The Transcriptional Regulator Np20 Is the Zinc Uptake Regulator in <i>Pseudomonas aeruginosa</i>

<div><p>Zinc is essential for all bacteria, but excess amounts of the metal can have toxic effects. To address this, bacteria have developed tightly regulated zinc uptake systems, such as the ZnuABC zinc transporter which is regulated by the Fur-like zinc uptake regulator (Zur). In <i>Pseudomonas aeruginosa</i>, a Zur protein has yet to be identified experimentally, however, sequence alignment revealed that the zinc-responsive transcriptional regulator Np20, encoded by <i>np20</i> (PA5499), shares high sequence identity with Zur found in other bacteria. In this study, we set out to determine whether Np20 was functioning as Zur in <i>P. aeruginosa</i>. Using RT-PCR, we determined that <i>np20</i> (hereafter known as <i>zur</i>) formed a polycistronic operon with <i>znuC</i> and <i>znuB</i>. Mutant strains, lacking the putative <i>znuA, znuB</i>, or <i>znuC</i> genes were found to grow poorly in zinc deplete conditions as compared to wild-type strain PAO1. Intracellular zinc concentrations in strain PAO-Zur (Δ<i>zur</i>) were found to be higher than those for strain PAO1, further implicating the <i>zur</i> as the zinc uptake regulator. Reporter gene fusions and real time RT-PCR revealed that transcription of <i>znuA</i> was repressed in a zinc-dependent manner in strain PAO1, however zinc-dependent transcriptional repression was alleviated in strain PAO-Zur, suggesting that the <i>P. aeruginosa</i> Zur homolog (ZurPA) directly regulates expression of <i>znuA</i>. Electrophoretic mobility shift assays also revealed that recombinant ZurPA specifically binds to the promoter region of <i>znuA</i> and does not bind in the presence of the zinc chelator <i>N,N</i>′,<i>N</i>-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN). Taken together, these data support the notion that Np20 is the <i>P. aeruginosa</i> Zur, which regulates the transcription of the genes encoding the high affinity ZnuABC zinc transport system.</p>