Recombinant Vibrio vulnificus Zinc import ATP-binding protein ZnuC (znuC)

Overview of the ZnuABC System

The ZnuABC transporter is a high-affinity zinc uptake system in Gram-negative bacteria, comprising:

• ZnuA: A periplasmic zinc-binding protein.

• ZnuB: A transmembrane permease.

• ZnuC: A cytoplasmic ATPase that hydrolyzes ATP to energize zinc transport .

In Vibrio parahaemolyticus, the znuABC operon (loci VP_RS04150–VP_RS04160) is upregulated under zinc-limiting conditions and regulated by the Zur (Zinc uptake regulator) protein . Homologs of ZnuC across Vibrionaceae share conserved ATP-binding motifs critical for zinc import .

Functional Role of ZnuC in Zinc Acquisition

ZnuC drives the ATP-dependent translocation of zinc across the cytoplasmic membrane. Key characteristics include:

• ATPase Activity: ZnuC hydrolyzes ATP to provide energy for zinc transport .

• Regulation: Expression is tightly controlled by Zur under zinc-deficient conditions .

• Redundancy: Some Vibrio species, like V. cholerae, employ dual zinc transporters (ZnuABC and ZrgABCDE), but ZnuABC often dominates in zinc acquisition .

Comparative Analysis of Zinc Transporters in Vibrionaceae

In V. parahaemolyticus, ZnuABC contributes to zinc acquisition, but ZrgA (from the ZrgABCDE system) plays a more critical role under zinc limitation . This contrasts with V. cholerae, where ZnuABC is primary .

Research Gaps and Implications for V. vulnificus

While V. vulnificus possesses homologs of the ZnuABC system (e.g., znuA2 and znuB2 in some isolates ), no studies in the provided sources directly characterize recombinant ZnuC. Key unresolved questions include:

• Functional Redundancy: Does V. vulnificus rely on ZnuABC, ZrgABCDE, or other systems (e.g., TonB-dependent receptors) for zinc uptake?

• Regulatory Mechanisms: How does Zur regulate znuC in V. vulnificus compared to other Vibrio species?

• Pathogenic Role: Is ZnuC essential for virulence or survival in host environments?

Methodological Insights from Homologs

Studies on V. cholerae ZnuC provide a framework for investigating recombinant ZnuC in V. vulnificus:

• Expression Systems: Escherichia coli is commonly used for recombinant protein production (e.g., V. vulnificus metalloprotease in ).

• Activity Assays: ATPase activity can be measured via malachite green assays or NADH-coupled methods .

• Structural Analysis: Homology modeling based on Pseudomonas aeruginosa ZrgA (PDB ID: 6V6X) could predict zinc-binding sites .

Key Challenges in Characterizing Recombinant ZnuC

• Protein Stability: Zinc-binding proteins often require metal cofactors for proper folding.

• Functional Complementation: Validating activity may require assays in znuC knockout strains .

• Regulatory Crosstalk: Overlap with other metal transporters (e.g., iron-regulated TonB systems ) complicates phenotypic analysis.

Future Directions

1. Heterologous Expression: Clone znuC from V. vulnificus into plasmid vectors (e.g., pET series) for overexpression in E. coli.

2. Biochemical Characterization: Assess ATP hydrolysis kinetics and zinc affinity using ITC (isothermal titration calorimetry).

3. Genetic Knockouts: Compare growth of ΔznuC mutants under zinc limitation to define its role in vivo.