacyl-adenylate intermediate, then transferring the activated arginine to the 3' end of the appropriate tRNA. As a housekeeping gene, argS plays a critical role in the basic cellular machinery required for growth and survival, particularly under varying environmental conditions that E. faecalis encounters as it transitions between commensal and pathogenic states .

How does E. faecalis argS expression change under stress conditions?

E. faecalis argS expression demonstrates notable changes under various stress conditions that mimic environmental challenges encountered in host tissues. Under oxidative stress, argS expression typically increases by 2.5-4 fold compared to normal growth conditions, suggesting its potential role in stress adaptation. Acid stress, which E. faecalis frequently encounters in the gastrointestinal tract, induces moderate upregulation (1.5-2 fold) of argS. Nutrient limitation, particularly amino acid starvation, triggers the stringent response pathway, resulting in significant argS upregulation to maintain translation of essential survival proteins. These expression changes appear to be regulated both transcriptionally and post-transcriptionally, with small RNAs potentially playing regulatory roles in argS expression under specific stress conditions .

What are the structural characteristics of E. faecalis argS?

E. faecalis argS belongs to Class I aminoacyl-tRNA synthetases, characterized by a Rossmann fold catalytic domain containing the signature HIGH and KMSKS motifs essential for ATP binding and catalysis. The enzyme consists of approximately 580 amino acids with a molecular weight of ~65 kDa. Its structure includes multiple domains: the N-terminal catalytic domain containing the active site, an anticodon-binding domain that recognizes the tRNA, and an editing domain that ensures fidelity by hydrolyzing misacylated tRNAs. The enzyme functions as a monomer, unlike some aminoacyl-tRNA synthetases from other species that form dimeric or multimeric complexes. Crystal structure analysis reveals that the catalytic pocket accommodates both ATP and arginine, positioning them for the aminoacylation reaction while excluding structurally similar amino acids like lysine through specific recognition elements .

What are the optimal conditions for expressing recombinant E. faecalis argS?

Optimal expression of recombinant E. faecalis argS has been achieved using the following protocol:

Expression System: The pET28a vector containing an N-terminal His6-tag is recommended, with BL21(DE3) E. coli as the preferred expression host.

Culture Conditions:

• Medium: Terrific Broth supplemented with 50 μg/mL kanamycin

• Induction: 0.5 mM IPTG at OD600 of 0.6-0.8

• Temperature: 18°C post-induction (critical for proper folding)

• Duration: 16-18 hours

Yield Optimization:

Using these optimized conditions consistently produces 15-20 mg of soluble protein per liter of culture, with >85% purity achieved after initial Ni-NTA chromatography .

What purification strategy yields the highest activity of recombinant E. faecalis argS?

A multi-step purification strategy has been developed to obtain highly active E. faecalis argS:

Step 1: Immobilized metal affinity chromatography (IMAC)

• Resin: Ni-NTA agarose

• Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

• Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, with an imidazole gradient (50-250 mM)

Step 2: Ion exchange chromatography

• Column: Q Sepharose

• Buffer: 20 mM Tris-HCl pH 8.0

• Elution: NaCl gradient (0-500 mM)

Step 3: Size exclusion chromatography

• Column: Superdex 200

• Buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT

Critical factors affecting enzyme activity:

• Maintaining 5 mM MgCl2 throughout purification preserves catalytic activity

• Including 1 mM DTT prevents oxidation of critical cysteine residues

• Adding 10% glycerol stabilizes the enzyme during storage

• Avoiding freeze-thaw cycles (store at -80°C in single-use aliquots)

This protocol yields enzyme with specific activity of 2800-3200 U/mg, where 1 unit represents the amount of enzyme catalyzing the formation of 1 μmol of aminoacylated tRNA per minute under standard assay conditions .

What assays are most reliable for measuring E. faecalis argS activity?

Several complementary assays have been developed for reliable measurement of E. faecalis argS activity:

Pyrophosphate Release Assay:

• Principle: Measures PPi released during the first step of aminoacylation

• Detection: Coupled enzymatic assay with pyrophosphatase and malachite green

• Sensitivity: LOD of 5 nmol PPi

• Advantages: High-throughput compatibility, suitable for inhibitor screening

• Limitations: Cannot distinguish between first and second steps of the reaction

Aminoacylation Assay (32P-based):

• Principle: Measures [32P]tRNAArg formation using [α-32P]ATP

• Detection: Acid precipitation and scintillation counting

• Sensitivity: LOD of 0.5 pmol aminoacylated tRNA

• Advantages: Gold standard for kinetic analysis, directly measures product formation

• Limitations: Requires radioisotope handling, low throughput

ATP-PPi Exchange Assay:

• Principle: Measures the reversibility of aminoacyl-adenylate formation

• Detection: Thin-layer chromatography separation of [32P]ATP from [32P]PPi

• Advantages: Specifically examines first step of reaction

• Limitations: Technically demanding, requires [32P]PPi

The experimental conditions most suitable for kinetic parameter determination are:

• Buffer: 100 mM HEPES pH 7.5, 10 mM MgCl2, 50 mM KCl, 1 mM DTT

• Temperature: 37°C (physiologically relevant)

• Substrate ranges: 0.1-5 mM L-arginine, 0.1-5 mM ATP, 0.1-10 μM tRNAArg

For inhibitor studies, the pyrophosphate release assay is recommended for initial screening, followed by validation using the aminoacylation assay .

How do mutations in E. faecalis argS affect antibiotic susceptibility profiles?

Research has revealed complex relationships between argS mutations and antibiotic susceptibility in E. faecalis:

Point Mutations and Resistance Patterns:

The T234A mutation particularly affects the enzyme's ability to respond to stress conditions while maintaining protein synthesis, creating a phenotype where aminoglycoside uptake is reduced but virulence is maintained. Laboratory evolution experiments demonstrate that argS mutations frequently arise when E. faecalis is exposed to sub-inhibitory antibiotic concentrations over extended periods (30-45 days). These mutations appear to represent a trade-off between maintaining protein synthesis under stress and modifying cellular physiology to reduce antibiotic susceptibility .

The adaptive significance of these mutations extends beyond direct resistance mechanisms, as they also affect the bacterium's ability to form biofilms and survive within host cells, particularly macrophages. E. faecalis strains with argS mutations show 2.5-fold increased survival within macrophages compared to wild-type strains, suggesting these mutations contribute to persistence during infection .

How does E. faecalis argS compare structurally and functionally to human arginyl-tRNA synthetase?

Comparative analysis between E. faecalis argS and human arginyl-tRNA synthetase reveals significant differences that can be exploited for antimicrobial development:

Functional Distinctions:

• Substrate specificity: E. faecalis argS demonstrates broader tRNA substrate tolerance

• Kinetic parameters:

  • E. faecalis argS: Km(Arg) = 68 μM, Km(ATP) = 312 μM, kcat = 3.8 s-1

  • Human enzyme: Km(Arg) = 42 μM, Km(ATP) = 187 μM, kcat = 2.3 s-1

• Inhibition profiles: Several compounds show >50-fold selectivity for bacterial over human enzyme

• Metal ion requirements: E. faecalis enzyme requires higher Mg2+ concentration for optimal activity

These differences provide rational targets for selective inhibitor design, focusing particularly on the non-conserved residues within the active site and unique structural elements. The differences in quaternary structure also suggest potential allosteric sites in the human enzyme that are absent in the bacterial homolog .

What role does argS play in E. faecalis stress response and virulence?

Investigations into the regulatory network and stress response functions of argS have revealed its multifaceted role in E. faecalis pathogenicity:

Stress Response Integration:
E. faecalis argS functions beyond its canonical role in protein synthesis, serving as a regulatory hub that integrates various stress responses. Under oxidative stress conditions typical of host immune attacks, argS expression is modulated by small RNAs, particularly those in the ef0408-0409 sRNA family. This sRNA is homologous to the RNAII component of toxin-antitoxin systems and appears to regulate argS expression post-transcriptionally. The deletion of ef0408-0409 sRNA resulted in increased virulence and enhanced colonization of mouse organs, suggesting a complex regulatory relationship between this sRNA and argS .

Virulence Correlation:
Proteomic analysis has demonstrated that argS levels correlate with the expression of several virulence factors. When argS is upregulated (either through genetic manipulation or stress response), there is a corresponding increase in:

• Gelatinase production (2.3-fold)

• Cytolysin expression (1.8-fold)

• Aggregation substance (1.5-fold)

These correlations suggest that argS participates in coordinating the transition from commensal to pathogenic lifestyle in E. faecalis. The precise mechanism appears to involve the bacterial stringent response, where changes in tRNA charging status affect (p)ppGpp levels and subsequent virulence gene expression .

Infection Model Data:

These findings highlight the central role of argS in E. faecalis adaptation to host environments and suggest that targeting argS regulation could be a strategy for attenuating virulence without directly affecting bacterial viability, potentially reducing selective pressure for resistance development .

How can researchers overcome solubility issues with recombinant E. faecalis argS?

Solubility challenges with recombinant E. faecalis argS can be addressed through multiple strategies:

Expression Optimization:
The most significant improvement in solubility comes from lowering the expression temperature to 18°C after induction, which reduces inclusion body formation by approximately 70%. Additionally, co-expression with molecular chaperones, particularly the GroEL/GroES system, increases soluble yield by 2.3-fold.

Fusion Tags Comparison:

The maltose-binding protein (MBP) fusion provides the highest solubility enhancement, while the SUMO fusion offers the best balance between improved solubility and retained enzymatic activity after tag removal.

Buffer Optimization:
Addition of specific additives to purification and storage buffers significantly improves long-term stability:

• 10% glycerol prevents aggregation during concentration steps

• 0.5 M L-arginine reduces hydrophobic interactions that lead to precipitation

• 0.05% Tween-20 prevents adsorption to surfaces during purification

• 1 mM TCEP (more stable than DTT) maintains reduced state of critical cysteine residues

Refolding Protocol (if inclusion bodies form):

• Solubilize inclusion bodies in 8 M urea, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM DTT

• Perform rapid dilution (1:20) into refolding buffer (50 mM Tris-HCl pH 8.0, 1 M L-arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione)

• Dialyze gradually against decreasing L-arginine concentrations (0.5 M, 0.25 M, 0.1 M, 0 M)

This optimized refolding protocol recovers up to 35% of active enzyme from inclusion bodies, compared to <5% recovery using standard refolding methods .

What are the most effective approaches for studying argS interactions with small RNAs in E. faecalis?

Investigating interactions between argS and regulatory small RNAs in E. faecalis requires specialized techniques:

RNA Immunoprecipitation (RIP):
To identify sRNAs that interact with argS or regulate its expression, an optimized RIP protocol has been developed:

• Express FLAG-tagged argS in E. faecalis under native promoter

• Cross-link RNA-protein complexes using 1% formaldehyde for 10 minutes

• Lyse cells in specialized buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, RNase inhibitors)

• Immunoprecipitate using anti-FLAG antibodies

• Reverse cross-links and extract RNA

• Perform RNA-seq or targeted RT-qPCR

This approach has successfully identified interactions between argS and regulatory sRNAs, particularly those encoded in the ef0408-0409 region. The ef0408-0409 sRNA appears to regulate argS expression through direct binding to the 5' UTR of the argS mRNA .

Electrophoretic Mobility Shift Assay (EMSA) Optimization:
For confirming direct RNA-RNA interactions between argS mRNA and regulatory sRNAs:

• Use 5% native polyacrylamide gels with 0.5× TBE buffer

• Include 10 mM MgCl2 in binding buffer to stabilize RNA structures

• Maintain temperature at 37°C during binding to mimic physiological conditions

• Include yeast tRNA (1 μg/μL) as a nonspecific competitor

• Use both 5' end-labeled and body-labeled RNA probes to detect different binding modes

Mutational Analysis Strategy:
To map interaction sites between argS mRNA and regulatory sRNAs:

• Perform computational prediction of potential interaction sites

• Generate series of 5-nucleotide mutants across predicted binding regions

• Test mutants in both EMSA and reporter gene assays

• Confirm with compensatory mutations that restore base-pairing

This combined approach has revealed that the ef0408-0409 sRNA binds to a sequence approximately 30-45 nucleotides upstream of the argS start codon, affecting translation efficiency rather than mRNA stability .

How might E. faecalis argS serve as a target for novel antimicrobial development?

The unique characteristics of E. faecalis argS present several promising avenues for antimicrobial development:

Target Validation Status:
E. faecalis argS has been validated as an essential gene through multiple approaches:

• Conditional knockdown strains show growth arrest when argS expression is repressed

• Transposon mutagenesis libraries show no viable insertions in critical argS domains

• Chemical inhibition of argS using rationally designed inhibitors shows bactericidal effects

Drug Discovery Approaches:

• Structure-Based Design: Crystal structures of E. faecalis argS have enabled computational docking of over 500,000 compounds, identifying three scaffolds with selective binding to bacterial over human enzyme.

• Fragment-Based Screening: NMR-based fragment screening has identified small molecules that bind to the ATP-binding pocket with millimolar affinity, providing starting points for medicinal chemistry optimization.

• Natural Product Derivatives: Several aminoquinolone alkaloids show selective inhibition of bacterial argS with IC50 values of 15-45 μM.

Preliminary Inhibitor Data:

Resistance Development Concerns:
Resistance studies suggest a relatively high barrier to resistance development:

• Serial passage experiments (30 days) failed to generate high-level resistance

• Observed mutations confer only 2-4 fold increases in MIC

• Resistant variants show significant fitness costs (30-45% reduction in growth rate)

These characteristics, combined with the structural differences from the human homolog, position E. faecalis argS as a promising antimicrobial target, particularly for treating multi-drug resistant infections where current therapies are failing .

What is the relationship between argS expression and small RNA regulation in E. faecalis pathogenicity?

The complex interplay between argS expression and small RNA regulation represents a frontier in understanding E. faecalis pathogenicity:

Regulatory Network Complexity:
Research has uncovered a sophisticated regulatory network where multiple small RNAs influence argS expression under different stress conditions. Of particular interest is the relationship between argS and the ef0408-0409 sRNA. Deletion of this sRNA results in a hypervirulent phenotype with enhanced colonization capabilities, suggesting it normally serves to repress certain virulence mechanisms. Proteomic studies revealed that this deletion affects the expression of 23 proteins, including argS, whose levels increased 1.8-fold in the mutant .

Stress-Dependent Regulation:
The interaction between argS and regulatory sRNAs appears to be stress-dependent:

• Under oxidative stress: ef0408-0409 sRNA levels decrease, allowing increased argS expression

• During amino acid limitation: A different set of sRNAs (ef1368-1369) regulate argS

• In biofilm formation: ef3314-3315 sRNA appears to coordinate argS expression with adhesin production

Virulence Implications in Infection Models:
Experimental infection models reveal that precisely controlled argS expression is critical for pathogenicity:

• Too little argS expression: Impaired growth and reduced virulence

• Too much argS expression: Enhanced initial virulence but reduced persistence

• Disrupted sRNA regulation: Altered infection dynamics and tissue tropism

Future Research Priorities:

• Detailed mapping of the complete sRNA regulatory network affecting argS

• Development of sRNA-targeting antisense oligonucleotides as potential therapeutics

• Investigation of argS regulatory mechanisms in clinical isolates from different infection sites

• Exploration of argS-sRNA interactions as biomarkers for virulence potential

This research direction holds promise for developing innovative therapeutic approaches that disrupt specific regulatory pathways rather than targeting essential bacterial functions directly, potentially reducing selective pressure for resistance development .

What are the best practices for working with recombinant E. faecalis argS in research applications?

Based on collective research experience, the following best practices are recommended for researchers working with recombinant E. faecalis argS:

Expression and Purification:

• Use the pET28a-SUMO vector system in BL21(DE3) E. coli for optimal expression

• Express at 18°C for 16-18 hours after induction with 0.5 mM IPTG

• Include 10% glycerol and 5 mM MgCl2 in all purification buffers

• Perform three-step purification: IMAC, ion exchange, and size exclusion chromatography

• Store purified enzyme at -80°C in single-use aliquots with 20% glycerol

Activity Assays:

• For routine activity checks: Use the malachite green-based pyrophosphate release assay

• For detailed kinetic studies: Use the aminoacylation assay with purified tRNAArg

• Always include positive controls (commercial aminoacyl-tRNA synthetases) and appropriate blanks

• Perform assays at 37°C to mimic physiological conditions

Structural Studies:

• Protein crystals form readily in 0.1 M HEPES pH 7.5, 10% PEG 8000, 8% ethylene glycol

• For NMR studies, uniform 15N-labeling can be achieved using M9 minimal medium with 15NH4Cl

• For cryo-EM, the addition of 0.05% CHAPS improves particle distribution

Common Pitfalls to Avoid:

• Prolonged storage at 4°C leads to significant activity loss (>50% after 72 hours)

• Multiple freeze-thaw cycles severely diminish activity

• Omitting reducing agents results in oxidation-induced aggregation

• Using imidazole concentrations >20 mM during binding to Ni-NTA causes premature elution

• Concentrating above 5 mg/mL without stabilizing additives triggers precipitation

By following these guidelines, researchers can achieve consistent results when working with this challenging but important enzymatic system .

How can argS research contribute to understanding E. faecalis adaptation mechanisms?

Research on E. faecalis argS provides valuable insights into bacterial adaptation strategies:

Integrative Understanding:
Studies of argS regulation and function have revealed how E. faecalis integrates multiple stress signals to coordinate appropriate responses. The enzyme serves as both a central player in protein synthesis and a regulatory node connecting nutrient availability, stress response, and virulence expression. This dual functionality allows E. faecalis to rapidly adapt to changing environments, particularly during the transition from commensal to pathogenic lifestyle .

Clinical Relevance:
The adaptive mechanisms uncovered through argS research have direct clinical implications:

• Antibiotic stress triggers specific argS regulatory changes that contribute to persistence

• Biofilm formation correlates with altered argS expression patterns

• Urinary tract adaptation involves specific argS-related gene expression changes

Comparative genomics analysis of clinical isolates has identified 19 candidate genes involved in E. faecalis adaptation to the urinary tract, several of which interact with argS-regulated pathways. These genes participate in core processes including sugar transport, cobalamin import, glucose metabolism, and post-transcriptional regulation of gene expression .

Translational Applications:
The knowledge gained from argS research is being applied in several areas:

• Development of diagnostic tools to predict virulence potential of clinical isolates

• Identification of novel drug targets at the intersection of stress response and virulence

• Design of combination therapies that target both growth and adaptation mechanisms

• Creation of attenuated strains for potential vaccine development

By continuing to investigate the multifaceted roles of argS in E. faecalis biology, researchers can develop more effective strategies for combating this increasingly problematic opportunistic pathogen, particularly in the context of rising antibiotic resistance and healthcare-associated infections .