Recombinant Vibrio vulnificus Valine--tRNA ligase (valS), partial

What is Valine--tRNA ligase (valS) and what is its function in Vibrio vulnificus?

Valine--tRNA ligase (EC 6.1.1.9), also known as Valyl-tRNA synthetase (ValRS), is an aminoacyl-tRNA synthetase that catalyzes the attachment of valine to its cognate tRNA molecule. This enzyme is essential for protein translation, ensuring the correct incorporation of valine into nascent polypeptide chains according to the genetic code. In V. vulnificus, valS functions similarly to other bacterial valS proteins but may have unique structural features that contribute to pathogen-specific translation dynamics .

How does the structure of V. vulnificus valS compare to other bacterial tRNA synthetases?

While the specific crystal structure of V. vulnificus valS has not been fully characterized in the provided search results, comparative analysis with other Vibrio species suggests conserved domains typical of Class I aminoacyl-tRNA synthetases. These include the catalytic domain containing the HIGH and KMSKS motifs essential for ATP binding and aminoacylation activity. Unlike some eukaryotic tRNA synthetases that contain additional N-terminal domains (as seen with Lysyl-tRNA synthetase in search result ), bacterial valS typically has a more streamlined structure focused on its catalytic function .

What expression systems are typically used for recombinant production of V. vulnificus valS?

The recombinant V. vulnificus valS is typically produced in yeast expression systems, as indicated in the product information . This choice likely reflects the need for eukaryotic post-translational modifications or improved protein folding compared to bacterial expression systems. When expressing V. vulnificus valS in research settings, careful consideration of tag placement is necessary, as tags may interfere with enzyme activity. The tag type is often determined during the manufacturing process based on optimal expression and purification results .

What are the recommended storage and handling conditions for recombinant V. vulnificus valS?

Recombinant V. vulnificus valS should be stored at -20°C/-80°C with glycerol as a cryoprotectant. The recommended glycerol concentration is typically 5-50% (with 50% being standard in many preparations). The shelf life is approximately 6 months for liquid preparations and 12 months for lyophilized preparations when stored properly.

For reconstitution, the protein should be centrifuged briefly to bring contents to the bottom of the vial, then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To prevent repeated freeze-thaw cycles, which can damage protein structure and function, working aliquots should be prepared and stored at 4°C for up to one week .

How can researchers validate the activity of recombinant V. vulnificus valS in experimental settings?

To validate enzymatic activity of recombinant V. vulnificus valS, researchers should employ the following methodological approach:

• Aminoacylation assay: Measure the ability of valS to charge tRNA^Val with radioactively labeled valine (typically [³H]-valine or [¹⁴C]-valine)

• ATP-PPi exchange assay: Evaluate the first step of the aminoacylation reaction by measuring the enzyme's ability to form valyl-adenylate intermediate

• Thermal shift assay: Assess protein stability and proper folding using differential scanning fluorimetry

• Kinetic parameters determination: Calculate Km and kcat values for ATP, valine, and tRNA^Val substrates using steady-state kinetics approaches similar to those described for transketolase enzymes in V. vulnificus

• Comparative activity analysis: Compare activity against other bacterial valS enzymes under various conditions (pH, temperature, salt concentration) to identify potential V. vulnificus-specific properties

What experimental approaches can be used to study the role of valS in V. vulnificus pathogenesis?

Several experimental approaches can be utilized to investigate valS's role in pathogenesis:

• Gene knockout/knockdown studies: Create valS-deficient or depleted V. vulnificus strains using CRISPR-Cas9 or antisense RNA techniques to study the effects on bacterial growth, virulence, and stress responses.

• Wound infection models: Utilize murine models of wound infection, similar to those described for studying V. vulnificus virulence factors, to evaluate the impact of valS mutations on bacterial colonization and tissue damage .

• Signature-tagged mutagenesis (STM): Apply STM methodology as described in search result to determine if valS is essential for in vivo proliferation during infection.

• Transcriptomic analysis: Compare gene expression profiles between wild-type and valS-mutant strains under various stress conditions (antibiotic exposure, iron limitation, oxidative stress) to identify potential regulatory networks involving valS.

• Complementation studies: Reintroduce functional valS to mutant strains to confirm phenotype restoration and validate experimental findings.

How might aminoacyl-tRNA synthetases like valS contribute to antibiotic resistance in V. vulnificus?

Aminoacyl-tRNA synthetases represent potential targets for antimicrobial development and may play roles in antibiotic resistance through several mechanisms:

• Altered translation fidelity: Mutations in valS could lead to decreased translation accuracy, potentially allowing adaptation to stressful environments, including antibiotic exposure.

• Stress response regulation: tRNA synthetases may participate in stress response pathways that confer resistance or tolerance to antibiotics, similar to how tRNA modifications affect antibiotic tolerance in V. cholerae (described in search result ).

• Moonlighting functions: Beyond their canonical role in translation, aminoacyl-tRNA synthetases may have secondary functions that contribute to pathogenesis or antibiotic resistance, as demonstrated by the non-canonical roles of Lysyl-tRNA synthetase in HIV-1 infection .

• Biofilm formation: tRNA synthetases could influence biofilm development, which enhances antibiotic resistance in many bacteria.

Research on V. vulnificus has shown growing resistance to certain antibiotics, making understanding potential resistance mechanisms through translation machinery critically important .

What is the potential relationship between environmental stressors and valS function in V. vulnificus?

V. vulnificus thrives in warm coastal waters with specific salinity ranges, suggesting adaptation mechanisms for environmental changes. The function of valS may be modulated by:

• Temperature effects: Rising water temperatures due to climate change may influence valS activity, potentially affecting V. vulnificus growth rates and virulence. Research indicates V. vulnificus prevalence increases with warming coastal waters .

• Salinity adaptation: valS function might be optimized for specific salinity conditions, as V. vulnificus abundance correlates with salinity parameters (search result ).

• Iron availability: Like other components of the translation machinery, valS function may be regulated by iron availability, which is a key environmental factor for V. vulnificus virulence.

• Oxidative stress response: Similar to findings with the tgt gene in V. cholerae (search result ), valS may participate in oxidative stress responses triggered by environmental factors or host defense mechanisms.

How might structural studies of V. vulnificus valS inform potential drug development strategies?

Structural analysis of V. vulnificus valS could reveal unique features that might be exploited for therapeutic development:

• Active site architecture: Detailed characterization of the valS active site could identify bacterial-specific features amenable to selective inhibition, similar to the approach taken with V. vulnificus transketolase (search result ).

• Allosteric sites: Beyond the active site, identification of allosteric regulatory sites could provide opportunities for inhibitor development with novel mechanisms of action.

• Species-specific structural elements: Comparative structural analysis between bacterial and human valS could reveal V. vulnificus-specific features that could be targeted to minimize off-target effects.

• Potential inhibitor binding sites: Computational docking studies, similar to those performed with transketolase inhibitors (search result ), could identify potential binding sites for small molecule inhibitors.

The recent structural determination of V. vulnificus transketolase at 2.1 Å resolution (search result ) provides a methodological template for similar structural studies with valS.

What are the key methodological challenges in studying V. vulnificus valS and how can they be addressed?

Several challenges exist in studying V. vulnificus valS:

• Protein stability issues: The enzyme may have stability challenges during purification and storage. This can be addressed through buffer optimization, addition of stabilizing agents, and expression with fusion partners that enhance solubility.

• Assay development: Developing sensitive assays for valS activity that can be used in high-throughput screening requires optimization of reaction conditions and detection methods. Fluorescence-based aminoacylation assays could provide alternatives to traditional radioactive methods.

• In vivo studies: Studying valS function in vivo is complicated by its essential nature. Conditional expression systems or partial activity mutants could provide workable compromises for in vivo functional studies.

• Structural analysis challenges: Obtaining crystal structures of full-length valS may be difficult due to size and flexibility. Domain-by-domain structural analysis or cryo-EM approaches could overcome these limitations.

How might valS interact with the MARTX toxin machinery and other virulence factors in V. vulnificus?

The multifunctional-autoprocessing RTX (MARTX) toxins are key virulence factors in V. vulnificus, and potential interactions with translation machinery could reveal novel pathogenesis mechanisms:

• Translational regulation of toxins: valS might preferentially support translation of virulence factors through codon usage patterns or specialized ribosomes.

• Environmental sensing: Similar to how tRNA modifications respond to environmental cues in V. cholerae (search result ), valS activity might coordinate with virulence factor expression in response to environmental triggers.

• Potential direct interactions: valS could potentially interact directly with virulence factors or their regulators, similar to how some tRNA synthetases have expanded functions beyond aminoacylation.

• Genetic proximity analysis: Genome analysis could reveal whether valS is located near virulence-associated genes, potentially indicating coordinated expression or functional relationships.

Research has shown that V. vulnificus rtxA1 gene variants encode toxins with different arrangements of effector domains (search result ), suggesting complex regulation of virulence that might involve translation machinery components.

What insights can comparative genomics provide about evolution and specialization of valS across Vibrio species?

Comparative genomic approaches can reveal evolutionary patterns and functional specialization:

Table 1: Comparative Analysis of tRNA Synthetase Conservation Across Vibrio Species

*Estimated conservation percentages based on typical sequence similarities between Vibrio species for essential genes

Comparative genomics could further reveal:

• Codon usage adaptation: Analysis of valS sequence in relation to V. vulnificus codon usage patterns might reveal adaptations for optimal translation of valine-rich proteins, particularly virulence factors.

• Horizontal gene transfer assessment: Determining whether valS shows evidence of horizontal gene transfer, similar to findings with rtxA1 gene variants (search result ).

• Selection pressure analysis: Identifying regions of valS under purifying or positive selection could highlight functionally critical domains versus adaptable regions.

• Synteny analysis: Examining conservation of gene order surrounding valS across Vibrio species could provide insights into regulatory relationships and functional associations.

What are the broader implications of studying V. vulnificus valS for understanding bacterial pathogenesis?

Studying V. vulnificus valS has implications beyond this specific organism:

• Translation as virulence regulator: Research on valS can illuminate how translation machinery adaptations contribute to pathogenesis across bacterial species.

• Environmental adaptation mechanisms: Understanding how valS functions under varying environmental conditions provides insights into bacterial adaptation to changing marine environments, particularly relevant as coastal water temperatures rise due to climate change.

• Novel therapeutic approaches: Insights from valS research could inform development of new antibacterial strategies targeting translation machinery, addressing the growing concern of V. vulnificus antibiotic resistance (search result ).

• Fundamental biology insights: Studies of bacterial tRNA synthetases continue to reveal unexpected functions beyond translation, expanding our understanding of bacterial physiology.

As V. vulnificus infections are expected to increase with warming coastal waters and rising antimicrobial resistance (search results ), fundamental research on translation machinery components like valS represents an important avenue for developing new intervention strategies.

How might future technological advances enhance our ability to study V. vulnificus valS?

Emerging technologies will likely accelerate valS research:

• Cryo-EM advancements: Improvements in cryo-electron microscopy resolution may enable detailed structural studies of valS without crystallization requirements.

• Single-molecule techniques: Methods to observe individual valS molecules during aminoacylation could provide unprecedented insights into reaction mechanisms and inhibition.

• AI-driven structure prediction: Tools like AlphaFold may enhance structural understanding of valS and facilitate virtual screening for inhibitors.

• CRISPR-based screening: High-throughput functional genomics approaches could identify genetic interactions between valS and other cellular components.

• Microfluidic approaches: Single-cell analysis of translation in V. vulnificus could reveal cell-to-cell variability in valS function and its consequences for virulence heterogeneity.