The nucleoid-associated protein subunit HupA positively regulates the Pqs system and pyocyanin production in <i>Pseudomonas aeruginosa</i>

<i>Pseudomonas aeruginosa,</i> a ubiquitous opportunistic pathogen, coordinates virulence gene expression primarily through three quorum-sensing (QS) systems: Las, Rhl, and Pqs. Despite the functions of QS systems in <i>P. aeruginosa</i> being relatively well-documented, the regulatory mechanisms modulating their activities remain largely unexplored. Here, we isolated and characterized a Las-null strain, PA_HN008, which overproduces pyocyanin (PYO) at the late growth stage due to its active RhlI-RhlR-PqsE pathway. Leveraging PYO production as a phenotypic marker, we conducted a genome-wide screening for upstream QS modulators and successfully identified PA5348 (designated as HupA), the α subunit of the nucleoid-associated protein HU, as a specific activator of the Pqs system and PYO production. RNA-seq and protein-DNA interaction assays further revealed that HupA directly binds to the promoters of <i>pqs</i> and <i>phz</i> genes, thereby inducing PqsE expression and PYO production. This study demonstrates that HupA acts as a novel transcriptional factor to control QS activity and virulence gene expression in <i>P. aeruginosa</i>, which not only reveals the regulatory function of the HU subunit HupA on virulence factor production but also uncovers the sequence-specific DNA-binding capability of HupA for stimulating gene transcription.IMPORTANCEThe nucleoid-associated protein HU is abundant and evolutionarily conserved in bacteria, which typically binds DNA in a sequence-nonspecific manner with high affinity to abnormal DNA structures during DNA damage and then contributes to DNA compaction, replication, recombination, and other physiological functions. However, the potential involvement of HU in regulating quorum sensing (QS), a critical cell-to-cell communication system governing bacterial virulence factor production, is poorly understood. In this study, we demonstrated that the α subunit of the HU protein, namely HupA, is a key regulator that activates the Pqs system and pyocyanin production through genome-wide screening in a clinical isolate PA_HN008. This study reveals the capability of the HU subunit to selectively bind target DNA sequences and stimulate QS activity as well as virulence factor production. Our findings provide novel insights into the biochemical properties of the HU subunit and its regulatory role in bacterial virulence.