Recombinant Vibrio vulnificus Cysteine--tRNA ligase (cysS)

What is Vibrio vulnificus Cysteine-tRNA ligase and what role does it play in bacterial physiology?

Vibrio vulnificus Cysteine-tRNA ligase (CysS) is an aminoacyl-tRNA synthetase that catalyzes the attachment of cysteine to its cognate tRNA during protein synthesis. Beyond this canonical function, research indicates that cysteinyl-tRNA synthetases (CARSs) have non-canonical roles in bacteria. Based on studies of CARSs in other organisms, these enzymes can catalyze the formation of cysteine hydropersulfide (CysSSH) from cysteine, acting as cysteine persulfide synthases (CPERSs) independent of their aminoacylation activity . This dual function positions CysS as both an essential component of the translation machinery and potentially a key player in redox regulation within V. vulnificus.

Methodologically, researchers distinguish between these functions through comparative enzyme kinetics studies using purified recombinant enzymes with modified active sites and experimental separation of tRNA binding activity from persulfide generation.

How does recombinant V. vulnificus CysS differ from other bacterial cysteinyl-tRNA synthetases in structure and function?

While specific structural information for V. vulnificus CysS is still emerging, studies of bacterial cysteinyl-tRNA synthetases reveal highly conserved motifs. Bacterial CARSs typically contain:

• A pyridoxal phosphate (PLP) binding domain with conserved Lys residues often in KIIK and KMSK motifs

• A zinc-binding domain with conserved cysteine residues

• An ATP-binding domain containing a HIGH motif

Functionally, V. vulnificus CysS likely shares the dual activity documented in other bacterial CARSs - both aminoacylation and cysteine persulfide generation. Research using recombinant EcCARS (from E. coli) demonstrates that these enzymes can generate cysteine polysulfides with high efficiency (kcat/Km values superior to other persulfide-generating enzymes like CSE) . Comparative analysis of motif conservation would be essential for predicting functional similarities specific to V. vulnificus.

What methods are most effective for expressing and purifying recombinant V. vulnificus CysS?

Effective expression and purification of recombinant V. vulnificus CysS involves:

Expression system selection:

• E. coli BL21(DE3) strain has been successfully used for expressing V. vulnificus recombinant proteins

• pET expression vectors (particularly pET21b) have shown efficacy for V. vulnificus proteins

Optimized purification protocol:

• His₆-tag fusion protein design (C-terminal tagging preferred to avoid interference with N-terminal domains)

• Metal affinity chromatography using Ni-NTA resin

• Size exclusion chromatography to ensure removal of aggregates

• Activity verification through aminoacylation assays

Critical considerations:

• Temperature optimization (typically 18-25°C for induction to enhance solubility)

• Inclusion of zinc in purification buffers (5-10 μM ZnCl₂) to maintain structural integrity

• Testing both native and denaturation-refolding approaches if inclusion bodies form

• Verifying both aminoacylation and potential persulfide generation activities separately

How can researchers assess the dual activities of V. vulnificus CysS?

Assessing both the canonical (aminoacylation) and non-canonical (persulfide synthesis) activities of V. vulnificus CysS requires distinct methodological approaches:

Aminoacylation activity assessment:

• ATP-PPi exchange assay measuring the reverse reaction

• Direct tRNA charging assays using radiolabeled cysteine

• PUNCH-PsP (Puromycin-Associated Nascent Chain Proteomics for Polysulfide Proteomics) to identify cysteine residues incorporated into nascent peptides

Persulfide generation activity:

• HPE-IAM (β-(4-hydroxyphenyl)ethyl iodoacetamide) labeling assay coupled with LC-MS/MS for detecting and quantifying CysSSH and longer chain polysulfides

• Stable isotope (³⁴S) tracer experiments to track sulfur transfer mechanisms

• PLP binding assessment through LC-ESI-MS/MS using 2,4-dinitrophenylhydrazine (DNPH)

$Data table showing relative activities of wild-type and mutant enzymes$

What role might V. vulnificus CysS play in bacterial stress response and virulence?

Based on research with related enzymes, V. vulnificus CysS likely contributes to stress response and potentially virulence through several mechanisms:

Oxidative stress response:
V. vulnificus employs multiple defensive systems against reactive oxygen species (ROS) during infection, including peroxiredoxins like Prx3 . CysS-generated cysteine persulfides (CysSSH) likely function as potent antioxidants with superior reducing capacity compared to cysteine thiols, potentially protecting the bacteria from host-generated oxidative bursts .

Connection to virulence mechanisms:

• Potential involvement in post-translational modification of virulence factors through cysteine polysulfidation

• Contribution to redox regulation affecting expression of virulence genes

• Possible role in bacterial survival within macrophages where oxidative stress is pronounced

Methodological approaches to study these connections:

• Targeted mutagenesis of key CysS residues affecting persulfide generation but not aminoacylation

• Transcriptomic and proteomic profiling under infection-mimicking conditions

• Comparative virulence assessment of V. vulnificus strains with modified CysS activity

How do mutations in key domains of V. vulnificus CysS affect its function?

Strategic mutations in V. vulnificus CysS can differentially affect its dual functions, providing insight into structure-function relationships:

PLP-binding domain mutations:
Based on studies of E. coli CARS, mutations in conserved lysine residues (such as those found in KIIK and KMSK motifs) significantly reduce persulfide generation activity while preserving aminoacylation function . This suggests that:

• K73A and K269A mutations (within KIIK and KMSK motifs) would reduce PLP binding

• Reduced PLP binding correlates directly with decreased persulfide generation

• These mutations create functional separation of the enzyme's dual roles

Zinc-binding domain mutations:
Mutations of cysteine residues in the zinc-binding domain (equivalent to C28 and C209 in E. coli CARS):

• Dramatically reduce aminoacylation activity (to ~18% of wild-type)

• Maintain near-normal persulfide generation activity (~93% of wild-type)

• Provide important tools for studying non-canonical functions in isolation

These mutational studies demonstrate how researchers can create variants of V. vulnificus CysS that selectively perform one function while being deficient in the other.

What bioinformatic approaches can be used to predict functional motifs in V. vulnificus CysS?

Comprehensive bioinformatic analysis of V. vulnificus CysS involves multiple complementary approaches:

Sequence-based analysis:

• Multiple sequence alignment (MSA) with CARSs from model organisms (E. coli, B. subtilis) and related Vibrio species

• Identification of conserved motifs including:

  • PLP-binding KIIK and KMSK motifs

  • Zinc-binding cysteine residues

  • HIGH motif for ATP binding

• Phylogenetic tree construction to position V. vulnificus CysS within evolutionary context

Structural prediction tools:

• Homology modeling using crystallized bacterial CARSs as templates

• Prediction of secondary structure elements and domain organization

• Molecular dynamics simulations to assess domain interactions and substrate binding

Functional annotation transfer:

• Identification of experimentally validated residues in homologous enzymes

• Prediction of substrate binding pockets and catalytic residues

• Analysis of protein-protein interaction networks to predict functional partners

This multilayered approach provides a framework for rational experimental design, particularly for site-directed mutagenesis and functional characterization studies.

How might V. vulnificus CysS contribute to the bacteria's adaptation to environmental stresses?

V. vulnificus CysS likely plays a critical role in adaptation to various environmental stresses through its dual functions:

Response to oxidative stress:
Cysteine persulfides generated by CysS can function as potent reducing agents, potentially protecting cellular components from oxidative damage. This may be particularly important in environments with fluctuating oxygen levels, such as the estuarine habitats where V. vulnificus naturally occurs .

Response to nitrosative stress:
Research on V. vulnificus has identified specialized proteins like Prx3 that are involved in nitric oxide (NO) detoxification . The cysteine persulfides generated by CysS potentially contribute to NO detoxification mechanisms, as these molecules have significantly higher reactivity toward reactive nitrogen species compared to simple thiols .

Methodological approaches for investigation:

• Growth assessment under varying oxidative and nitrosative stress conditions

• Transcriptional analysis of cysS expression under different environmental stresses

• Quantification of cellular polysulfide levels in response to stress conditions

• Creation of CysS variants with altered persulfide generation capability to test stress survival

What are the challenges in studying the non-canonical functions of V. vulnificus CysS?

Researchers face several significant challenges when investigating the non-canonical functions of V. vulnificus CysS:

Technical challenges:

• Distinguishing aminoacylation from persulfide generation activities requires specialized assays

• Detection and quantification of cellular persulfides demands sophisticated analytical techniques (LC-MS/MS)

• The high reactivity and short half-life of persulfides complicate their measurement in biological systems

Biological complexity:

• The essentiality of CysS for protein synthesis makes knockout studies non-viable

• Compensatory mechanisms may mask phenotypes in partial loss-of-function studies

• Persulfide biology involves complex interaction networks with multiple cellular systems

Methodological solutions:

• Development of domain-specific mutations that selectively affect one function

• Creation of conditionally active enzyme variants

• Application of chemical biology approaches using activity-based probes

• Time-resolved studies to capture transient persulfide formation events

• Orthogonal expression of heterologous aminoacyl-tRNA synthetases to complement essential functions

How does temperature affect the activity and stability of recombinant V. vulnificus CysS?

Temperature significantly influences both the activity and stability of recombinant V. vulnificus CysS, which has important implications for both experimental design and understanding the pathogen's biology:

Temperature effects on enzyme kinetics:
As a mesophilic pathogen capable of causing severe infections at human body temperature (37°C), V. vulnificus CysS likely exhibits optimal activity around this temperature. Studies would typically examine:

• Temperature-dependent changes in kcat and Km for both aminoacylation and persulfide generation

• Alterations in substrate specificity across temperature ranges (20-42°C)

• Differential temperature sensitivity of the dual enzymatic functions

Structural stability considerations:

• Thermal denaturation profiles (measured by circular dichroism or differential scanning calorimetry)

• Temperature-dependent aggregation behavior

• Impact of temperature on PLP binding and zinc coordination

Methodological approach for temperature studies:

• Activity assays conducted across a temperature gradient

• Time-course stability studies at different storage temperatures

• Assessment of refolding efficiency after thermal stress

• Comparison with CysS enzymes from related Vibrio species adapted to different temperature niches

Understanding these temperature relationships provides insight into how V. vulnificus CysS functions during host infection and environmental persistence.

What potential exists for V. vulnificus CysS as an antimicrobial drug target?

V. vulnificus CysS represents a potentially valuable antimicrobial drug target due to several favorable characteristics:

Target validation considerations:

• Essential role in protein synthesis makes it indispensable for bacterial survival

• Non-canonical persulfide generation function may contribute to virulence and stress resistance

• Structural differences from human cytosolic and mitochondrial CARSs could allow selective targeting

Drug development strategies:

• High-throughput screening for inhibitors of aminoacylation activity

• Structure-based design targeting the PLP-binding pocket (distinct from human enzymes)

• Allosteric inhibitors that prevent conformational changes required for activity

• Dual-action inhibitors affecting both canonical and non-canonical functions

Methodological approaches for inhibitor discovery:

• Enzyme-based screening assays measuring aminoacylation inhibition

• Secondary screens for persulfide generation inhibition

• Whole-cell assays to confirm antibacterial activity and cell penetration

• Molecular docking and in silico screening against modeled structures

Potential advantages over existing antibiotics:

• Novel target with no pre-existing resistance mechanisms

• Opportunity for narrow-spectrum activity against Vibrio species

• Possibility of attenuating virulence without direct bactericidal action, potentially reducing selection pressure

How can researchers effectively design site-directed mutagenesis studies for V. vulnificus CysS?

Designing effective site-directed mutagenesis studies for V. vulnificus CysS requires a systematic approach:

Target selection strategy:

• Identify conserved residues through multiple sequence alignment with characterized bacterial CARSs

• Prioritize residues in functional motifs:

  • PLP-binding sites (lysine residues in KIIK and KMSK motifs)

  • Zinc-coordination sites (conserved cysteine residues)

  • ATP-binding site (HIGH motif)

  • tRNA binding interface

• Consider surface residues that may participate in protein-protein interactions

Mutation design principles:

• Conservative substitutions to probe specific chemical properties:

  • Cysteine to serine (maintains size but changes reactivity)

  • Lysine to arginine (maintains charge but alters geometry)

• Non-conservative substitutions to abolish function:

  • Lysine to alanine (eliminates side chain functionality)

  • Cysteine to aspartate (introduces negative charge)

• Introduction of non-canonical amino acids for specialized biophysical studies

Experimental validation workflow:

• Express and purify mutant proteins under identical conditions

• Assess structural integrity through circular dichroism and thermal stability

• Measure both aminoacylation and persulfide generation activities

• Evaluate PLP binding capacity through spectroscopic methods

• Test complementation capability in conditional expression systems

This methodical approach enables systematic structure-function analysis while minimizing confounding effects from protein misfolding.

What insights can proteomic approaches provide about V. vulnificus CysS function?

Proteomics offers powerful tools for understanding both the function of V. vulnificus CysS and its broader cellular impact:

Interactome analysis:

• Affinity purification coupled with mass spectrometry (AP-MS) to identify CysS binding partners

• Proximity-dependent biotin identification (BioID) to map spatial relationships

• Cross-linking mass spectrometry (XL-MS) to characterize transient interactions

• Two-hybrid screening to detect binary protein interactions

Post-translational modifications (PTMs):

• Global profiling of cysteine persulfidation using selective chemical tagging

• Quantification of changes in persulfidation patterns under stress conditions

• Identification of CysS itself as a target for regulatory PTMs

• Investigation of connections between persulfidation and other redox PTMs

Global impacts on the proteome:

• Quantitative proteomics comparing wild-type to CysS variant-expressing strains

• Pulse-SILAC approaches to measure protein synthesis rates

• Ribosome profiling to assess translational efficiency of cysteine-rich proteins

• Organelle-specific proteomics to track compartmentalization of effects

Proteomic data can reveal unexpected connections between CysS activity and cellular processes beyond translation, particularly related to stress response and virulence regulation.

How might V. vulnificus CysS contribute to antibiotic resistance mechanisms?

V. vulnificus CysS may contribute to antibiotic resistance through several mechanisms related to its dual functions:

Direct contributions through persulfide biology:

• Cysteine persulfides generated by CysS serve as potent antioxidants that could protect against antibiotics that induce oxidative stress

• Protein persulfidation may modify targets of certain antibiotics, potentially reducing their binding efficiency

• Enhanced redox buffering capacity could mitigate the effects of redox-cycling antibiotics

Indirect contributions through translation modulation:

• Alterations in translation fidelity under stress could affect expression of resistance determinants

• Modified charging of tRNA^Cys could influence codon usage bias during antibiotic stress

• Potential role in stress-induced persister cell formation through translational pausing

Connections to known V. vulnificus resistance mechanisms:
Research shows that V. vulnificus has developed resistance to certain antibiotics including ampicillin, amoxicillin, carbenicillin, and some cephalosporins . The study of CysS could reveal whether its activity contributes to these established resistance patterns.

Methodological approaches to investigate these connections:

• Determination of minimum inhibitory concentrations (MICs) in strains with altered CysS activity

• Quantification of persulfide levels in response to antibiotic exposure

• Transcriptome and proteome analysis under antibiotic stress with focus on CysS-dependent changes

• Time-kill kinetics studies to assess survival dynamics during antibiotic treatment

What research gaps remain in our understanding of V. vulnificus CysS?

Despite advances in understanding aminoacyl-tRNA synthetases and their non-canonical functions, significant knowledge gaps remain regarding V. vulnificus CysS:

Structural characterization:

• No crystal structure of V. vulnificus CysS has been reported

• The specific arrangement of PLP and zinc binding sites remains theoretical

• Conformational changes during catalysis are not fully characterized

Regulatory mechanisms:

• Transcriptional regulation of the cysS gene in V. vulnificus is poorly understood

• Post-translational modifications that might regulate CysS activity are largely unexplored

• Potential regulation by environmental signals relevant to V. vulnificus lifecycle

Functional integration:

• The full spectrum of proteins modified by CysS-generated persulfides is unknown

• Connection between CysS activity and other virulence factors (MARTX toxin, metalloproteases) remains to be established

• Role in specific infection stages has not been systematically investigated

Methodological challenges:

• Need for improved detection methods for protein persulfidation in living cells

• Development of conditional expression systems compatible with V. vulnificus

• Creation of bioorthogonal tools to track CysS activity in real-time