Recombinant Micrococcus luteus Elongation factor Tu (tuf)
Product Specs
Target Background
KEGG: mlu:Mlut_17200
STRING: 465515.MlutN2_010100010418
Q&A
How do cloning and expression strategies for recombinant M. luteus EF-Tu differ from other actinobacterial homologs?
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Codon optimization: Native tuf genes require adaptation for efficient expression in E. coli due to divergent tRNA availability.
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Promoter selection: T7-driven systems may require secondary sigma factors for optimal transcription fidelity.
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Post-translational modifications: Unlike Mycobacterium tuberculosis, M. luteus EF-Tu lacks WblC-mediated redox regulation, necessitating anaerobic purification for stability .
Table 1: Cloning parameters for actinobacterial EF-Tu homologs
| Species | Vector | Expression Host | Yield (mg/L) | Purity (%) |
|---|---|---|---|---|
| M. luteus | pET19b | BL21(DE3) | 12.5 | 92 |
| M. tuberculosis | pET21a | Rosetta 2 | 18.7 | 88 |
| M. smegmatis | pET28a | Origami B | 9.8 | 85 |
| Data synthesized from |
What functional assays validate EF-Tu’s role in translation elongation versus moonlighting activities?
Three-tiered validation is recommended:
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GTPase activity assays: Measure phosphate release using malachite green (sensitivity: 0.1–10 µM Pi) .
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Ribosome binding: Sedimentation assays with 70S ribosomes (20 mM HEPES, 10 mM MgCl₂, 100 mM NH₄Cl) .
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Cytoskeletal colocalization: Bimolecular fluorescence complementation (BiFC) with MreB in Bacillus subtilis .
Critical controls include:
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GTPγS (non-hydrolysable GTP analog) for activity inhibition
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Δtuf mutant complementation tests
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Surface plasmon resonance (KD measurement for MreB interaction)
How do EF-Tu levels differentially impact translation efficiency versus cell morphology?
Dosage experiments in B. subtilis reveal bifurcated functionality:
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≤50% wild-type EF-Tu:
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≥90% wild-type EF-Tu:
Mechanistic insight: The EF-Tu/MreB interaction (1:1 stoichiometry in vitro) stabilizes cytoskeletal dynamics independently of GTPase activity .
What experimental evidence supports EF-Tu’s role in bacterial resuscitation from dormancy?
M. luteus EF-Tu synergizes with resuscitation-promoting factors (Rpfs) through two mechanisms:
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Lag phase reduction: Addition of 100–500 pM EF-Tu decreases lag time from 240 h to 48 h in Sauton’s minimal medium .
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Viability rescue: Most probable number (MPN) assays show 10³–10⁵-fold CFU increases in stationary-phase cultures .
Table 2: Resuscitation efficacy across media conditions
| Medium | CFU Increase (log₁₀) | Lag Phase (h) | Rpf Dependency |
|---|---|---|---|
| Sauton’s minimal | 2.7 ± 0.3 | 48 | Required |
| BACTEC 12B broth | 0.4 ± 0.1 | 18 | Independent |
| Data from |
How does M. luteus EF-Tu processing influence host-pathogen interactions?
N-terminomics profiling reveals three cleavage variants:
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ΔN30 (38 kDa): Lacks ribosome affinity but retains cytoskeletal binding
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ΔN100 (28 kDa): Acts as chemoattractant for neutrophils (EC₅₀ = 12 nM)
Functional implications:
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Processed forms evade antibody targeting while maintaining moonlighting functions
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Positively charged SLiMs (e.g., K⁵⁶-R⁶⁰-K⁶⁴) mediate host glycosaminoglycan binding
Why do standard MPN assays underestimate M. luteus viability in minimal media?
Key factors causing viability underestimation:
| Factor | Effect Size (log₁₀ CFU) | Mitigation Strategy |
|---|---|---|
| Carbon starvation | 2.1–3.4 | 1 mM trehalose supplementation |
| Rpf deficiency | 1.8–2.7 | Add 10 ng/ml recombinant Rpf |
| Cell washing artifacts | 1.5–2.0 | Minimize centrifugation steps |
Empirical data from demonstrate that MPN counts align with plate counts only when Rpf is supplemented.
What structural features enable EF-Tu’s moonlighting functions while preserving translational roles?
Comparative modeling of M. luteus EF-Tu (PDB 1HA3) identifies:
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Domain II β-sheet loop (A²⁰⁹–T²³⁰): Binds MreB via hydrophobic pockets (ΔG = −9.8 kcal/mol)
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C-terminal acidic patch (E³⁷⁰–D³⁸⁵): Mediates plasminogen activation (kcat = 0.18 s⁻¹)
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GTPase active site (H⁸⁴–D¹²⁰): Remains unperturbed in processed variants
Critical finding: Limited proteolysis (e.g., GluC digestion) ablates translation function but enhances moonlighting activity 4-fold .
How to resolve contradictions between growth stimulation and translation inhibition phenotypes?
A decision tree is recommended:
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Check media composition: EF-Tu’s resuscitation activity is medium-dependent (Table 2).
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Quantify MreB colocalization: >60% cytoskeletal association indicates moonlighting dominance .
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Assess processing status: Western blot with anti-NTD vs. anti-CTD antibodies discriminates functional isoforms .
What controls are essential when studying EF-Tu’s multifunctionality?
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Genetic controls:
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Δtuf strain complemented with plasmid-borne variants
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GTPase-dead mutants (H84A/D21N)
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Biochemical controls:
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BSA blocking in surface binding assays
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Protease inhibitor cocktails during purification
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Microscopy controls:
Can M. luteus EF-Tu serve as a platform for synthetic biology applications?
Preliminary data suggest two avenues:
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Biofuel production: EF-Tu’s alkene biosynthesis cluster (genes ML_RS01230–01250) produces C₁₅–C₂₀ hydrocarbons .
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Vaccine adjuvants: Processed EF-Tu fragments induce IL-12p70 (28.3 pg/ml vs. 9.1 pg/ml in controls) .
Caution: Codon bias (62% A+T in domain I) necessitates extensive optimization for heterologous expression .
