The sprotein PhrS-P, encoded by the dual-function small RNA PhrS, augments aminoglycoside resistance in Pseudomonas aeruginosa

PhrS is a dual-function small RNA of Pseudomonas aeruginosa (Pae). The base-pairing small regulatory RNA PhrS-R positively regulates pyocyanin (PYO) biosynthesis. PhrS-R stimulates the production of the Pseudomonas quinolone signal (PQS) by indirectly activating the translation of pqsR mRNA, encoding the quorum sensing (QS) regulator PqsR (MvfR), and by inhibiting the synthesis of the anthranilate degradation regulator AntR. Furthermore, PhrS-R was shown to promote biofilm formation, swarming motility, and CRISPR-Cas immunity. In addition, PhrS encodes a 37 amino acid long and highly conserved small protein (PhrS-P), the function of which remained unknown. Here, we provide evidence that PhrS-P is an inner membrane protein, which does not contribute to the known physiological roles of its RNA counterpart. RNA-Seq based transcriptome analyses revealed that multiple genes of the AmgRS regulon are differentially expressed depending on PhrS-P. AmgR is the response regulator of the AmgRS two-component system (TCS), which affects aminoglycoside and zinc resistance in Pae. In vivo co-purification studies coupled to mass spectrometry identified the sensor kinase AmgS as an interacting partner of PhrS-P. A PhrS-P deficient mutant strain exhibited reduced fitness when exposed to sub-inhibitory concentrations of the aminoglycosides tobramycin and amikacin, as well as zinc. Based on our findings, we present a working model wherein PhrS-P positively regulates the AmgRS TCS activity, leading to increased aminoglycoside resistance.