A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism

Abstract

The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 infects both plants and animals. Previously, using plants to screen directly for P. aeruginosa virulence-attenuated mutants, we identified a locus, pho34B12, relevant in mammalian pathogenesis. Here, nonsense point mutations in the two opposing ORFs identified in the pho34B12 locus revealed that one of them, mvfR (multiple virulence factor Regulator), is able to control all of the phenotypes that mutant phoA34B12 displays. Both genetic and biochemical evidence demonstrate that the mvfR gene encodes a LysR-like transcriptional factor that positively regulates the production of elastase, phospholipase, and of the autoinducers, 3oxo-dodecanoyl homoserine lactone (PAI I) and 2-heptyl-3-hydroxy-4-quinolone (PQS), as well as the expression of the phnAB operon, involved in phenazine biosynthesis. We demonstrate that the MvfR protein is membrane-associated and acts as a transcriptional activator until cells reach stationary phase, when a unique negative feedback mechanism is activated to signal the down-regulation of the MvfR protein. This work reveals an unprecedented virulence mechanism of P. aeruginosa and identifies a unique indispensable player in the P. aeruginosa quorum-sensing cascade.

Figure 1

Coomassie blue stain of exoproteins isolated from wild-type (lane 1) and mutant strains, pho34B12 (lane 2), ORF1* (lane 3), and ORF2* (lane 4). Extracellular proteins were isolated from equal amounts of wild-type and mutant bacterial cells grown in Luria–Bertani media (OD₆₀₀ = 2.5–3.0), separated on a 4–20% gradient polyacrylamide gel, and stained with Coomassie brilliant blue R-250.

Figure 2

(A) The expression of the mvfR-lacZ transcriptional fusion in wild-type PAO1 and mutant strains lasR⁻, rhlR⁻, and lasR⁻rhlR⁻. Plasmid containing mvfR-lacZ transcriptional fusion was introduced into strains PAO1, lasR⁻, rhlR⁻, and lasR⁻rhlR⁻. β-galactosidase activity was measured in these strains at OD₆₀₀ = 2.5–3.0. (B) The expression of lasR-lacZ and rhlR-lacZ in wild-type PA14 and mutant strain ORF2*. Plasmids containing either lasR-lacZ or rhlR-lacZ were introduced into wild-type PA14 and mutant ORF2*. β-galactosidase activity was measured in these strains at OD₆₀₀ = 2.5–3.0.

Figure 3

The MvfR protein binds specifically to the promoter of the phnAB operon. Lane 1, radio-labeled P1 (a 51-bp sequence 185-bp upstream of the start codon of phnAB operon) only; lane 2, blank; lanes 3–8, MvfR + radio-labeled P1 + x-fold cold P1. Lane 3, x = 0; lane 4, x = 10; lane 5, x = 20; lane 6, x = 40; lane 7, x = 80; lane 8, x = 160; lane 9, blank; lanes 10–12, MvfR + radio-labeled P1 + y-fold cold P2 (a 51-bp sequence 460-bp upstream of the start codon of phnA/B operon). Lane 10, y = 20; lane 11, y = 40; lane 12, y = 80; and lane 13, MvfR + radio-labeled P1 + anti-MvfR polyclonal Ab.

Figure 4

(A) Cellular localization of the MvfR protein at different growth phases. Plasmid containing the MvfR-GST translational fusion was introduced into PA14 strain. Protein extracts from cell fractionations of both PA14 and transformant strains grown to cell density as indicated were prepared, separated on a 10% polyacrylamide gel, and blotted onto Immobilon-P [poly(vinylidene difluoride) (PVDF)] membranes. A mAb against GST was used to detect the MvfR-GST fusion. The numbers to the right indicate the cell density (OD₆₀₀). WT, wild-type PA14 strain; GST, PA14 strain containing MvfR-GST translational fusion; P, periplasmic; C, cytoplasmic; S, secreted; M, membrane; O, outer; I, inner. (B) The expression of mvfR and phnAB at different growth phases. β-galactosidase activities in PA14 strain containing either mvfR-lacZ or phnAB-lacZ transcriptional fusion were measured at the growth phases as indicated on the graph.

Figure 5

Translocation and cleavage of MvfR in response to extracellular signals from wild-type PA14 (B) and mutant strain ORF2* (C). Both PA14 strain (WT) and PA14 strain containing MvfR-GST translational fusion (GST) were grown in Luria–Bertani media until OD₆₀₀ reached 2.5–3.0. Then the cells were harvested and treated for 1 h with cell-free cultures of PA14 and ORF2* strains grown to stationary phase (OD₆₀₀ > 5.0). Protein preps from supernatant (s) and membrane (m) fractions of those treated (B and C) and untreated (A) cells were separated on a 10% polyacrylamide gel and blotted onto Immobilon-P [poly(vinylidene difluoride) (PVDF)] membranes. A mAb against GST was used to detect the MvfR-GST fusion.