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ABSTRACT Pseudomonas aeruginosa thrive to survive in harsh conditions imposed by the host. We have previously described two Zn 2+ -transporting members of the Cation Diffusion Facilitator family, YiiP and CzcD, inhibiting susceptibility to imipenem by decreasing the expression of the outer membrane porin OprD (A. Salusso and D. Raimunda, Front Cell Infect Microbiol 7:84, 2017, https://doi.org/10.3389/fcimb.2017.00084 ). Here, we provide evidence that a protein encoded in yiiP ’s operon, PA3962, is fundamental to the coupling of these processes. Immunodetection assay indicates that PA3962 locates in membranes. Supporting a role in oprD regulation, a PA3962 insertional mutant has a significant increase of oprD expression levels and imipenem sensitivity, which is suppressed by gene complementation but not in the presence of Zn 2+ , as opposed to the YiiP mutant. We identified two pairs of conserved acidic residues in a hydrophobic juxtamembrane domain. Metal binding specificity and stoichiometry were explored in wild-type and mutant versions of these. Zn 2+ appears as the cognate metal of PA3962, with residues D40, D47, and D65 required for its coordination. As the periplasmic Zn 2+ -sensor CzcS regulates oprD expression, the interaction with it was analyzed in vitro . Interaction was Zn 2+ -dependent, and mutations of D47A or D65A abolished it. We propose a role for PA3962, hereafter periplasmic metallochaperone of YiiP (PmcY), in the context of Zn 2+ signaling pathways in P. aeruginosa . The relay YiiP–PmcY supplies Zn 2+ and activates CzcS/CzcR, down-regulating the transcription of the imipenem-permeable OprD. This mechanism would allow P. aeruginosa to put a brake on unspecific mechanisms for micronutrient uptake, with potential xenobiotic entry, while the cytosolic Zn 2+ quota is still sufficient.
Mihelj et al. (Fri,) studied this question.