Abstract
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 Pseudomonas aeruginosa, a Zur protein has yet to be identified experimentally, however, sequence alignment revealed that the zinc-responsive transcriptional regulator Np20, encoded by np20 (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 P. aeruginosa. Using RT-PCR, we determined that np20 (hereafter known as zur) formed a polycistronic operon with znuC and znuB. Mutant strains, lacking the putative znuA, znuB, or znuC genes were found to grow poorly in zinc deplete conditions as compared to wild-type strain PAO1. Intracellular zinc concentrations in strain PAO-Zur (Δzur) were found to be higher than those for strain PAO1, further implicating the zur as the zinc uptake regulator. Reporter gene fusions and real time RT-PCR revealed that transcription of znuA was repressed in a zinc-dependent manner in strain PAO1, however zinc-dependent transcriptional repression was alleviated in strain PAO-Zur, suggesting that the P. aeruginosa Zur homolog (ZurPA) directly regulates expression of znuA. Electrophoretic mobility shift assays also revealed that recombinant ZurPA specifically binds to the promoter region of znuA and does not bind in the presence of the zinc chelator N,N′,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN). Taken together, these data support the notion that Np20 is the P. aeruginosa Zur, which regulates the transcription of the genes encoding the high affinity ZnuABC zinc transport system.
Highlights
Zinc is an essential trace element required for virtually all forms of life
ZurPA is Highly Similar to zinc uptake regulator (Zur) Found in Other Bacteria If the protein encoded by np20 is the P. aeruginosa Zur, it would be expected to share sequence similarity with Zur proteins found in other bacteria
The occurrence of the putative C and M zinc binding sites in ZurPA and the high overall similarity between ZurPA and Zur proteins from other bacteria strongly suggested that the protein encoded by np20 is the P. aeruginosa Zur (ZurPA)
Summary
The importance of the metal is demonstrated by the fact that 4% to 10% of all proteins found throughout the domains of life are zinc metalloproteins [1]. Zinc has been shown to protect against harmful free radical formation which can occur through normal aerobic metabolism within a cell [2]. Even though the intracellular zinc concentrations may be 1000-higher than the extracellular environment, there is typically no free zinc ions found inside the cell [3]. This suggests that the cell possesses means to actively take in the metal and to prevent excess free zinc from accumulating within the bacterium
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