Differential Regulation of the Phenazine Biosynthetic Operons by Quorum Sensing in Pseudomonas aeruginosa PAO1-N

Abstract

The Pseudomonas aeruginosa quorum sensing (QS) network plays a key role in the adaptation to environmental changes and the control of virulence factor production in this opportunistic human pathogen. Three interlinked QS systems, namely las, rhl, and pqs, are central to the production of pyocyanin, a phenazine virulence factor which is typically used as phenotypic marker for analysing QS. Pyocyanin production in P. aeruginosa is a complex process involving two almost identical operons termed phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), which drive the production of phenazine-1-carboxylic acid (PCA) which is further converted to pyocyanin by two modifying enzymes PhzM and PhzS. Due to the high sequence conservation between the phz1 and phz2 operons (nucleotide identity > 98%), analysis of their individual expression by RNA hybridization, qRT-PCR or transcriptomics is challenging. To overcome this difficulty, we utilized luminescence based promoter fusions of each phenazine operon to measure in planktonic cultures their transcriptional activity in P. aeruginosa PAO1-N genetic backgrounds impaired in different components of the las, rhl, and pqs QS systems, in the presence or absence of different QS signal molecules. Using this approach, we found that all three QS systems play a role in differentially regulating the phz1 and phz2 phenazine operons, thus uncovering a higher level of complexity to the QS regulation of PCA biosynthesis in P. aeruginosa than previously appreciated.ImportanceThe way the P. aeruginosa QS regulatory networks are intertwined creates a challenge when analysing the mechanisms governing specific QS-regulated traits. Multiple QS regulators and signals have been associated with the production of phenazine virulence factors. In this work we designed experiments where we dissected the contribution of specific QS switches using individual mutations and complementation strategies to gain further understanding of the specific roles of these QS elements in controlling expression of the two P. aeruginosa phenazine operons. Using this approach we have teased out which QS regulators have either indirect or direct effects on the regulation of the two phenazine biosynthetic operons. The data obtained highlight the sophistication of the QS cascade in P. aeruginosa and the challenges in analysing the control of phenazine secondary metabolites.

PqsE Is Essential for RhlR-Dependent Quorum Sensing Regulation in Pseudomonas aeruginosa

“…Since the absence of LasR seems to impose the requirement for PqsE to achieve efficient RhlR activity, we overexpressed pqsE in a lasR-null background. As previously shown (43), the constitutive expression of PqsE augments and advances the transcription of phzA1 (Fig. 5B).…”

Bacteria-Host Crosstalk: Sensing of the Quorum in the Context of <b><i>Pseudomonas aeruginosa</i></b> Infections

“…All three QS systems, Las, Rhl, and PQS, play a role in the regulation of PYO production (Fig. 1) [48], and a recent study uncovered a higher level of its complexity [60]. …”

Effects of Signal Disruption Depends on the Substrate Preference of the Lactonase

“…Studies show that the currently understood hierarchical system can be altered in clinical isolates of P. aeruginosa which have lived in human lungs for years (Bjarnsholt et al, 2010;Feltner et al, 2016;Chen et al, 2019;Kostylev et al, 2019). There is redundancy in the transcriptional control of certain gene products but certain virulence factors essential to pathogenicity are under strictly regulated QS control; for example, pyocyanin production is a product of a complex metabolic pathway positively controlled by the transcriptional regulator, RhlR (Nadal Jimenez et al, 2012;Higgins et al, 2018). In addition, some of these strains can lose the ability to respond to QS: they are called social cheaters (Sandoz et al, 2007;Popat et al, 2012;Mukherjee and Bassler, 2019).…”