Abstract
Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation–PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host.
Highlights
Bacterial pathogens must acquire iron from their host for survival and virulence where because of its reactivity, it is tightly regulated and sequestered in iron-binding proteins, such as transferrin and ferritin, or in heme and iron–sulfur cluster–containing proteins
Through chromatin immunoprecipitation–PCR (ChIP–PCR), EMSA, and fluorescence anisotropy (FA), we show that apo-PhuS binds with high affinity to the promoter of prrF1 but not that of prrF2
We propose that PhuS has a dual function integrating extracellular heme metabolism into the iron-homeostasis networks through transcriptional modulation of the PrrF/ PrrH sRNAs
Summary
The heme-binding protein PhuS transcriptionally regulates the Pseudomonas aeruginosa tandem sRNA prrF1,F2 locus.
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