PhuS conformational dynamics are essential for DNA binding and heme-responsive control of the prrF operon in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa chronic infections, heme is a primary source of the essential micronutrient iron. The cytoplasmic heme-binding protein, PhuS, regulates extracellular heme flux through its interaction with the iron-regulated heme oxygenase (HemO). Additionally, in its apo-state, PhuS modulates iron homeostasis by transcriptionally regulating the prrF1,2 sRNA genes. These two functions are mutually exclusive and dependent on the conformational rearrangement of PhuS upon heme binding and coordination. Herein, we characterize a PhuS R25A variant that shows similar heme-binding kinetics and transfer of heme to HemO as PhuS WT, while DNA-binding to the prrF1 promoter is completely lost, successfully uncoupling the two functions. HDX-MS analysis revealed an overall decrease in conformational dynamics of apo- and holo-PhuS R25A compared with their WT counterparts, demonstrating the importance of conformational flexibility for DNA binding. qRT-PCR and Northern blot analysis comparing the phuSR25A allelic mutant strain to the PAO1 WT showed a significant decrease in PrrF and PrrH levels and revealed PhuS-dependent differences in regulation over PrrF1 and PrrF2, altering the relative ratio of these two sRNAs in a heme-specific manner that is distinct from iron. By removing its DNA-binding function, we elucidated the direct effects of PhuS binding on PrrF expression, separate from its effects on heme transfer and utilization. The contrasting effects on gene expression of the tandem sRNAs PrrF1 and PrrF2 in iron and heme and the resulting distinct mRNA profiles may allow the bacteria a fitness advantage in establishing chronic infection.