Cu+-importing Phenotypes of ΔCuiT strain and the Expression Optimization of P1B-ATPases in Pseudomonas aeruginosa

Xu, Cheng

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Cu+-importing Phenotypes of ΔCuiT strain and the Expression Optimization of P1B-ATPases in Pseudomonas aeruginosa

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Copper is required as cofactor for many enzymes in bacteria, but excess copper can be toxic due to the ability to produce free radicals and disrupting Fe-S clusters in specific proteins. Therefore, bacteria need to maintain copper homeostasis. In Pseudomonas aeruginosa, the expression levels of various proteins are changed to alleviate copper stress conditions, and this suggests that the proteins must play a role in maintaining copper homeostasis. The roles of some upregulated proteins have been described recently. Chaperone CopZs distribute copper in the cytoplasm, while ATPases CopAs efflux the excess copper to the periplasm. However, the biochemical properties of CopAs have not been characterized yet. Downregulated transmembrane proteins are likely acting as copper importers, but these have received little attention. To address these questions, we focused our research on the copper-importing phenotypes of the putative CuiT importer and optimizing the expression of the P1B-ATPases. The copper-importing phenotypes of CuiT were investigated by three methods (growth rate, cell survival, and copper uptake assays). Results showed that ΔCuiT strain accumulated less copper than WT within 10 min of incubation, while longer incubation (more than 12 hours) in the other two experimental approaches showed no phenotype. The optimizations of the sequences and expression conditions of both ΔN-CopA1 and ΔN-CopA2 were investigated. The results suggested that the expression of codon-optimized ΔN- CopA1 and ΔN-CopA2 was significantly improved, and the optimum conditions for ΔN-CopA1 and ΔN-CopA2 expression were obtained. In summary, the work here provides initial data supporting the copper-importing role of CuiT and optimum parameters for expressing for ΔN- CopA1 and ΔN-CopA2.

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