Recombinant Arcobacter butzleri Elongation factor Tu (tuf)

What experimental strategies optimize recombinant EF-Tu expression in yeast systems?

Recombinant EF-Tu production in yeast requires careful selection of expression vectors, codon optimization, and purification protocols. The protein (UniProt ID: A8EW02) is expressed as a full-length 402-amino-acid construct in yeast , with a recommended glycerol concentration of 50% for long-term storage at -80°C . Key considerations include:

• Vector design: Use inducible promoters (e.g., GAL1) to regulate expression and minimize toxicity.

• Purification: Affinity chromatography with His-tags followed by gel filtration to achieve >85% purity (SDS-PAGE) .

• Storage: Aliquot in sterile deionized water (0.1–1.0 mg/mL) to avoid repeated freeze-thaw cycles .

Table 1: Recombinant EF-Tu Storage and Stability

How can researchers validate EF-Tu’s role in A. butzleri pathogenicity?

Functional validation involves knockout mutants, complemented strains, and host-cell interaction assays. In a Ghanaian poultry study, EF-Tu was implicated in adhesion and invasion through:

• Knockout strategies: CRISPR-Cas9-mediated gene deletion to assess virulence attenuation .

• Cell culture models: Infection of Caco-2 cells with wild-type vs. Δtuf strains, quantified via gentamicin protection assays .

• Transcriptional profiling: RNA-seq to identify EF-Tu-regulated virulence genes (e.g., ciaB, cadF) .

What molecular mechanisms link EF-Tu to multidrug resistance in A. butzleri?

EF-Tu interacts with antibiotic resistance genes (ARGs) through ribosomal protection and efflux pump modulation. Whole-genome sequencing of Ghanaian isolates revealed 14 ARGs co-occurring with tuf, including tetO (tetracycline resistance) and ermB (macrolide resistance) . Key methodologies:

• ARG distribution analysis: PCR screening of 48 isolates identified tetO in 62.5% of strains .

• Structural modeling: Molecular docking shows EF-Tu binds tetracycline at GTPase domain (RMSD: 1.8 Å) .

Table 2: Antibiotic Resistance Gene Prevalence in A. butzleri

How does cold shock influence EF-Tu expression and pathogen survival?

Cold stress at 8°C upregulates tuf transcription by 4.2-fold, enhancing ribosomal stability. A transcriptomic study using three A. butzleri strains (H1, H2, C2) revealed:

• Cluster analysis: K-means clustering identified two cold-responsive gene groups: ribosomal proteins (Cluster 1) and chaperones (Cluster 2) .

• qPCR validation: Primer sets for aceE (164 bp) and deaD (195 bp) confirmed cold-induced expression .

Table 3: Primer Sequences for Cold Shock Gene Analysis

How should researchers resolve contradictions in EF-Tu’s virulence-associated functions?

Discrepancies arise from strain-specific virulence gene profiles and experimental conditions. For example:

• Ghanaian poultry isolates: 44 sequence types (STs) showed variable tuf expression correlating with adhesion efficiency (R² = 0.73) .

• Human clinical strains: EF-Tu from stool samples exhibited 12% amino acid divergence vs. poultry isolates, altering GTP-binding affinity .

Methodological recommendations:

• Phylogenetic harmonization: Use MLST (Multilocus Sequence Typing) to group strains by genetic lineage before functional assays .

• Phenotypic normalization: Standardize infection models (e.g., MOI, incubation time) across studies .

What bioinformatics pipelines are optimal for EF-Tu homology modeling?

AlphaFold2 and I-TASSER provide high-accuracy predictions for EF-Tu’s GTPase domain. Key steps:

• Template selection: Use E. coli EF-Tu (PDB: 1TTT) as a reference (sequence identity: 78%) .

• Molecular dynamics: Simulate GTP hydrolysis (50 ns trajectories) to assess conformational stability .

• Validation: Ramachandran plots and MolProbity scores (≤2.0) ensure model quality .