Characterization of zinc ion uptake mediated by cntRLMN operon in Pseudomonas aeruginosa

Abstract:

[Objective] We characterized the function of cntRLMN in zinc ion uptake of Pseudomonas aeruginosa PAO1 (ATCC15692). [Methods] On the basis of the mutant strain ΔznuBC, we built ΔcntRLMN using homologous recombination. We also constructed its complementary strain and lacZ fusion report strain through plasmid conjugation transfer. Transcriptional regulation of Zur protein on cntRLMN was studied by beta-galactoside enzyme activity assay. In vitro binding of Zur protein to cnt promoter and mutated fragments of cnt promoter was tested using EMSA. The zinc ion uptake function of cntR, cntL, cntN and other genes in the cntRLMN operon was examined through growth curve analysis. Finally, the influence of cntRLMN mutation on the virulence of P. aeruginosa was studied using the infection model of the wax moth larva. [Results] The enzyme activity analysis of lacZ transcription and fusion showed that cntRLMN was negatively regulated by Zur protein, and its expression was induced by zinc ion starvation in a Zur-dependent manner. The EMSA results showed that the promoter of cntRLMN could bind His-Zur to form a DNA-protein complex, where the binding site was GCGTTATAGTATATCAT. The growth curve and the infection experiment of the wax moth larva showed that ZnuBC and CntRLMN were functionally complementary; when grown in Zn-restricted culture, the toxicity of P. aeruginosa to the wax moth larve was significantly inhibited only in the znuBC and cntRLMN double-deleted strain. [Conclusion] The cntRLMN, which plays an important role in toxicity of P. aeruginosa, is likely an alternative zinc ion uptake system directly and negatively regulated by the Zur protein.