Recombinant Xylella fastidiosa Copper homeostasis protein CutC (cutC)
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Target Background
KEGG: xft:PD_0586
Q&A
What experimental approaches confirm CutC’s copper-binding function in Xylella fastidiosa?
To validate CutC’s copper-binding capacity, researchers employ:
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Recombinant protein expression: Heterologous expression in E. coli followed by affinity chromatography purification .
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In vitro binding assays: Use of inductively coupled plasma mass spectrometry (ICP-MS) or isothermal titration calorimetry (ITC) to quantify Cu(I/II) binding .
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Structural analysis: X-ray crystallography (e.g., PDB IDs 5A0U, 5A0Z) to identify copper-coordinating residues like Cys156 and His220 in the GRE domain .
Table 1: Structural Parameters of CutC from Crystallography Studies
| Parameter | Choline-Bound (5A0U) | Choline-Free (5A0Z) |
|---|---|---|
| Space Group | P2₁2₁2₁ | P2₁ |
| Resolution (Å) | 2.4 | 3.0 |
| R-factor | 0.19 | 0.23 |
| Cu-Binding Residues | Cys156, His220 | Conserved |
How does cutC deletion alter Xylella fastidiosa’s copper stress response?
Comparative studies between wild-type (WT) TemeculaL and ΔcutC mutants reveal:
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Increased copper sensitivity: ΔcutC shows 3.2-fold reduced viability after 24-hour exposure to 0.5 mM CuSO₄ .
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Virulence attenuation: In greenhouse trials, ΔcutC reduces disease incidence by 40% in grapevines but exacerbates symptoms when exogenous copper is applied .
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Spatiotemporal symptom modulation: ΔcutC infections show delayed acropetal movement, suggesting CutC facilitates systemic colonization under copper stress .
What controls are essential for in planta copper homeostasis experiments?
Robust experimental design requires:
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Copper quantification: Atomic absorption spectroscopy of xylem sap to baseline endogenous copper levels .
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Genetic controls: Complemented ΔcutC strains to confirm phenotype reversibility .
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Environmental controls: Standardized hydroponic copper regimes (e.g., 0–1.0 mM CuSO₄) to avoid confounding soil variability .
How can researchers resolve contradictions in copper’s dual role as a stressor and virulence enhancer?
Conflicting observations arise from:
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Dose-dependent effects: Low copper (≤0.2 mM) upregulates cutC expression and enhances biofilm formation, while high copper (>0.5 mM) induces oxidative stress .
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Host-specific responses: Copper’s antimicrobial activity in Citrus vs. its nutritional role in Vitis xylem .
Methodological resolution:
What strategies address genetic variability in recombinant CutC studies?
Intersubspecific recombination between X. fastidiosa subspecies introduces allelic heterogeneity. Mitigation approaches include:
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MLST-based screening: Use of 7-locus multilocus sequence typing to confirm strain purity .
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Recombination-aware phylogenies: Tools like ClonalFrameML to identify IHR (intersubspecific homologous recombination) regions in cutC loci .
Table 2: Phenotypic Comparison of WT vs. ΔcutC in Copper Stress
| Trait | WT TemeculaL | ΔcutC Mutant |
|---|---|---|
| LC50 (CuSO₄, 24 hr) | 1.8 mM | 0.56 mM |
| Biofilm formation | 2.1-fold increase | No change |
| Systemic spread (days) | 14–21 | 28–35 |
How can quasi-experimental designs improve cutC functional studies in field conditions?
When randomization is impractical (e.g., established vineyards), use:
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Non-equivalent group designs: Compare naturally copper-rich vs. copper-deficient plots, controlling for soil pH and organic matter .
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Regression discontinuity: Apply copper treatments at threshold xylem concentrations (e.g., 0.3 µM Cu) to isolate cutC’s role in detoxification .
What structural insights guide mutagenesis studies of CutC’s GRE domain?
The GRE domain (residues 305–932) contains:
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Radical SAM motif: Cys172, Cys176, and Cys179 coordinate the [4Fe-4S] cluster essential for choline lyase activity .
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Substrate-binding pocket: Trp318 and Phe414 stabilize choline via π-cation interactions .
Targeted mutagenesis protocol:
Methodological Recommendations
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Copper shock assays: Standardize exposure times to ≤2 hours to isolate acute stress responses .
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In planta imaging: Employ confocal microscopy with Cu-specific fluorophores (e.g., Phen Green SK) to map CutC’s spatial activity .
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Data contradiction analysis: Apply causal inference frameworks like Rubin’s model to disentangle copper’s direct vs. host-mediated effects .
