Recombinant Vibrio vulnificus Histidine--tRNA ligase (hisS)

What is the structure and function of Vibrio vulnificus Histidine--tRNA ligase (hisS)?

Histidine--tRNA ligase (HisRS) is an essential aminoacyl-tRNA synthetase that catalyzes the attachment of histidine to its cognate tRNA. The V. vulnificus hisS protein consists of 422 amino acids with a molecular weight of approximately 30 kDa. The protein contains characteristic motifs of class II aminoacyl-tRNA synthetases, including the catalytic core domain responsible for ATP binding and aminoacylation reactions .

The primary function of hisS is to maintain translational fidelity by ensuring the correct incorporation of histidine during protein synthesis. This enzyme specifically:

• Activates histidine with ATP to form histidyl-adenylate

• Transfers the activated histidine to the 3' end of tRNA^His

• Ensures accurate charging of tRNA^His for protein translation

Which expression systems are available for producing recombinant V. vulnificus hisS?

Recombinant V. vulnificus hisS can be produced in multiple expression systems, each with distinct advantages depending on research requirements:

The selection of expression system should align with your specific research objectives and downstream applications .

How is the purity of recombinant V. vulnificus hisS typically assessed?

The purity of recombinant V. vulnificus hisS is typically assessed using multiple complementary techniques:

• SDS-PAGE analysis: Purified protein should appear as a single distinctive band at approximately 30 kDa, similar to other bacterial NATs (N-acetyltransferases) .

• Western blot confirmation: Using anti-His tag monoclonal antibodies to verify the identity of the purified protein .

• Gel filtration chromatography: The chromatogram of purified protein should display a single peak, confirming homogeneity and absence of aggregates or contaminants .

• Mass spectrometry: For precise molecular weight determination and confirmation of the full-length protein or any modifications.

A purity level of >85% is generally considered acceptable for most research applications .

How does the expression of V. vulnificus hisS vary under different environmental conditions relevant to pathogenesis?

The expression of V. vulnificus hisS, like many virulence-associated proteins, may be regulated by environmental conditions that mimic host environments. Research approaches to study this include:

• Comparative transcriptomics: Similar to studies on TonB systems in V. vulnificus CMCP6, RT-PCR and qRT-PCR can be used to compare hisS expression under in vitro conditions versus in vivo (animal models) . Select appropriate reference genes like gyrA that show consistent expression across experimental conditions.

• Environmental triggers: Examine hisS expression under varying conditions:

  • Iron availability (given that iron acquisition is critical for V. vulnificus virulence)

  • Temperature shifts (37°C human body temperature vs. marine temperatures)

  • Osmolarity changes (seawater vs. human tissue environments)

  • Oxygen levels (aerobic vs. microaerobic conditions)

• Phase variation analysis: Since V. vulnificus can switch between opaque, translucent, and rugose phenotypes with different virulence properties, examine whether hisS expression differs between these variants .

For quantitative analysis, normalize hisS expression to established reference genes and employ the ΔΔCt method for calculating relative gene expression .

What role might hisS play in the stress response and pathogenesis of Vibrio vulnificus?

While direct evidence for hisS involvement in V. vulnificus pathogenesis is limited, several research approaches can explore this connection:

• Stress response connection: tRNA synthetases can function beyond translation in bacterial stress responses. The V. vulnificus stressosome responds to oxygen levels and modulates iron metabolism . Investigate whether hisS participates in these pathways through:

  • Co-immunoprecipitation with stressosome components (RsbR, RsbS)

  • Transcriptional analysis under oxidative stress conditions

• In vivo expression technology (IVET): Similar to previously identified in vivo-expressed (ive) genes in V. vulnificus , determine if hisS is preferentially expressed during infection.

• Mutant construction and virulence assessment: Generate hisS mutants and evaluate impacts on:

  • Growth in iron-limited conditions

  • Resistance to oxidative stress

  • Adhesion to epithelial cells

  • Cytotoxicity (LDH release assays)

  • Virulence in mouse models

• Transposon insertion sequencing (TIS): Use approaches similar to those employed for identifying V. vulnificus genes required for survival in human serum to determine if hisS is essential under specific host conditions .

What are the optimal conditions for measuring the enzymatic activity of recombinant V. vulnificus hisS?

Designing robust enzymatic assays for hisS requires careful consideration of multiple parameters:

• Buffer composition and pH:

  • Test phosphate or Tris buffers at pH range 7.0-8.5

  • Include 5-10 mM MgCl₂ (essential cofactor)

  • Add 1-5 mM ATP

  • Consider including 0.1-1 mM DTT to maintain reduced thiols

• Temperature optimization:

  • V. vulnificus grows optimally in warm seawater (9-31°C)

  • Test activity at temperatures ranging from 25-37°C

  • Consider assessing thermal stability at different temperatures as was done for NAT enzymes

• Metal ion dependencies:

  • Test divalent cations (Mg²⁺, Mn²⁺)

  • Screen for inhibitory effects of Zn²⁺ and Cu²⁺, which were shown to inhibit other V. vulnificus enzymes

• Kinetic parameter determination:

  • Measure initial velocities at varying substrate concentrations

  • Plot data using Michaelis-Menten kinetics

  • Calculate Km and Vmax values to compare with other bacterial HisRS enzymes

• Aminoacylation assay options:

  • Radioactive assay: [³H]-histidine incorporation into tRNA

  • Colorimetric pyrophosphate release assay

  • HPLC-based detection of aminoacylated tRNA

How can recombinant V. vulnificus hisS be used as a tool to screen for novel antimicrobial compounds?

Developing hisS-targeted antimicrobial screening platforms involves several methodological approaches:

• High-throughput enzymatic inhibition assays:

  • Adapt aminoacylation assays to microplate format

  • Primary screen: pyrophosphate release assay using malachite green detection

  • Secondary confirmation: direct measurement of aminoacylated tRNA^His

• Structure-based drug design:

  • Generate homology models of V. vulnificus hisS based on crystal structures of related bacterial HisRS

  • Identify unique binding pockets compared to human HisRS

  • Perform virtual screening of chemical libraries

  • Validate hits through biochemical assays

• Whole-cell assays with sensitized strains:

  • Generate V. vulnificus strains with reduced hisS expression

  • Screen compounds for enhanced activity against sensitized strains

  • Confirm mechanism through enzyme inhibition assays

• Target validation approaches:

  • Determine compound effects on protein synthesis using isotope-labeled amino acid incorporation

  • Examine hisS binding using thermal shift assays or surface plasmon resonance

  • Evaluate resistance development and map resistance mutations

This approach leverages the essential nature of hisS for bacterial survival while targeting structural differences from human HisRS.

What are the common challenges in expressing and purifying active V. vulnificus hisS?

Researchers frequently encounter several challenges when working with recombinant hisS:

• Solubility issues:

  • Problem: Inclusion body formation in E. coli expression systems

  • Solutions:

    • Lower induction temperature (16-20°C)

    • Use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

    • Consider alternative expression hosts (similar to approaches used for other V. vulnificus proteins)

• Stability concerns:

  • Problem: Poor colloidal stability at higher concentrations

  • Solutions:

    • Maintain at lower concentrations (1 mg/mL) to prevent aggregation

    • Determine Tagg (aggregation temperature) using static light scattering

    • Optimize buffer conditions with stabilizing additives (glycerol, arginine)

• Activity preservation:

  • Problem: Loss of enzymatic activity during purification

  • Solutions:

    • Include reducing agents to protect catalytic cysteine residues

    • Avoid exposure to heavy metals (Cu²⁺, Zn²⁺) which inhibit enzyme activity

    • Perform activity assays at each purification step

• Contaminant removal:

  • Problem: Co-purifying bacterial tRNAs or host proteins

  • Solutions:

    • Include high-salt washes during affinity purification

    • Add nuclease treatment steps

    • Implement additional chromatography steps (ion exchange, size exclusion)

How can V. vulnificus hisS be used to study bacterial adaptation to host environments?

Investigating bacterial adaptation using hisS as a model requires sophisticated experimental approaches:

• Comparative sequence analysis:

  • Analyze hisS sequence variations among different V. vulnificus strains (clinical vs. environmental)

  • Compare with other Vibrio species to identify conserved regions and species-specific adaptations

  • Correlate variations with strain virulence classifications (similar to SUKU_G1/G2/G3 classification systems)

• Expression analysis under host-mimicking conditions:

  • Design experiments using conditions that mimic:

    • Iron limitation (using chelators)

    • Serum exposure (similar to TIS screens)

    • Oxidative stress (H₂O₂ treatment)

    • Host cell co-culture systems

  • Employ RT-qPCR to quantify expression changes

• Functional evolution assessment:

  • Compare kinetic parameters of hisS from different V. vulnificus lineages

  • Examine temperature and pH optima variations

  • Assess resistance to oxidative inactivation between variants

• Recombinant strain construction:

  • Generate cross-complementation strains with hisS variants

  • Evaluate fitness in various stress conditions

  • Assess virulence changes in infection models

This approach provides insights into how essential housekeeping genes like hisS may adapt to support pathogen survival in diverse environments.

What controls and validation steps are critical when using recombinant V. vulnificus hisS in research?

Ensuring experimental rigor when working with recombinant hisS requires comprehensive controls:

• Protein quality controls:

  • Purity assessment: SDS-PAGE, Western blot with anti-His antibody

  • Homogeneity verification: Size exclusion chromatography showing single peak

  • Mass spectrometry confirmation of intact protein

  • Activity verification: Aminoacylation of tRNA^His

• Negative controls for enzymatic assays:

  • Heat-inactivated enzyme

  • Catalytic site mutants (identify and mutate critical residues)

  • Reaction without ATP or tRNA substrates

  • Reactions with non-cognate amino acids

• Species specificity validation:

  • Compare activity with human HisRS

  • Test cross-species tRNA aminoacylation efficiency

  • Identify species-specific inhibitors

• In vivo validation approaches:

  • Complementation of E. coli hisS temperature-sensitive mutants

  • Construction of conditional hisS mutants in V. vulnificus

  • In vitro translation assays using purified components

• Data reporting standards:

  • Document complete methods including expression system used

  • Report protein concentration determination method

  • Include enzyme batch variation assessments

  • Provide detailed buffer compositions and reaction conditions

How can structural studies of V. vulnificus hisS contribute to understanding aminoacyl-tRNA synthetase evolution?

Structural biology approaches to studying V. vulnificus hisS can yield valuable evolutionary insights:

• Comparative structural analysis:

  • Determine X-ray crystal structure of V. vulnificus hisS

  • Compare with structures from other bacteria and eukaryotes

  • Analyze domain architecture and catalytic site organization

  • Map conservation patterns onto the structural model

• Phylogenetic structure-function correlations:

  • Integrate structural data with phylogenetic analyses

  • Identify clade-specific structural adaptations

  • Correlate structure with habitat-specific challenges (marine vs. host environment)

• Domain architecture analysis:

  • Examine if V. vulnificus hisS contains additional domains beyond the core catalytic domain

  • Investigate potential moonlighting functions like those observed in other tRNA synthetases

  • Analyze if structural adaptations relate to the pathogenic lifestyle

• Molecular dynamics simulations:

  • Model protein flexibility and substrate binding

  • Compare dynamics between marine bacteria and terrestrial pathogens

  • Identify potential allosteric sites specific to Vibrio species

These approaches would advance understanding of how essential enzymes evolve while maintaining their critical cellular functions in different bacterial lifestyles.

How does V. vulnificus hisS interact with the bacterial stress response system and virulence regulation?

Investigating the potential dual role of hisS in translation and virulence regulation requires multi-faceted approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with key stress response proteins

  • Bacterial two-hybrid assays to screen for interactions with:

    • Stressosome components (RsbR, RsbS, RsbT)

    • Virulence regulators (HlyU, ToxRS system)

  • Crosslinking mass spectrometry to capture transient interactions

• Transcriptome analysis:

  • Compare wild-type and hisS-depleted strains

  • Examine changes in virulence gene expression

  • Analyze under normal and stress conditions

  • Focus on known virulence factors (VVH, MARTX, RtxA1)

• Metabolome changes:

  • Assess how hisS depletion affects central metabolism

  • Monitor iron metabolism and siderophore production

  • Examine amino acid pools and their relation to virulence factor synthesis

• Stress response integration:

  • Test if hisS functions in oxygen sensing like the stressosome

  • Examine interaction with iron regulation pathways

  • Investigate potential role in phase variation between opaque and translucent forms

This research would reveal whether hisS has evolved additional regulatory functions beyond its canonical role in translation, similar to other dual-function aminoacyl-tRNA synthetases.

What are the prospects for developing V. vulnificus hisS inhibitors as potential therapeutic agents?

Developing hisS inhibitors requires systematic approaches to drug discovery and development:

• Target validation:

  • Confirm essentiality through conditional knockout studies

  • Demonstrate growth inhibition upon hisS depletion

  • Validate across different V. vulnificus strains and growth conditions

• Inhibitor discovery strategies:

  • High-throughput screening of chemical libraries

  • Fragment-based drug discovery

  • Structure-based virtual screening

  • Natural product library screening (marine-derived compounds)

• Selectivity assessment:

  • Compare inhibition of bacterial vs. human HisRS

  • Evaluate activity against other bacterial species

  • Determine inhibition mechanism (competitive, non-competitive, allosteric)

• Lead optimization considerations:

  • Structure-activity relationship studies

  • Physicochemical property optimization

  • Cell penetration enhancement

  • Stability in biological fluids

• Proof-of-concept studies:

  • In vitro growth inhibition of V. vulnificus

  • Efficacy in infection models

  • Combination studies with established antibiotics

  • Resistance development assessment

This research direction is particularly valuable given the high mortality rate of V. vulnificus infections and increasing antibiotic resistance concerns.

How can systems biology approaches integrate V. vulnificus hisS function with broader pathogenesis networks?

Systems-level investigation of hisS requires integrated multi-omics approaches:

• Network reconstruction:

  • Map genetic interactions through synthetic genetic arrays

  • Identify functional connections through correlation analysis of multi-omics data

  • Position hisS within essential gene networks identified through TIS screens

• Temporal dynamics during infection:

  • Track transcriptional, proteomic, and metabolomic changes during:

    • Host cell adhesion

    • Invasion processes

    • Biofilm formation stages

    • Response to host defense mechanisms

  • Monitor hisS expression alongside virulence factors (VVH, MARTX, TonB systems)

• Computational modeling:

  • Construct kinetic models of translation processes

  • Develop integrated regulatory networks connecting translation to virulence

  • Simulate effects of hisS perturbation on cellular physiology

  • Predict critical nodes in pathogenesis networks

• Multi-strain comparative analysis:

  • Compare systems-level organization between:

    • Clinical vs. environmental isolates

    • High vs. low virulence strains

    • Different phase variants (opaque, translucent, rugose)

  • Correlate with genomic features like the presence of specific genomic islands

This integrated approach would position hisS within the broader context of V. vulnificus pathogenesis mechanisms, potentially revealing unexpected connections between basic cellular processes and virulence.