Recombinant Vibrio vulnificus Tyrosine--tRNA ligase 1 (tyrS1)

What is the primary function of tyrS1 in Vibrio vulnificus?

Tyrosine-tRNA ligase 1 (tyrS1) belongs to the aminoacyl-tRNA synthetase family and catalyzes the ATP-dependent attachment of tyrosine to its cognate tRNA molecules. This charged tRNA (tyrosyl-tRNA) is then used by ribosomes during translation to incorporate tyrosine residues into growing polypeptide chains. In V. vulnificus, as in other bacteria, this enzyme ensures the fidelity of protein synthesis by specifically recognizing tyrosine and tRNA^Tyr. Similar to observations in V. cholerae, tyrS1 likely plays a critical role in the efficiency of decoding tyrosine codons (TAT and TAC), thereby contributing to accurate and efficient translation of the bacterial proteome .

How does tyrS1 specificity for tyrosine impact bacterial protein synthesis?

Tyrosine-tRNA ligase 1 exhibits high specificity for tyrosine through its amino acid binding pocket, which recognizes the phenol ring structure unique to this amino acid. This specificity is crucial for translational fidelity, as misincorporation of incorrect amino acids can lead to protein misfolding and dysfunction. The enzyme's specificity involves a two-step reaction: first activating tyrosine with ATP to form tyrosyl-AMP, then transferring the activated tyrosine to the 3' end of tRNA^Tyr. Research in related Vibrio species suggests that the efficiency of this process may be particularly important for proteins with high tyrosine content or biased tyrosine codon usage, similar to how tRNA modifications in V. cholerae affect decoding efficiency at specific codons .

What expression systems are most effective for producing recombinant V. vulnificus tyrS1?

For recombinant expression of V. vulnificus tyrS1, E. coli-based expression systems typically offer the highest yield and experimental tractability. The pET expression system under T7 promoter control using BL21(DE3) or its derivatives as host strains is commonly employed for aminoacyl-tRNA synthetases. To optimize expression, researchers should consider codon optimization for E. coli if significant codon bias exists between V. vulnificus and the expression host. Including a cleavable N-terminal His6-tag facilitates purification via nickel affinity chromatography. Expression at lower temperatures (16-25°C) often enhances proper folding and solubility. For studies requiring post-translational modifications specific to Vibrio species, a homologous expression system using an attenuated V. vulnificus strain may be necessary, though typically with lower yields than heterologous systems .

What assays are most reliable for measuring tyrS1 enzymatic activity?

Several established methods can be used to assess tyrS1 enzymatic activity with varying degrees of sensitivity and throughput:

• ATP-PPi exchange assay: This measures the first step of the aminoacylation reaction by tracking the incorporation of [32P]PPi into ATP, indicating amino acid activation.

• tRNA aminoacylation assay: This directly measures the formation of tyrosyl-tRNA using:

  • Radiometric detection with [14C] or [3H]-labeled tyrosine

  • Acid gel electrophoresis to separate charged from uncharged tRNAs

  • Coupled enzyme assays where pyrophosphate release is linked to colorimetric or fluorescent readouts

• Kinetic analysis: Determines Km and kcat values for each substrate (tyrosine, ATP, and tRNA^Tyr) through initial velocity measurements at varying substrate concentrations.

For comparative analysis of wild-type and mutant enzymes, maintaining consistent experimental conditions is crucial, as factors like pH, temperature, and ionic strength significantly affect aminoacylation activity .

How can site-directed mutagenesis inform structure-function relationships in tyrS1?

Site-directed mutagenesis provides powerful insights into the structure-function relationship of tyrS1. Key targets for mutagenesis include conserved catalytic residues in the HIGH and KMSKS motifs characteristic of Class I aminoacyl-tRNA synthetases, residues implicated in tyrosine recognition, tRNA binding interface residues, and potential regulatory regions. PCR-based methods like QuikChange are suitable for single mutations, while Gibson Assembly can be used for larger insertions or deletions. Conservative substitutions that maintain charge or size properties can assess specific chemical contributions to function.

Mutant characterization should include kinetic parameters comparison, thermal stability analysis using differential scanning fluorimetry, binding affinity measurements for substrates, and structural analysis where possible. Interpreting mutagenesis results requires considering potential long-range effects on protein structure and dynamics, not just local interactions. Computational modeling prior to mutagenesis can provide valuable insights into which residues may be most informative to target .

Is there evidence of genetic recombination in the tyrS1 gene across different V. vulnificus strains?

While direct evidence of recombination in tyrS1 is not explicitly reported in the search results, V. vulnificus is known to undergo significant genetic recombination in other genes, particularly virulence factors. For example, the rtxA1 gene, which encodes the MARTX(Vv) toxin, shows four distinct variants arising from recombination events with rtxA genes carried on plasmids or with the rtxA gene of Vibrio anguillarum . By analogy, it's reasonable to hypothesize that tyrS1 might also experience recombination events, particularly if this provides selective advantages under specific environmental conditions.

To investigate potential recombination in tyrS1, researchers should sequence the gene from multiple clinical and environmental V. vulnificus isolates and employ computational methods such as recombination detection programs and analysis of sequence mosaicism. The presence of mobile genetic elements near the tyrS1 locus would provide additional evidence supporting potential recombination .

How might tyrS1 sequence variation correlate with V. vulnificus biotypes and virulence?

To establish correlations between tyrS1 sequence variation and V. vulnificus biotypes/virulence, researchers should perform comparative genomic analysis across Biotype 1 strains (primarily human pathogens), Biotype 2 strains (primarily eel pathogens), and Biotype 3 strains (associated with wound infections). Analysis should focus on non-synonymous mutations that may affect enzyme function, promoter region variations that might affect expression levels, and the presence of specific tyrS1 alleles in clinical versus environmental isolates.

Similar to observations with the rtxA1 toxin gene, where the most common variant in clinical-type V. vulnificus surprisingly encodes a toxin with reduced potency compared to strains from market oysters, tyrS1 variants might show unexpected relationships to virulence . This could indicate selection for specific tyrS1 variants in different environmental niches or during host adaptation, potentially influencing translational regulation during infection.

What evolutionary pressures might shape tyrS1 diversity in environmental versus clinical isolates?

Several evolutionary pressures could shape tyrS1 diversity across V. vulnificus populations:

• Environmental adaptations: Temperature fluctuations in marine environments may select for enzymes with different thermal optima, while salinity variations could drive selection for protein stability variants.

• Host interactions: Adaptation to different host environments (human vs. marine animals) and selection for optimal function at human body temperature (37°C) in clinical isolates may drive evolutionary changes.

• Antibiotic pressure: Similar to the observed upregulation of tgt in response to aminoglycosides in V. cholerae, tyrS1 might be under selection related to antibiotic exposure .

• Genetic drift and bottlenecks: Population restrictions during host infection could drive stochastic changes, as could seasonal population fluctuations in marine environments.

Analysis of these pressures would require combining population genomics with experimental validation to understand how tyrS1 variants contribute to environmental persistence and virulence potential .

Could tyrS1 be involved in translational regulation of virulence factors?

The potential involvement of tyrS1 in translational regulation of virulence factors is supported by evidence from related systems. In V. cholerae, tRNA modifications affect the efficiency of decoding at tyrosine codons (TAT and TAC), demonstrating that tRNA-related enzymes can influence translational regulation . By extension, tyrS1 activity or expression changes could create a regulatory circuit where mRNAs with specific tyrosine codon usage patterns are preferentially translated.

This regulatory mechanism would be particularly impactful for virulence factors with biased tyrosine codon usage. Research in V. cholerae identified "modification tunable transcripts" that are subject to translational regulation based on tRNA modification status . Similar mechanisms might exist in V. vulnificus, where tyrS1 activity could influence the translation efficiency of specific virulence factors, potentially providing a rapid response to environmental changes without requiring de novo transcription.

How might environmental conditions impact tyrS1 expression and function?

Environmental conditions likely impact tyrS1 expression and function through multiple mechanisms. Research in related Vibrio species shows that tRNA modification enzymes respond to environmental cues. For example, in V. cholerae, the tgt gene (involved in tRNA modification) is upregulated in response to sub-inhibitory concentrations of aminoglycoside antibiotics and during the stringent response, while being repressed by the carbon catabolite regulator CRP .

By analogy, V. vulnificus tyrS1 expression might be regulated in response to:

• Antibiotic exposure, similar to tgt regulation in V. cholerae

• Nutrient availability, potentially through CRP-like regulation

• Temperature shifts when transitioning between marine environments and human hosts

• Iron limitation, a common stress in both environments and hosts

• Osmotic pressure changes during host colonization

These environmental responses could fine-tune translation efficiency for specific subsets of the proteome based on tyrosine codon usage patterns, providing adaptability during environmental transitions and host infection .

How might inhibiting tyrS1 affect V. vulnificus pathogenicity?

Inhibiting tyrS1 could impact V. vulnificus pathogenicity through several mechanisms. The primary effect would be disruption of protein synthesis, potentially with stronger impacts on tyrosine-rich virulence proteins. This could result in bacteriostatic or bactericidal effects depending on the inhibition level and whether the bacterium can compensate through alternate mechanisms.

The development of selective tyrS1 inhibitors could be explored for therapeutic potential, potentially in combination with existing antibiotics. Assessing the likelihood of resistance development would be an important consideration in such studies .

What structural features are likely to distinguish V. vulnificus tyrS1 from other bacterial tyrosine-tRNA synthetases?

While specific structural information for V. vulnificus tyrS1 is not provided in the search results, aminoacyl-tRNA synthetases often display species-specific adaptations that could be relevant. Key structural features likely to distinguish V. vulnificus tyrS1 include:

• Tyrosine binding pocket architecture, which may be optimized for function under the environmental conditions V. vulnificus encounters

• tRNA recognition elements, particularly the anticodon binding domain

• Species-specific surface features that might interact with other cellular components

• Insertion or deletion regions that differ from homologs in other species

These distinguishing features could be identified through crystal or cryo-EM structure determination followed by structural alignment with homologs from other bacteria. Computational approaches such as homology modeling and molecular dynamics simulations could also provide insights into unique structural properties related to the marine environment or human infection process .

What computational approaches can predict substrate binding and catalytic mechanisms of tyrS1?

Several computational approaches can provide insights into tyrS1 function:

• Molecular docking studies: Docking of tyrosine, ATP, and tRNA to the enzyme can identify key binding interactions and predict binding energies. These studies can be validated by comparison with known aminoacyl-tRNA synthetase structures.

• Molecular dynamics simulations: These can reveal conformational changes during substrate binding, identify water networks in the active site, and investigate protein flexibility. Similar approaches have been used to study RNA-protein interactions in other systems .

• Quantum mechanics/molecular mechanics (QM/MM) calculations: These can model the reaction mechanism in detail, identify transition states, and calculate activation barriers for wild-type and mutant enzymes.

• Machine learning approaches: These can predict residue importance based on evolutionary conservation and aid in virtual screening for potential inhibitors.

These computational approaches should be integrated with experimental validation through enzyme kinetics, mutagenesis, and structural studies for maximum reliability and biological relevance .

How do post-translational modifications potentially affect tyrS1 function?

Post-translational modifications (PTMs) could significantly impact tyrS1 function, though specific information about PTMs in V. vulnificus tyrS1 is not provided in the search results. Potential effects include:

• Regulation of enzymatic activity through reversible modifications such as phosphorylation, which could modulate catalytic efficiency or substrate binding

• Altered subcellular localization or protein-protein interactions

• Changed stability or turnover rates

• Modification of substrate specificity or preference

In bacteria, aminoacyl-tRNA synthetases can be modified in response to stress conditions, potentially linking translational regulation to environmental adaptation. For example, in V. cholerae, translational reprogramming occurs under antibiotic stress, suggesting that enzymes involved in translation might be regulated post-translationally .

Mass spectrometry-based approaches would be optimal for identifying and characterizing PTMs, including bottom-up proteomics for site localization and top-down proteomics for comprehensive proteoform characterization. Functional validation through site-directed mutagenesis of modified residues would be essential to determine their biological significance.

How does V. vulnificus tyrS1 likely compare to homologs in V. cholerae and other Vibrio species?

A comprehensive comparative analysis of tyrS1 across Vibrio species would reveal evolutionary relationships and functional adaptations. While specific comparative data isn't provided in the search results, insights can be drawn from related systems. The queuosine modification of tRNA-Tyrosine in V. cholerae demonstrates that tRNA biology can vary significantly between related species and under different conditions .

Sequence analysis would identify conserved catalytic residues versus variable regions that might confer species-specific properties. Genomic context comparison could reveal conserved operonic structures or regulatory elements. Functional comparison through enzymatic activity assays under identical conditions would determine differences in catalytic efficiency, substrate specificity, and response to environmental factors like temperature and salinity.

These comparative studies could identify species-specific adaptations related to environmental niche or pathogenicity mechanisms, potentially revealing how tyrS1 has evolved to support the distinct lifestyles of different Vibrio species .

Are there functional differences in tRNA synthetases between pathogenic and non-pathogenic Vibrio species?

While the search results don't provide direct comparisons of tRNA synthetases between pathogenic and non-pathogenic Vibrio species, related findings suggest potential functional differences. In V. cholerae, the tgt enzyme (involved in tRNA modification) is regulated in response to environmental stresses and affects translational efficiency at specific codons . Similar mechanisms might distinguish pathogenic from non-pathogenic Vibrio species.

Potential functional differences could include:

• Altered substrate specificity or catalytic efficiency optimized for host environments

• Differential regulation in response to host-associated stresses

• Unique protein-protein interactions in pathogenic species

• Contribution to stress tolerance or virulence factor production

Experimental comparisons would require recombinant expression and purification of tyrS1 from multiple species, followed by detailed kinetic parameter determination and expression pattern analysis under various stress conditions. Cross-species functional complementation tests could assess the interchangeability of these enzymes, potentially revealing pathogen-specific adaptations .

Could tyrS1 sequence or structure be used for Vibrio species identification in clinical or environmental samples?

The potential of tyrS1 as a molecular marker for Vibrio species identification depends on several factors:

• Sequence conservation patterns: Ideal markers have conserved regions for primer binding and variable regions for species discrimination. While specific data for tyrS1 isn't provided in the search results, aminoacyl-tRNA synthetases typically contain both highly conserved catalytic domains and variable regions.

• Detection methods: Species-specific PCR assays targeting variable regions of tyrS1 could be developed, along with high-resolution melt analysis for closely related species. LAMP (Loop-mediated isothermal amplification) could enable field detection.

• Advantages as a marker: Single-copy essential genes like tyrS1 often provide reliable phylogenetic signals compared to multi-copy genes. Additionally, functional genes can reveal ecological adaptations that 16S rRNA analysis might miss.

For comparison, V. vulnificus genetic variation has been studied in other genes like rtxA1, revealing distinct variants that correlate with source (clinical vs. environmental) . If tyrS1 shows similar variation patterns, it could serve as a useful marker for identification and potentially for source tracking in epidemiological investigations.

What makes tyrS1 a potential target for antimicrobial development against V. vulnificus?

Several features make tyrS1 a promising antimicrobial target:

• Essential function: Protein synthesis depends on correctly charged tRNAs, and limited redundancy in aminoacyl-tRNA synthetase function makes tyrS1 likely essential for bacterial survival.

• Selectivity potential: Structural differences between bacterial and human tyrosyl-tRNA synthetases offer opportunities for designing selective inhibitors with reduced toxicity compared to broad-spectrum targets.

• Precedent: Other aminoacyl-tRNA synthetases have been successfully targeted for antimicrobial development, such as mupirocin targeting isoleucyl-tRNA synthetase.

• Pathogen importance: V. vulnificus is associated with high mortality rates in certain infections, with the bacterium linked to approximately 1% of all food-related deaths, predominantly from contaminated seafood consumption . This highlights the need for new therapeutic approaches.

The potential for resistance development would need careful assessment, particularly in light of the genetic variation and recombination observed in other V. vulnificus genes like rtxA1 .

How might inhibitors of tyrS1 be designed based on structural and mechanistic insights?

Rational design of tyrS1 inhibitors would involve multiple complementary approaches:

• Structure-based design strategies: These include fragment-based screening targeting the active site, virtual screening of compound libraries against crystal structures, and structure-activity relationship development from initial hits.

• Mechanistic considerations: Different inhibitor classes could include competitive inhibitors of ATP or tyrosine binding, transition state analogs mimicking the aminoacylation reaction, allosteric inhibitors affecting enzyme dynamics, and irreversible inhibitors targeting catalytic residues.

• Selectivity optimization: This would involve targeting non-conserved residues near the active site, structure-guided modifications to enhance bacterial selectivity, and counter-screening against human tyrosyl-tRNA synthetase.

This approach aims to develop inhibitors with high potency, selectivity, and appropriate drug-like properties. The genetic variation observed in V. vulnificus virulence factors suggests that resistance monitoring would be an important component of any therapeutic development program .

What experimental approaches can evaluate tyrS1 inhibitors' effects on V. vulnificus pathogenicity?

A comprehensive evaluation of tyrS1 inhibitors would include:

• In vitro assessment: Enzymatic inhibition assays (IC50, Ki determination), growth inhibition in culture (MIC, MBC determination), time-kill curves at various inhibitor concentrations, and resistance development frequency analysis.

• Cellular effect characterization: Impact on protein synthesis rates using labeled amino acids, effects on virulence factor production, morphological changes via electron microscopy, and transcriptomic/proteomic responses to inhibition.

• In vivo efficacy studies: Mouse models of V. vulnificus infection similar to those used to study MARTX(Vv) toxin , dosing optimization studies, combination studies with existing antibiotics, and pharmacokinetic/pharmacodynamic analysis.

• Safety assessment: Cytotoxicity in human cell lines, off-target effects on human protein synthesis, and preliminary toxicity studies in animal models.

This systematic approach would provide comprehensive data on the potential of tyrS1 inhibitors as therapeutic agents against V. vulnificus infections, which are associated with significant mortality, particularly in immunocompromised individuals .

How can ribosome profiling be used to study the impact of tyrS1 mutations on translation?

Ribosome profiling offers powerful insights into translation dynamics affected by tyrS1 mutations:

• Experimental design: Compare wild-type V. vulnificus with strains carrying tyrS1 mutations under various conditions, including those mimicking the infection environment. Time-course experiments can capture dynamic changes in translation.

• Technical approach: The method involves nuclease digestion of ribosome-protected mRNA fragments, followed by deep sequencing of these fragments to provide a genome-wide snapshot of translation at codon resolution.

• Data analysis: Codon-level resolution of ribosome positioning can identify pause sites at tyrosine codons, differential translation efficiency, and metagene analysis around tyrosine codons.

This approach could reveal how tyrS1 mutations affect translation of specific mRNAs, particularly those with biased tyrosine codon usage. Similar translational reprogramming has been observed with tRNA modifications in V. cholerae, where queuosine modification of tRNA-Tyrosine affects decoding at tyrosine TAT and TAC codons .

What RNA structural analysis techniques can inform tyrS1-tRNA interactions?

Several techniques can provide insights into the structural basis of tyrS1-tRNA interactions:

• SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension): This chemical probing method has been successfully used to analyze RNA structure within virus-like particles and could be adapted to study tyrS1-tRNA complexes . SHAPE reagents modify RNA based on flexibility, providing structural information at nucleotide resolution.

• Cross-linking studies: UV or chemical cross-linking followed by mass spectrometry can identify points of contact between tyrS1 and its tRNA substrate.

• Cryo-EM: Recent advances in cryo-EM resolution make it possible to visualize aminoacyl-tRNA synthetase-tRNA complexes at near-atomic resolution.

• X-ray crystallography: Co-crystallization of tyrS1 with tRNA substrates can provide atomic-level details of the interaction.

• Computational modeling: Molecular dynamics simulations can complement experimental approaches by predicting dynamic aspects of the interaction.

These approaches could identify key recognition elements and conformational changes during aminoacylation, similar to studies of tRNA-protein complexes in other systems .

How can single-molecule techniques advance our understanding of tyrS1 dynamics?

Single-molecule techniques offer unique insights into tyrS1 function that bulk methods cannot provide:

• Single-molecule FRET (smFRET): Strategic placement of fluorophore pairs on tyrS1 can monitor conformational changes during catalysis, substrate binding, and product release. This approach has been used successfully to study the dynamics of other RNA-protein interactions .

• Optical tweezers: This technique can measure forces during tRNA accommodation and investigate mechanical stability of enzyme-substrate complexes.

• Atomic Force Microscopy: Surface preparation for single-molecule imaging allows visualization of tyrS1-tRNA interactions, measurement of unbinding forces, and observation of oligomerization states.

These approaches would provide unprecedented insights into the dynamic behavior of tyrS1, revealing transient states and heterogeneity that might be critical for understanding its biological function in V. vulnificus and developing effective inhibitors. The structural analysis methods used to study RNA in other systems demonstrate the feasibility of applying these techniques to tyrS1-tRNA interactions .