Fig 1.
The Pseudomonas aeruginosa LuxR/I-type QS circuit.
Schematic of the acyl-homoserine lactone (AHL) based QS network: LasR/I (white) and RhlR/I (gray). The LasI autoinducer is 3OC12-HSL (squares) and the RhlI autoinducer is C4-HSL (circles). The two black horizontal lines represent the cytoplasmic membrane, regulatory genes are shown in the open arrows, and bent arrows represent promoters. The alternative ligand (represented by the stars) refers to the putative ligand(s) that binds to RhlR and enables RhlI-independent RhlR function.
Fig 2.
P. aeruginosa ΔrhlI and ΔrhlR mutants have distinct colony biofilm phenotypes.
A) Pyocyanin production was measured at OD695 in cell-free culture fluids prepared from the WT, ΔrhlR mutant, ΔrhlI mutant, ΔrhlR mutant complemented with the rhlR gene under its native promoter on pUCP18 (prhlR), and the ΔrhlI mutant supplied with exogenous 10 μM C4-HSL. Error bars represent SD for three biological replicates. B) Colony biofilm phenotypes of the strains in panel A and the ΔrhlR ΔrhlI double mutant. Again, 10 μM C4-HSL was added to the ΔrhlI mutant. Scale bar is 2 mm. C) Colony biofilm surface area quantitation for the indicated strains over 5 days. Error bars represent SEM of three independent experiments.
Fig 3.
P. aeruginosa has distinct RhlR and RhlI regulons.
A) Venn diagram showing differentially expressed genes in the ΔrhlI and ΔrhlR P. aeruginosa PA14 strains at high cell density (denoted Planktonic Culture; OD600 = 2.0) and in colony biofilms grown on Congo red agar medium for 5 days (denoted Colony Biofilm). Blue circles represent differentially expressed genes in the ΔrhlR P. aeruginosa strain compared to the WT strain. Orange circles indicate differentially expressed genes in the ΔrhlI P. aeruginosa strain compared to the WT strain. Numbers of cDNA reads for annotated genes were compared to the WT strain under the same conditions. Statistically significant genes (P < 0.001) that changed >2-fold are shown (see S1 and S2 Tables). RNA-seq was performed on three biological replicates per strain and per condition. B) Relative expression of the representative phenazine biosynthesis genes phzA1 (black), phzF2 (dark gray), phzM (light gray), and phzH (checkered). Data are normalized to 16S RNA measured by qRT-PCR in WT, ΔrhlR, and ΔrhlI strains in high cell density (OD600 = 2.0) planktonic culture (top panel) and in colony biofilms on Congo red agar medium after 5 days (bottom panel). Error bars represent SD of three replicates. AU denotes arbitrary units. C) Colony biofilm morphology of the WT, Δphz, and the ΔrhlR Δphz, and ΔrhlI Δphz mutants. Scale bar is 2 mm.
Fig 4.
RhlR-activated genes exhibit varying levels of RhlI dependence.
Relative expression of the chiC, rhlA, and hcnA genes normalized to 16S RNA measured by qRT-PCR in the WT, ΔrhlR, and ΔrhlI strains at high cell density (OD600 = 2.0). Class I, II, III denote RhlI-dependent, partially dependent, and independent genes, respectively. AU denotes arbitrary units. Error bars represent SD of three replicates.
Fig 5.
Cell-free culture fluids from the ΔrhlI mutant activate RhlR-dependent gene expression.
rhlA expression was measured using a chromosomally encoded PrhlA- mNeonGreen transcriptional reporter. Gray bars represent rhlA reporter activity when rhlR was induced in the ΔlasR ΔlasI ΔrhlR ΔrhlI (i.e., Δ4 PBAD-rhlR) strain with 0.1% L-arabinose in the presence of 1% DMSO, 10 μM C4-HSL, 10 μM 3OC12-HSL, or 50 μM PQS. DMSO was used as the solvent for C4-HSL, 3OC12-HSL, and PQS. The rhlA reporter activity was set to 100% when 10 μM C4-HSL was added. In the cultures represented by the black bars, PrhlA-mNeonGreen was monitored in response to 20% (v/v) of the indicated cell-free culture fluids subjected to the specified treatments. Error bars represent SEM for three biological replicates.
Fig 6.
The P. aeruginosa ΔrhlI mutant is virulent and the ΔrhlR mutant is avirulent in animal infection models.
A) C. elegans were applied to lawns of E. coli OP50 (open squares), WT P. aeruginosa PA14 (closed diamonds), ΔrhlR mutant (closed triangles), and ΔrhlI mutant (closed circles). Error bars represent SEM of three independent replicates. B) Real-time monitoring of WT P. aeruginosa PA14 P1-lux and isogenic mutants in the acute pneumonia model. BALB/c mice infected with WT, ΔrhlR, and ΔrhlI strains were imaged at 24 and 48 h using an IVIS CCD camera following intratracheal infection. Imaging was performed from the ventral side of representative mice while the animals were under isoflourane anesthesia. The color bars indicate the intensity of the bioluminescence output, with red and blue denoting the high and low signals, respectively. Note that the color scales on the various mouse bioluminescence imaging panels are not the same. (C) Bacterial load in lung homogenates of mice infected intratracheally with WT P. aeruginosa PA14, and the ΔrhlR and ΔrhlI mutants. Each symbol represents a single mouse. The data are pooled from two independent experiments. Data were analyzed using the Mann-Whitney U test. *** P <0.001 and ns means not significant.