Table 1.
Bacterial strains and plasmids.
Table 2.
PCR primers used in this study.
Fig 1.
Structures of two phz operons in P. aeruginosa PAO1 and its derivatives and two types of plasmid fusions with the truncated lacZ.
(A) phz1 (light grey arrows) and phz2 (heavy grey arrows) indicate two phenazine operons of phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, respectively. aacC1 (horizontally striped arrow) and aph (vertically striped arrow) indicate the gentamycin and kanamycin resistance cassettes inserted into chromosome, respectively. lacZ (black arrow) indicates the truncated β-galactosidase gene inserted and fused in frame with the first several codons of phzA1 or phzA2 and their upstream region in the chromosome. The translational plasmid fusion (B) and the transcriptional plasmid fusion (C) were generated in plasmids pME6015 and pME6522, respectively. MCS stands for the multi-cloning site.
Fig 2.
Bacterial growth curves of P. aeruginosa PAO1 and its derivatives in LB and GA medium.
Each of the wild-type strain PAO1 and its derivatives was respectively inoculated in 150 ml of LB medium (A) or GA medium (B). Optical density 600 nm was determined at 12 hour intervals. All experiments were performed in triplicate, and each value was presented as the average ± standard deviation. * indicates P > 0.05, two-tailed paired Student t test.
Fig 3.
PCA produced by P. aeruginosa PAO1 and its derivatives in LB medium.
All strains including the wild-type strain PAO1 (solid circle), the single-deletion mutant Δphz1 (solid square) and Δphz2 (solid triangle), the double-deletion mutant Δphz1phz2 (solid diamond), the Δphz1 mutant complemented with pME10Z1 (open square) and the Δphz2 mutant harboring pME10Z2 (open triangle) were grown in LB broth. All experiments were performed in triplicate, and each value was presented as the average ± standard deviation.
Fig 4.
PCA and PYO produced by P. aeruginosa PAO1 and its derivatives in GA medium.
PCA (A) and PYO (B) were biosynthesized by the wild-type strain PAO1 (solid circle) and its derivatives, the single-deletion mutant Δphz1 (solid square) and Δphz2 (solid triangle), the double-deletion mutant Δphz1phz2 (solid diamond), the Δphz1 mutant harboring pME10Z1 (open square) and the Δphz2 mutant containing pME10Z2 (open triangle) in GA medium. All experiments were performed in triplicate, and each value was presented as the average ± standard deviation.
Fig 5.
Translational fusion constructs pME15Z1 (phz1′-′lacZ) and pME15Z2 ((phz2′-′lacZ) generated to study regulation between two phenazine-producing loci.
(A) β-Galactosidase activities were produced by pME15Z1 in the double-deletion mutant Δphz1phz2 (solid circle), the single-deletion mutant Δphz1 (solid square), Δphz2 (solid triangle), and in the wild-type PAO1 (solid diamond). pME6015 in mutant Δphz1 (open square) served as the negative control. (B) β-Galactosidase activities were produced by pME15Z2 in the Δphz1phz2 mutant (solid circle), the Δphz1 mutant (solid square), the Δphz2 mutant (solid triangle), and the wild-type PAO1 (solid diamond). pME6015 in the single-deletion mutant Δphz2 (open triangle) served as the negative control. All experiments were performed in triplicate, and each value was presented as the average ± standard deviation.
Fig 6.
Enhancement of expression of one phz operon in the absence of the other operon.
(A) Expression of the translational fusion phz1-lacZ in chromosome in the presence of the phz2 operon (in the mutant ΔphzA1Z, solid squares) or in the absence of the phz2 operon (in the mutant ΔphzA1Zphz2, solid circles). (B) Expression of the translational fusion phz2-lacZ in chromosome in the presence of the phz1 operon (in the mutant ΔphzA2Z, open squares) or in the absence of the phz1 operon (in the mutant Δphz1phzA2Z, open circles). β-Galactosidase activities determined in the single-deletion mutant Δphz1 (solid triangles) or Δphz2 (open triangles) were used as the negative controls. Each point was the mean of three measurements ± standard deviation.
Fig 7.
The transcription level assay of one phz operon in the absence or in the presence of the other one phz operon.
(A) β-Galactosidase activities were produced by pME22Z1 in the double-deletion mutant Δphz1phz2 (black column), the single-deletion mutant Δphz1 (grey column) and mutant Δphz2 (light grey column). pME6522 in the mutant Δphz1 (white column) served as the negative control. (B) β-Galactosidase activities were produced by pME22Z2 in the mutant Δphz1phz2 (grey column), Δphz1 (light grey column) and Δphz2 (black column). pME6522 in the Δphz2 mutant (white column) served as the negative control. All experiments were performed in triplicate, and each value was presented as the average ± standard deviation. *indicates P > 0.05, **indicates P < 0.01, two-tailed paired Student t test.
Fig 8.
Effects of exogenous phenazines on expression of the phz operon.
(A) β-Galactosidase activities expressed in the mutant ΔphzA1Zphz2 in the presence of exogenous PYO or PCA. (B) β-Galactosidase activities expressed in the mutant Δphz1phzA2Z in the presence of exogenous PYO or PCA. a-e, PYO was added to samples with final concentration of 0.08, 0.16, 0.32, 0.64 and 1.28 μg/ml; f-j, PCA was added with 0.025, 0.25, 0.5,1.0, and 2.0 μg/ml. All values were measured after 24 hour of addition. Data reported were the means of triplicate experiments ± standard deviations. * indicates P > 0.05, two-tailed paired Student t test.