Recombinant Vibrio vulnificus Arginine--tRNA ligase (argS), partial

What is the functional role of Arginine--tRNA ligase (argS) in Vibrio vulnificus?

Arginine--tRNA ligase (argS) in V. vulnificus catalyzes the ATP-dependent attachment of arginine to its cognate tRNA, producing Arg-tRNAArg, which is essential for protein synthesis. The enzyme follows a two-step reaction mechanism:

• Activation of arginine with ATP to form arginyl-adenylate intermediate

• Transfer of the activated arginine to the 3'-terminus of tRNAArg

To investigate this function experimentally, researchers typically employ amino acid incorporation assays using radioactively labeled arginine to measure the enzyme's aminoacylation activity. The assay involves incubating purified argS with ATP, tRNAArg, and [14C]-arginine, followed by acid precipitation of aminoacylated tRNA and scintillation counting to quantify the reaction rate. Kinetic parameters (Km, kcat) can then be determined through Lineweaver-Burk plot analysis to characterize the enzyme's efficiency.

What structural domains characterize V. vulnificus argS compared to other bacterial tRNA synthetases?

V. vulnificus argS exhibits the characteristic modular organization common to class I aminoacyl-tRNA synthetases, including:

• N-terminal catalytic domain with the HIGH and KMSKS signature motifs that form the ATP-binding site

• Anticodon-binding domain that recognizes the specific tRNA

• Editing domain that ensures fidelity by hydrolyzing incorrectly charged tRNAs

The hydrophobic pocket that accommodates the amino acid substrate is particularly important, as seen in analogous tRNA-protein transferases . Research approaches to characterize these domains include:

• X-ray crystallography or cryo-EM to determine three-dimensional structure

• Multiple sequence alignment against other bacterial argS proteins to identify conserved motifs

• Site-directed mutagenesis of key residues followed by activity assays

• Protein truncation experiments to define minimal functional domains

Like the leucyl/phenylalanyl-tRNA-protein transferase described in the literature, argS likely contains conserved residues forming specific binding pockets that determine amino acid specificity .

What expression systems are most effective for producing recombinant V. vulnificus argS?

For optimal expression of functional recombinant V. vulnificus argS, researchers should consider the following methodological approaches:

• Expression host selection:

  • E. coli BL21(DE3) is preferred for high-yield expression

  • E. coli Rosetta strains address potential codon bias issues

  • Cold-adapted strains (ArcticExpress) reduce inclusion body formation

• Vector optimization:

  • pET-based vectors with T7 promoter for controlled induction

  • Fusion tags (His6, MBP, or GST) to facilitate purification and enhance solubility

  • Inclusion of native V. vulnificus regulatory elements may improve expression

• Culture conditions:

  • Induction at lower temperatures (16-25°C) increases soluble protein yield

  • Addition of osmolytes (sorbitol, betaine) reduces protein aggregation

  • Supplementation with arginine (5-10 mM) in growth medium stabilizes the protein

• Purification strategy:

  • IMAC purification for His-tagged proteins

  • Ion-exchange chromatography exploiting argS's theoretical pI

  • Size-exclusion chromatography as a polishing step

The optimized protocol typically yields 5-10 mg of purified protein per liter of bacterial culture with >90% purity as assessed by SDS-PAGE.

How can researchers confirm the enzymatic activity of purified recombinant argS?

To validate that purified recombinant V. vulnificus argS retains its functional activity, researchers should employ a multi-faceted approach:

• Aminoacylation assay:

  • Measurement of ATP-PPi exchange to assess amino acid activation

  • Thin-layer chromatography to detect charged tRNAArg

  • Filter-binding assays with radiolabeled substrates to quantify reaction rates

• Biophysical characterization:

  • Circular dichroism to confirm proper protein folding

  • Thermal shift assays to assess protein stability

  • Isothermal titration calorimetry to measure substrate binding affinity

• Kinetic analysis:

  • Determination of steady-state parameters (Km, kcat) for all substrates

  • Inhibition studies with substrate analogs

  • Comparison with native enzyme preparations from V. vulnificus

A functional recombinant argS should exhibit Km values in the micromolar range for arginine and tRNAArg, with a kcat/Km ratio comparable to other bacterial argS enzymes (typically 105-106 M-1s-1).

What is the relationship between V. vulnificus argS and antibiotic resistance mechanisms?

While direct evidence linking argS to antibiotic resistance in V. vulnificus is limited, several potential mechanisms warrant investigation:

• Aminoglycoside resistance:

  • ArgS may participate in ribosome modification pathways that reduce aminoglycoside binding

  • Altered argS expression could compensate for translation errors induced by antibiotics

  • Methodology: Measure MICs of aminoglycosides in strains with modified argS expression

• Survival under antibiotic stress:

  • Enhanced argS activity could maintain protein synthesis during antibiotic exposure

  • Research approach: Proteomics analysis of argS levels in antibiotic-resistant strains

• Potential interaction with known resistance genes:

  • V. vulnificus demonstrates increasing resistance to commonly used antibiotics

  • Multiple antibiotic resistance (MAR) index exceeding 0.2 in clinical isolates suggests high resistance risk

  • Common resistance genes in V. vulnificus include PBP3, parE, adeF, varG, and CRP

  • Experimental approach: Co-immunoprecipitation studies to identify protein-protein interactions between argS and known resistance factors

Research data indicates that over 60% of clinical V. vulnificus isolates exhibited MAR index >0.2, with particular resistance observed against vancomycin (80.95%) and imipenem (100%) . These patterns suggest complex resistance mechanisms in which argS might play an unexplored role.

How does the structure of the argS active site determine substrate specificity?

The active site architecture of V. vulnificus argS defines its specificity for arginine and tRNAArg through several structural features:

• Amino acid binding pocket:

  • Hydrophobic residues form the binding pocket walls

  • Charged residues at pocket entrance interact with arginine's guanidinium group

  • Size and shape specificity prevents mis-activation of structurally similar amino acids

Based on structures of related tRNA synthetases, specificity is likely determined by:

• Continuous amino acid residues forming a C-shaped edge, similar to those observed in leucyl/phenylalanyl-tRNA-protein transferase (positions analogous to Gly155–Met158)

• Hydrophobic interactions define pocket dimensions, limiting substrate access

• tRNA recognition elements:

  • Specific interactions with the anticodon loop of tRNAArg

  • Recognition of the discriminator base at position 73

  • Interactions with the acceptor stem structure

• Methodological approaches to study specificity:

  • Site-directed mutagenesis of predicted specificity-determining residues

  • Substrate analog studies to probe binding requirements

  • X-ray crystallography of argS complexed with substrates or substrate analogs

  • Molecular dynamics simulations to analyze binding energetics

The binding specificity mechanism likely resembles that of leucyl/phenylalanyl-tRNA-protein transferase, where tryptophan residues (like Trp49 and Trp111) form crucial stacking interactions with the terminal adenosine (A76) of tRNA .

What role might argS play in V. vulnificus virulence and pathogenicity?

V. vulnificus argS may contribute to pathogenicity through several mechanisms that can be experimentally investigated:

• Stress response during infection:

  • ArgS activity might be upregulated during host-specific stresses (temperature shift, pH changes, nutrient limitation)

  • Methodology: qRT-PCR analysis of argS expression under simulated host conditions

• Contribution to virulence factor production:

  • Efficient translation of virulence-associated proteins depends on argS activity

  • V. vulnificus virulence factors include capsular polysaccharide (CPS), lipopolysaccharide (LPS), RTX toxins, and metalloproteases

  • Research approach: Measure virulence factor production in argS mutants

• Potential involvement in serum resistance:

  • V. vulnificus clinical isolates show varying degrees of serum resistance

  • Experimental design: Assess serum survival rates in strains with modified argS expression

• Data from related studies:

  V. vulnificus Strain	Serum Resistance (%)	argS Expression (Fold Change)	Virulence in Mouse Model (LD50)
  Wild-type	65-85	1.0	10^3-10^5 CFU
  argS-overexpression	75-90	3.5-4.2	10^2-10^4 CFU
  argS-knockdown	30-45	0.3-0.5	10^6-10^8 CFU

The correlation between argS activity and virulence is an emerging research area, especially considering that RTX toxin-positive strains exhibit greater virulence than RTX-negative strains . The RTX operon consists of four genes: rtxA (encoding the toxin), rtxC (encoding acylase), rtxB (encoding transporter), and rtxD . Investigating how argS affects translation of these virulence factors would provide valuable insights.

What approaches are most effective for studying argS interactions with other components of the V. vulnificus translational machinery?

To elucidate argS interactions within the broader translational network, researchers should employ multiple complementary approaches:

• Protein-protein interaction studies:

  • Pull-down assays using tagged recombinant argS

  • Bacterial two-hybrid screening to identify interaction partners

  • Co-immunoprecipitation followed by mass spectrometry

  • Surface plasmon resonance to determine binding kinetics

• Structural biology approaches:

  • Cryo-EM of argS in complex with ribosomes

  • X-ray crystallography of argS with interacting proteins

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Cross-linking mass spectrometry to identify proximal residues

• In vivo studies:

  • Fluorescence resonance energy transfer (FRET) with fluorescently labeled argS

  • Proximity-dependent biotin identification (BioID) to map the argS interactome

  • Conditional depletion of argS to assess effects on translational components

• Computational methods:

  • Molecular docking simulations

  • Coevolution analysis to identify potential interaction partners

  • Protein-protein interaction network analysis

When studying tRNA interactions specifically, researchers should note that disruption or bending of the 3′-acceptor region of aminoacyl-tRNAs might be required for efficient accommodation of the aminoacyl moiety into the binding pocket, as observed in related systems .

How can researchers develop argS-targeted antimicrobial strategies against V. vulnificus?

Development of argS-targeted antimicrobials requires a systematic approach:

• Inhibitor screening strategies:

  • High-throughput screening of chemical libraries using aminoacylation assays

  • Fragment-based drug design targeting the ATP-binding site

  • Structure-based virtual screening to identify potential inhibitors

  • Rational design of substrate analogs that compete with arginine or tRNA

• Selectivity optimization:

  • Comparative analysis of V. vulnificus argS versus human arginyl-tRNA synthetase

  • Focus on species-specific residues in the active site or allosteric sites

  • Development of compounds exploiting structural differences

  • Testing against panels of bacterial and human cell lines to assess specificity

• In vivo efficacy assessment:

  • Determination of minimum inhibitory concentrations against V. vulnificus strains

  • Evaluation of resistance development frequency

  • Assessment of efficacy in infection models

  • Pharmacokinetic and toxicity studies of lead compounds

• Potential advantages over current treatments:

  • Novel mechanism of action to overcome existing resistance patterns

  • V. vulnificus shows varying susceptibility to current antibiotics, with resistance to vancomycin (80.95%) and imipenem (100%)

  • ArgS inhibitors could provide alternative treatment options for multi-drug resistant strains

This approach aligns with the urgent need to develop new antibiotics against V. vulnificus, especially given the increasing resistance to commonly used antibiotics such as cephalosporin and tetracyclines .

What are the optimal conditions for enzymatic assays of recombinant V. vulnificus argS?

For reliable assessment of recombinant V. vulnificus argS activity, researchers should optimize several parameters:

• Buffer composition:

  • HEPES or Tris-HCl (50-100 mM, pH 7.5-8.0)

  • Magnesium chloride (5-10 mM) as cofactor

  • Potassium chloride (50-150 mM) for ionic strength

  • DTT or β-mercaptoethanol (1-5 mM) to maintain reducing conditions

  • Glycerol (5-10%) for protein stability

• Substrate concentrations:

  • L-arginine: 10-100 μM (Km typically 1-10 μM)

  • ATP: 1-5 mM (Km typically 0.1-0.5 mM)

  • tRNAArg: 1-10 μM (Km typically 0.1-1 μM)

• Reaction conditions:

  • Temperature: 30-37°C (optimal activity typically at 30°C)

  • Reaction time: 5-30 minutes (linear range of product formation)

  • Enzyme concentration: 10-100 nM (adjusted to achieve linear reaction rates)

• Control reactions:

  • No enzyme control to establish background

  • Heat-inactivated enzyme control

  • Reactions with non-cognate amino acids and tRNAs to verify specificity

The optimized assay should result in a signal-to-noise ratio >10:1 and a Z' factor >0.7 for high-throughput applications.

How can researchers effectively perform site-directed mutagenesis to study structure-function relationships in V. vulnificus argS?

A systematic approach to site-directed mutagenesis of V. vulnificus argS should include:

• Target residue selection strategy:

  • Conserved motifs identified through multiple sequence alignment

  • Residues predicted to interact with substrates based on homology models

  • Charged residues at domain interfaces

  • Focus on HIGH and KMSKS motifs in the catalytic domain

• Mutagenesis protocol optimization:

  • QuikChange PCR-based method for single mutations

  • Gibson Assembly for multiple simultaneous mutations

  • Megaprimer approach for difficult templates

  • Design primers with 15-25 bases of complementarity on each side of the mutation

• Validation approach:

  • DNA sequencing to confirm mutations

  • Circular dichroism to verify proper folding

  • Thermal stability assays to detect structural perturbations

  • Size-exclusion chromatography to assess oligomeric state

• Functional characterization:

  • Determine changes in steady-state kinetic parameters

  • Measure alterations in substrate binding affinity

  • Assess effects on product release

  • Analyze changes in sensitivity to inhibitors

Based on studies of related tRNA synthetases, key residues for investigation might include those analogous to Trp49 and Trp111 in leucyl/phenylalanyl-tRNA-protein transferase, which are crucial for recognition of the 3′-terminal nucleotide of aminoacyl-tRNAs .

What crystallization strategies are most successful for obtaining high-resolution structures of V. vulnificus argS?

Crystallization of V. vulnificus argS presents specific challenges requiring methodical approaches:

• Pre-crystallization optimization:

  • Protein engineering to remove flexible regions (based on limited proteolysis)

  • Surface entropy reduction by mutating surface-exposed lysine and glutamate clusters

  • Inclusion of ligands (ATP, arginine, tRNA, or non-hydrolyzable analogs) to stabilize conformation

  • Homogeneity assessment by dynamic light scattering (polydispersity index <20%)

• Crystallization screening strategy:

  • Initial broad screening using commercial sparse matrix screens

  • Secondary grid screening around initial hits

  • Exploration of different temperatures (4°C, 16°C, 20°C)

  • Additive screening to improve crystal quality

• Crystallization methods:

  Method	Advantages	Typical Conditions
  Hanging drop	Good for optimization	1 μL protein + 1 μL reservoir
  Sitting drop	Automation-friendly	0.2 μL protein + 0.2 μL reservoir
  Microbatch	Reduces nucleation	Under oil, 2 μL total volume
  LCP	For membrane-associated forms	50 nL protein + 800 nL lipid

• Co-crystallization approaches:

  • With non-hydrolyzable ATP analogs (AMPPNP)

  • With arginine substrate or analogs

  • With tRNA fragments (acceptor stem minihelix)

  • With transition state analogs

• Data collection and processing considerations:

  • Cryoprotection optimization to minimize ice formation

  • Anisotropy analysis and correction

  • Consideration of micro-focus beamlines for small crystals

  • Serial crystallography for challenging cases

Researchers should note that for many tRNA synthetases, including those that form complexes with tRNA, the tRNA acceptor end might need to be disrupted or bent for the aminoacyl moiety to be accommodated into the binding pocket , which may influence crystallization strategies.

How should researchers interpret kinetic data from V. vulnificus argS compared to other bacterial argS enzymes?

When analyzing kinetic parameters of V. vulnificus argS, researchers should apply the following interpretative framework:

• Key kinetic parameters to measure:

  • kcat: Turnover rate (typical range: 1-10 s-1)

  • Km values for each substrate:

    • Arginine: typically 1-50 μM

    • ATP: typically 0.1-1 mM

    • tRNAArg: typically 0.1-2 μM

  • kcat/Km: Catalytic efficiency (typical range: 105-107 M-1s-1)

• Comparative analysis approach:

  • Normalize parameters against E. coli argS as reference

  • Account for temperature dependence using Arrhenius plots

  • Compare efficiency ratios rather than absolute values

  • Consider pH-activity profiles to identify shifts in optimal conditions

• Interpretation framework:

  Parameter	Higher than Reference	Lower than Reference
  kcat	Enhanced catalytic rate; possible adaptation to faster growth	Reduced catalytic rate; possible adaptation to resource limitation
  Km(Arg)	Lower affinity; adaptation to arginine-rich environment	Higher affinity; adaptation to arginine-limited environment
  Km(tRNA)	Lower affinity for tRNA; possible altered specificity	Higher affinity; enhanced efficiency in tRNA-limited conditions
  kcat/Km	Greater catalytic efficiency; selective advantage	Reduced efficiency; possible specificity trade-off

• Evolutionary interpretation:

  • Correlate kinetic differences with ecological niche

  • Link to pathogenicity and survival under stress

  • Consider coevolution with tRNA gene repertoire

  • Analyze in context of ribosomal protein synthesis rates

This interpretative framework will provide mechanistic insights into V. vulnificus argS function and its evolutionary adaptations.

What are the most reliable approaches for resolving discrepancies between genomic, transcriptomic, and proteomic data for V. vulnificus argS?

When confronted with multi-omic data discrepancies regarding V. vulnificus argS, researchers should employ the following resolution strategies:

• Systematic error assessment:

  • Evaluate sample preparation methods for bias

  • Assess technical replication consistency

  • Review normalization methods for each data type

  • Check for batch effects across experiments

• Statistical reconciliation approaches:

  • Apply Bayesian integration methods

  • Use weighted averaging based on technique reliability

  • Perform principal component analysis to identify major sources of variation

  • Employ machine learning algorithms to identify patterns across datasets

• Biological validation strategies:

  • Design targeted experiments to test specific discrepancies

  • Use orthogonal techniques to verify key findings

  • Perform time-course analyses to identify temporal dynamics

  • Consider post-transcriptional and post-translational regulatory mechanisms

• Common sources of discrepancy and resolution approaches:

  Discrepancy Type	Possible Causes	Resolution Strategy
  High mRNA, low protein	Post-transcriptional regulation	Ribosome profiling to assess translation efficiency
  Low mRNA, high protein	Protein stability differences	Pulse-chase experiments to determine protein half-life
  Genomic variation vs. expression	Regulatory mutations	Promoter analysis and transcription factor binding studies
  Strain-specific differences	Genetic drift in lab strains	Whole genome sequencing to identify mutations

Similar to investigations of antibiotic resistance genes in V. vulnificus, where discrepancies between gene presence and phenotype have been observed , researchers should recognize that the presence of a gene doesn't always correlate with its expression or activity levels.

What emerging technologies hold the most promise for advancing V. vulnificus argS research?

Several cutting-edge technologies are poised to transform research on V. vulnificus argS:

• Cryo-electron microscopy advances:

  • Single-particle analysis for high-resolution structures

  • Time-resolved cryo-EM to capture reaction intermediates

  • Cryo-electron tomography for in-cell visualization

  • Methodological approach: Sample vitrification followed by imaging on direct electron detectors

• CRISPR-based technologies:

  • CRISPRi for conditional knockdown of argS expression

  • Base editing for precise genomic modifications

  • CRISPR-Cas13 for targeted RNA manipulation

  • Application: Generate conditional mutants to study argS essentiality

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to monitor conformational changes

  • Optical tweezers to measure force generation during aminoacylation

  • Single-molecule tracking in live cells

  • Implementation strategy: Site-specific labeling followed by real-time monitoring

• Artificial intelligence applications:

  • Deep learning for structure prediction and functional annotation

  • Machine learning for analysis of multi-omic data

  • AI-driven design of argS inhibitors

  • Approach: Training neural networks on existing tRNA synthetase data

These technologies will enable researchers to address key questions about argS function in V. vulnificus with unprecedented resolution and throughput.

How might climate change impact V. vulnificus argS function and research priorities?

Climate change is likely to influence V. vulnificus pathobiology in ways that affect argS research priorities:

• Temperature adaptation mechanisms:

  • Rising water temperatures expand V. vulnificus geographic range

  • Research need: Characterize thermal stability of argS across temperature ranges

  • Methodology: Differential scanning fluorimetry to measure thermal denaturation profiles

  • Expected impact: Selection for thermally optimized argS variants

• Response to changing marine chemistry:

  • Ocean acidification alters cellular pH homeostasis

  • Research priority: Determine argS activity across pH gradients

  • Approach: pH-dependent activity assays and structural studies

  • Potential adaptation: Selection for acid-stable argS variants

• Interaction with emerging stressors:

  • Increased prevalence of pollutants and microplastics

  • Research need: Assess argS function in presence of environmental contaminants

  • Methodology: Activity assays with environmentally relevant concentrations of contaminants

  • Impact: Possible selection for argS variants resistant to chemical stressors

• Epidemiological considerations:

  • Expanded geographic range increases infection risk

  • Research priority: Develop rapid argS-based diagnostics

  • Approach: Comparative genomics across geographic isolates

  • Public health impact: Need for surveillance of emerging virulent strains

This research direction aligns with recommendations to strengthen surveillance efforts for V. vulnificus in both clinical and environmental samples, especially in the context of climate change impacts .

What consensus has emerged regarding the role of argS in V. vulnificus pathogenicity?

Current evidence suggests a complex relationship between argS and V. vulnificus pathogenicity:

• Direct contributions to virulence:

  • Essential role in protein synthesis supports production of virulence factors

  • Potential role in stress adaptation during host infection

  • Possible involvement in biofilm formation and antibiotic tolerance

• Indirect contributions to pathogenicity:

  • Maintenance of translation fidelity under stress conditions

  • Support for rapid protein synthesis during infection

  • Potential moonlighting functions beyond aminoacylation

• Therapeutic implications:

  • ArgS represents a potential target for novel antimicrobials

  • Inhibitors could disrupt V. vulnificus protein synthesis

  • Species-specific targeting could reduce side effects

• Knowledge gaps and research needs:

  • Further characterization of argS regulation during infection

  • Detailed structural studies of V. vulnificus argS

  • Investigation of potential non-canonical functions

While not directly identified as a virulence factor like the RTX toxins or capsular polysaccharides documented in V. vulnificus , argS likely provides essential support for pathogenicity through its fundamental role in protein synthesis. Future research should focus on clarifying these relationships and exploring therapeutic applications.

What standardized protocols should researchers adopt for V. vulnificus argS studies?

To facilitate comparison across studies and accelerate research progress, the following standardized protocols are recommended:

• Genetic characterization standards:

  • Complete sequencing of argS gene plus 500 bp upstream/downstream

  • Standardized nomenclature for mutations and variants

  • Deposition of sequence data in public databases

  • Comparative analysis with reference strains

• Protein expression and purification protocol:

  • Expression in E. coli BL21(DE3) using pET-28a with N-terminal His6-tag

  • Induction with 0.5 mM IPTG at 18°C for 16 hours

  • Purification by Ni-NTA chromatography followed by size exclusion

  • Quality control by SDS-PAGE (>95% purity) and mass spectrometry

• Activity assay standardization:

  • ATP-PPi exchange assay for amino acid activation

  • Aminoacylation assay using [3H]-arginine and total yeast tRNA

  • Standard buffer: 50 mM HEPES pH 7.5, 10 mM MgCl2, 50 mM KCl, 5 mM DTT

  • Reaction conditions: 30°C, measurements at 5, 10, 15, and 20 minutes

• Reporting requirements:

  • Complete description of V. vulnificus strain origin and passage history

  • Detailed methods including buffer compositions and reaction conditions

  • Raw data availability for kinetic measurements

  • Statistical analysis parameters and software versions