Recombinant Vibrio vulnificus Elongation factor Tu 1 (tuf1)

What is the role of Elongation Factor Tu 1 in Vibrio vulnificus bacterial physiology?

Elongation Factor Tu 1 (tuf1) in Vibrio vulnificus plays a critical role in protein biosynthesis by delivering aminoacyl-tRNA to the ribosome during the elongation phase of translation. Research has demonstrated that tufA (encoding EF-Tu) is continuously expressed in V. vulnificus under various environmental conditions, including in natural estuarine waters . This consistent expression suggests that tuf1 is essential for the bacterium's survival in diverse environments. Studies examining gene expression in situ have confirmed that tufA continues to be expressed alongside other genes such as katG, rpoS, wza, and wzb, even when the bacterium is subjected to environmental stressors .

How does the structure of V. vulnificus tuf1 compare to EF-Tu from other bacterial pathogens?

The structure of V. vulnificus Elongation Factor Tu 1 follows the conserved three-domain architecture typical of bacterial EF-Tu proteins. While specific structural data for V. vulnificus EF-Tu is limited in the provided search results, comparative analysis with other Vibrio species suggests high sequence conservation. The protein consists of domain I (containing the GTP/GDP binding pocket), domain II (involved in aminoacyl-tRNA binding), and domain III (which completes the aminoacyl-tRNA binding surface). The structural conservation of EF-Tu across bacterial species makes it a potential target for broad-spectrum antimicrobial development, though species-specific structural elements in V. vulnificus tuf1 may exist to accommodate its unique pathogenic lifestyle.

What is the expression pattern of tuf1 in V. vulnificus during different growth phases and environmental conditions?

Research indicates that V. vulnificus maintains consistent expression of tufA (encoding EF-Tu) across various environmental conditions. In situ studies conducted in natural estuarine waters have shown that clinical isolates (C7184k/o), environmental isolates (Env1), and strain 707o all expressed tufA continuously throughout a 108-hour incubation period . This consistent expression pattern contrasts with some other genes like vvhA (encoding hemolysin), which showed differential expression among strains. The table below demonstrates the expression pattern of tufA alongside other genes during the in situ study:

The consistent expression of tufA across all time points and strains indicates its fundamental importance to bacterial survival, regardless of the strain's origin (clinical vs. environmental) .

What are the optimal expression systems for producing recombinant V. vulnificus tuf1 protein?

For optimal expression of recombinant V. vulnificus tuf1, E. coli-based expression systems have proven most effective, particularly BL21(DE3) or its derivatives. When designing expression constructs, researchers should consider codon optimization for E. coli, as V. vulnificus may utilize different codon preferences. The pET expression system with a T7 promoter typically yields high expression levels when induced with IPTG (0.5-1.0 mM) at 30°C for 4-6 hours. Adding an N-terminal His-tag facilitates downstream purification while minimizing interference with protein function, as the N-terminus of EF-Tu is not directly involved in GTP binding or aminoacyl-tRNA interactions.

A methodological approach should include: (1) PCR amplification of the tuf1 gene from V. vulnificus genomic DNA using high-fidelity polymerase; (2) cloning into a suitable expression vector; (3) transformation into an expression host; (4) optimization of induction conditions through small-scale expression trials; and (5) scale-up for protein production.

What purification challenges are specific to recombinant V. vulnificus tuf1, and how can they be overcome?

Purification of recombinant V. vulnificus tuf1 presents several specific challenges. The protein may form inclusion bodies when overexpressed, particularly at higher temperatures. To address this, researchers should:

• Optimize expression conditions by lowering temperature to 18-25°C during induction

• Include solubility enhancers such as 1% glucose in the culture medium

• Co-express molecular chaperones like GroEL/GroES to assist proper folding

• Use lysis buffers containing 10% glycerol and 1-5 mM magnesium chloride to stabilize the protein structure

For purification, a multi-step approach is recommended:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged constructs

• Ion exchange chromatography to separate EF-Tu from nucleotides and other contaminants

• Size exclusion chromatography as a final polishing step to achieve >95% purity

During all purification steps, the presence of GDP/GTP (1 mM) and magnesium ions (5 mM) helps maintain protein stability and activity.

How can researchers assess the proper folding and activity of purified recombinant tuf1?

Assessment of proper folding and activity of purified recombinant V. vulnificus tuf1 requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Intrinsic tryptophan fluorescence to evaluate tertiary structure

  • Thermal shift assays to determine protein stability

  • Limited proteolysis to confirm proper domain organization

• Functional activity assays:

  • GTP binding assay using fluorescent GTP analogs

  • GTPase activity measurement using malachite green phosphate detection

  • Aminoacyl-tRNA binding assay using fluorescence anisotropy

  • In vitro translation assay to confirm participation in protein synthesis

A properly folded and active tuf1 should demonstrate GTPase activity that is enhanced by aminoacyl-tRNA binding and should participate effectively in in vitro translation systems. Researchers should compare the activity metrics with those of EF-Tu from model organisms like E. coli to benchmark performance.

What techniques can be used to study the GTP/GDP binding kinetics of V. vulnificus tuf1?

Multiple complementary techniques can characterize the GTP/GDP binding kinetics of V. vulnificus tuf1:

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding thermodynamics (ΔH, ΔS, ΔG)

  • Determines stoichiometry and binding constants (KD)

  • Requires 0.5-1 mg of purified protein per experiment

  • Optimal buffer conditions: 50 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl2

• Surface Plasmon Resonance (SPR):

  • Measures real-time binding kinetics (kon and koff rates)

  • Requires immobilization of His-tagged tuf1 on NTA sensor chips

  • Can compare different nucleotides (GTP, GDP, GMP, GTP analogs)

  • Buffer recommendation: 10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.005% surfactant P20

• Fluorescence-based methods:

  • FRET using fluorescent GTP analogs (BODIPY-GTP, mant-GTP)

  • Stopped-flow kinetics to measure rapid association/dissociation

  • Intrinsic tryptophan fluorescence changes upon nucleotide binding

When analyzing results, researchers should determine the key kinetic parameters (KD, kon, koff) and compare them with those of other bacterial EF-Tu proteins to identify any distinctive features of V. vulnificus tuf1 that might relate to its pathogenicity or survival strategies.

How does aminoacyl-tRNA binding capacity of V. vulnificus tuf1 compare to that of other bacterial species?

The aminoacyl-tRNA binding capacity of V. vulnificus tuf1 can be assessed through several methodical approaches:

• Quantitative binding assays:

  • Filter binding assays using radiolabeled aminoacyl-tRNAs

  • Fluorescence anisotropy with fluorescently labeled tRNAs

  • Microscale thermophoresis for determination of binding constants

• Comparative analysis framework:

  • Side-by-side testing with EF-Tu from model organisms (E. coli, B. subtilis)

  • Testing with aminoacyl-tRNAs from different sources to assess specificity

  • Evaluation across different environmental conditions (pH, temperature, salinity)

While specific comparative data for V. vulnificus tuf1 is limited in the provided search results, researchers should examine binding preferences across different aminoacyl-tRNA species, focusing on whether V. vulnificus tuf1 exhibits unique selectivity patterns that might influence translation efficiency under stress conditions. Given that V. vulnificus maintains tufA expression during environmental stress , its tuf1 protein might possess distinctive aminoacyl-tRNA binding properties adapted to marine environments.

What role does post-translational modification play in the function of V. vulnificus tuf1?

Post-translational modifications (PTMs) significantly influence EF-Tu function across bacterial species, and V. vulnificus tuf1 likely follows similar patterns. Key experimental approaches to investigate PTMs include:

• Mass spectrometry-based PTM mapping:

  • Bottom-up proteomics with enrichment strategies for specific PTMs

  • Top-down proteomics to analyze intact proteoforms

  • Targeted analysis of known EF-Tu modification sites (methylation, phosphorylation, acetylation)

• Functional impact assessment:

  • Site-directed mutagenesis of identified PTM sites

  • Activity comparisons between modified and unmodified forms

  • Structure analysis of modified vs. unmodified protein

• Environmental regulation of PTMs:

  • Analysis of modifications under different growth conditions

  • Comparison between in vitro grown cultures and in situ samples

While specific information about V. vulnificus tuf1 PTMs is not detailed in the search results, researchers should focus on identifying modifications that might be unique to V. vulnificus or correlate with its pathogenic potential. The continuous expression of tufA in diverse environments suggests that PTMs might play a role in modulating tuf1 function according to environmental conditions.

Can recombinant V. vulnificus tuf1 be used as a biomarker for detection of pathogenic strains?

Recombinant V. vulnificus tuf1 has potential as a biomarker for pathogenic strain detection, though with important considerations:

• Advantages as a biomarker:

  • Constitutive expression across environmental conditions as evidenced by in situ studies

  • High abundance in bacterial cells makes detection more sensitive

  • Potential strain-specific sequence variations between clinical and environmental isolates

• Development of detection methods:

  • Generation of specific antibodies against recombinant tuf1 for immunoassays

  • PCR-based detection targeting strain-specific variants of the tuf1 gene

  • Mass spectrometry identification of tuf1 peptide markers for strain differentiation

• Validation approach:

  • Comparative analysis across diverse strain collections (clinical and environmental)

  • Testing in complex environmental samples

  • Correlation with established virulence markers

Unlike vvhA (hemolysin) which shows differential expression between clinical and environmental strains , tufA expression appears consistent across different V. vulnificus strains. Therefore, detection systems should focus on strain-specific sequence variations rather than expression differences. Researchers should also develop multiplex approaches incorporating both tuf1 and other virulence markers for improved specificity in pathogenic strain identification.

How does tuf1 expression correlate with expression of known virulence factors during infection?

Understanding the correlation between tuf1 and virulence factor expression requires comprehensive investigation:

• Temporal expression analysis:

  • RNA-seq or qRT-PCR time course studies during infection model progression

  • Correlation analysis between tufA expression and virulence genes like rtxA1, vvhA, and vvpE

  • Evaluation during different infection phases (adhesion, invasion, dissemination)

• In situ expression patterns:

  • Available data shows that tufA and vvhA expression patterns differ in some strains: while tufA is consistently expressed across all tested strains, vvhA shows strain-dependent expression patterns

  • The table below summarizes expression patterns observed in natural estuarine waters:

• Regulatory connections:

  • Investigation of shared regulatory elements between tufA and virulence genes

  • Effect of HlyU (known to regulate rtxA1 ) on tufA expression

  • Impact of environmental signals (iron availability, temperature, quorum sensing) on co-expression

The continuous expression of tufA alongside variable expression of virulence factors suggests that while tuf1 provides the translation capacity necessary for virulence, its expression alone does not determine virulence factor production, which is controlled by additional regulatory mechanisms.

What are the potential applications of V. vulnificus tuf1 in the development of novel antimicrobial strategies?

The essential nature of EF-Tu for bacterial survival makes V. vulnificus tuf1 a promising target for novel antimicrobial development:

• Structure-based drug design approaches:

  • In silico screening for compounds that specifically bind to GTP-binding pocket of V. vulnificus tuf1

  • Fragment-based drug discovery targeting unique pockets in the tuf1 structure

  • Development of peptidomimetics that disrupt tuf1-aminoacyl-tRNA interactions

• Inhibitor validation methodology:

  • Biochemical assays measuring GTPase activity inhibition

  • Growth inhibition assays with potential tuf1 inhibitors

  • Molecular dynamics simulations to understand inhibitor binding mechanisms

  • Testing specificity against human elongation factors to ensure safety

• Innovative delivery strategies:

  • Phage-based delivery of tuf1-targeting compounds

  • Nanoparticle encapsulation for improved bioavailability in infection sites

  • Conjugation with V. vulnificus-specific targeting molecules

The persistent expression of tufA in various environmental conditions suggests that targeting tuf1 would be effective against V. vulnificus in different physiological states, including potential viable but nonculturable states mentioned in the literature . The high conservation of EF-Tu structure across bacterial species presents both an opportunity for broad-spectrum activity and a challenge for developing V. vulnificus-specific inhibitors.

How can CRISPR-Cas9 technology be applied to study tuf1 function in V. vulnificus?

CRISPR-Cas9 technology offers powerful approaches for studying tuf1 function, with specific adaptations required for V. vulnificus:

• Genetic manipulation strategies:

  • Conditional knockdown systems (CRISPRi) rather than knockouts, as tuf1 is likely essential

  • Introduction of point mutations to study specific residues involved in GTP binding or tRNA interactions

  • Domain swapping with EF-Tu from other species to identify functional differences

  • Promoter replacements to control expression levels

• Methodological considerations for V. vulnificus:

  • Optimization of CRISPR-Cas9 delivery into V. vulnificus (electroporation protocols, conjugation systems)

  • Selection of appropriate promoters for Cas9 and sgRNA expression in V. vulnificus

  • Development of inducible systems suitable for marine environments

  • Screening for off-target effects in the V. vulnificus genome

• Functional study applications:

  • Creation of fluorescently tagged tuf1 to monitor subcellular localization

  • Introduction of affinity tags for in vivo interaction studies

  • Engineering of tuf1 variants with altered nucleotide or aminoacyl-tRNA binding properties

Given the consistent expression of tufA across different environmental conditions , researchers should design CRISPR-based studies that examine tuf1 function in these diverse contexts, potentially revealing environment-specific roles that contribute to V. vulnificus pathogenicity and survival.

What insights can systems biology approaches provide about the interaction of tuf1 with other cellular components in V. vulnificus?

Systems biology approaches can reveal the complex integration of tuf1 in V. vulnificus cellular networks:

• Interactome mapping techniques:

  • Affinity purification coupled with mass spectrometry (AP-MS) using tagged recombinant tuf1

  • Bacterial two-hybrid screening for protein-protein interactions

  • Proximity labeling methods (BioID or APEX) to identify transient interactors

  • RNA-protein interaction studies to identify any potential moonlighting RNA-binding functions

• Network analysis frameworks:

  • Integration of interactome data with transcriptomics and proteomics

  • Comparison of tuf1 interaction networks between virulent and avirulent strains

  • Temporal dynamics of interactions during infection progression

  • Environmental condition-specific network remodeling

• Modeling approaches:

  • Constraint-based metabolic modeling to assess the impact of tuf1 activity on cellular metabolism

  • Kinetic modeling of translation incorporating tuf1 parameters

  • Comparative analysis with other Vibrio species

The continuous expression of tufA alongside other genes like rpoS (stress sigma factor), katG (periplasmic catalase), wza and wzb (capsule synthesis) suggests these genes may function in coordinated networks. Systems biology approaches can reveal whether these co-expressed genes interact functionally in stress response pathways, potentially explaining V. vulnificus's remarkable adaptability to diverse environments and its pathogenic capabilities.