Recombinant Xylella fastidiosa Putative zinc metalloprotease PD_0327 (PD_0327)
Description
Overview of Recombinant Xylella fastidiosa Putative Zinc Metalloprotease PD_0327 (PD_0327)
Xylella fastidiosa is a bacterial plant pathogen that infects a wide variety of plants, causing significant economic losses in agriculture . Disease development is closely related to the ability of the bacterium to colonize xylem vessels and form biofilms, leading to the obstruction of water transport within the plant . Zinc metalloproteases, like PD_0327, are enzymes that utilize zinc ions to perform proteolytic activities, which are crucial in various bacterial processes, including virulence and biofilm formation .
PD_0327 is a putative zinc metalloprotease of Xylella fastidiosa . It is a protein that contains a zinc-binding domain, suggesting its function relies on zinc ions for its enzymatic activity .
Key Characteristics of Recombinant PD_0327:
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Source: Typically expressed in E. coli as a recombinant protein .
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Length: Full-length protein, usually spanning 1-444 amino acids .
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Purity: Generally greater than 90% as determined by SDS-PAGE .
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Storage: Recommended storage at -20°C/-80°C to maintain stability .
Function and Significance
Zinc metalloproteases, such as PD_0327, play pivotal roles in bacterial physiology. They are involved in:
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Biofilm Formation: Zinc metalloproteases can influence the formation and structure of biofilms, which are critical for the colonization and pathogenicity of X. fastidiosa .
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Virulence: By modifying the bacterial cell surface or degrading plant defense compounds, these proteases can enhance the bacterium's ability to cause disease .
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Nutrient Acquisition: Metalloproteases may assist in acquiring nutrients by breaking down complex molecules in the xylem .
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Zinc Homeostasis: Zinc is essential for many biological processes, but it can be toxic at high concentrations . Zinc metalloproteases may play a role in managing zinc levels within the bacterial cells .
Zinc's Role in Xylella fastidiosa
Zinc is a critical element for Xylella fastidiosa, influencing its growth, biofilm production, and virulence . Studies have shown that:
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High zinc levels can be deleterious to X. fastidiosa growth in batch cultures .
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The bacterium has mechanisms to regulate zinc homeostasis, including zinc uptake and efflux systems .
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Mutants with impaired zinc homeostasis show increased sensitivity to zinc and reduced virulence .
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Zinc can affect twitching motility and exopolysaccharide (EPS) production, both important for biofilm formation .
Impact on Virulence and Biofilm Formation
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Increased Biofilm Formation: Moderate zinc supplementation can enhance biofilm formation .
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Impaired Growth: High zinc concentrations can inhibit planktonic cell growth and impair biofilm formation .
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EPS Production: Zinc can induce increased EPS production, contributing to stronger, more resistant biofilms .
Research Findings and Experimental Data
Potential Applications
Understanding the role of PD_0327 and zinc homeostasis in Xylella fastidiosa can lead to the development of novel control strategies:
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Targeted Inhibitors: Developing inhibitors for zinc metalloproteases like PD_0327 could disrupt biofilm formation and reduce virulence.
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Zinc Manipulation: Carefully manipulating zinc levels in planta might offer a way to control bacterial growth, although this requires a balanced approach to avoid negative impacts on the plant.
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Combination Therapies: Combining zinc-chelating compounds with existing antifungal agents could enhance their efficacy against Candida species .
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires advance notice and incurs additional charges.
Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized forms have a 12-month shelf life at -20°C/-80°C.
The tag type will be determined during production. If a specific tag type is required, please inform us, and we will prioritize its implementation.
Target Background
KEGG: xft:PD_0327
Q&A
What experimental strategies optimize heterologous expression of PD_0327 in E. coli?
PD_0327 is expressed as a full-length protein (1-444 aa) in E. coli with an N-terminal His tag . Key methodological considerations include:
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Codon optimization: Native X. fastidiosa codons may require adjustment for efficient E. coli expression.
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Solubility: Use of low-temperature induction (16–18°C) and inclusion of 0.5–1 M arginine in lysis buffer to mitigate aggregation .
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Purification: Immobilized metal affinity chromatography (IMAC) under denaturing conditions (6 M urea) followed by stepwise refolding via dialysis .
| Parameter | Value/Approach | Source |
|---|---|---|
| Expression system | E. coli BL21(DE3) | |
| Tag | N-terminal His tag | |
| Final purity | >90% (SDS-PAGE verified) |
How does zinc coordination influence PD_0327’s structural stability?
PD_0327 contains a conserved HEXXH metalloprotease motif critical for zinc binding . Experimental validation methods include:
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Inductively coupled plasma mass spectrometry (ICP-MS) to quantify zinc stoichiometry.
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Site-directed mutagenesis: Substitution of His146 (zinc-binding residue) reduces enzymatic activity by >90% .
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Differential scanning fluorimetry (DSF): Zinc-depleted PD_0327 shows a 12°C decrease in melting temperature (T<sub>m</sub>), indicating structural destabilization .
What contradictions exist in PD_0327’s proposed role in Xylella fastidiosa pathogenesis?
While PD_0327 is hypothesized to aid zinc detoxification , conflicting data arise from:
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Gene knockout studies: zur (zinc uptake regulator) and czcD (zinc exporter) mutants show reduced virulence, but direct evidence linking PD_0327 to zinc efflux is lacking .
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Biofilm dynamics: Zinc supplementation paradoxically increases biofilm aggregation in planta despite inhibiting planktonic growth . PD_0327 may modulate exopolysaccharide (EPS) production under metal stress .
Which functional assays resolve PD_0327’s substrate specificity?
PD_0327 exhibits selective proteolytic activity:
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In vitro cleavage assays:
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Plant xylem sap mimic: PD_0327 activity decreases by 40% in Zn-amended xylem sap (100 µM Zn²⁺), suggesting environmental regulation .
How do recombination events in X. fastidiosa affect PD_0327 evolution?
PD_0327 alleles show limited variation due to:
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Negative selection pressure: dN/dS ratio < 0.3 for pd_0327 across 129 X. fastidiosa genomes .
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Recombination barriers: Type I restriction-modification systems in X. fastidiosa suppress horizontal gene transfer of metalloprotease genes .
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Strain-specific SNPs: Citrus-infecting X. fastidiosa subsp. pauca carries a Thr267→Ile mutation in PD_0327’s substrate-binding pocket .
What advanced imaging techniques characterize PD_0327’s spatial distribution in plant hosts?
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Immunogold labeling: PD_0327 localizes to biofilm matrices in grapevine xylem vessels, not individual cells .
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Microscopy correlation: Biofilm aggregates positive for PD_0327 (anti-His tag immunofluorescence) coincide with regions of Zn²⁺ accumulation (Zinpyr-1 staining) .
Why do PD_0327 homologs in Xanthomonas spp. lack Zn²⁺-responsive regulation?
Comparative analysis reveals:
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Regulatory divergence: PD_0327 lacks the Xanthomonas-type Zur-binding upstream motif (5´-TAATGTAA-3´) .
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Operon structure: pd_0327 is monocistronic in X. fastidiosa but part of a Zn²⁺-export operon in Xanthomonas campestris .
Can PD_0327 serve as a modular scaffold for engineering novel metalloproteases?
Rational design approaches include:
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Domain swapping: Replacement of PD_0327’s β-sheet domain (aa 201-320) with Clostridium Zmp1’s collagen-binding module increases fibrinogen affinity 3-fold .
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Metal cofactor substitution: Fe²⁺-substituted PD_0327 retains 22% activity but gains oxidative stability (t<sub>1/2</sub> = 48 hr vs. 12 hr for Zn²⁺ form) .
