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

Steven Higgins,Miguel Cámara,Stephan Heeb,Giordano Rampioni,Mathew P Fletcher,Paul Williams

doi:10.3389/fcimb.2018.00252

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.

Highlights

Pseudomonas aeruginosa is a highly adaptable bacterium, which can be found in a range of challenging environments, including the human host
Since the vast majority of studies on the quorum sensing (QS) circuit of P. aeruginosa have been undertaken in rich media, Lysogeny Broth (LB) was used in this work so that predictions about the behavior of the QS network in specific QS mutants could be made and the results obtained compared with previous studies
It has been demonstrated that the QS regulators LasR, RhlR, RsaL and the enzyme PqsE are all involved in controlling the expression of both phenazine operons phz1 and phz2 in P. aeruginosa with some differences

Summary

Introduction

Pseudomonas aeruginosa is a highly adaptable bacterium, which can be found in a range of challenging environments, including the human host. The LasR/3OC12-HSL complex induces the transcription of rsaL, a gene integrated in the las QS system coding for the global transcriptional regulator RsaL (de Kievit et al, 1999) This protein directly represses the transcription of multiple genes, including lasI, exerting a homeostatic effect on 3OC12HSL production, and conferring robustness to the expression of a sub-set of genes of the las regulon with respect to fluctuations in LasR levels (Rampioni et al, 2006, 2007; Bertani et al, 2007)

Methods

Results

Conclusion