Fig 1.
Genomic context and transcriptional features of P. aeruginosa pesA gene.
A) Schematic overview of the PAPI-1 region of PA14. The pesA gene is indicated as white arrowhead. B) Sequence of the pilM2–PA14_59370 intergenic region of PA14. The pesA sequence is in bold. The 5′-end of PesA is indicated with +1. A predicted Rho-independent transcription terminator is highlighted in black. The predicted stop codons of pilM2 and PA14_59370 are highlighted in gray. C) PesA expression is induced in stationary phase. Wild-type PA14 was inoculated in BHI at an OD600 of 0.2 and grown for 20 h at 37°C with agitation. At the indicated OD600, culture samples were taken and processed for total RNA extraction and analysis by Northern blot. Black arrow indicates the main band of pesA of ≈ 260 nt; white arrows indicate bands of higher molecular weight of pesA present especially at early exponential phase (OD600 = 0.2). D) Northern blot analysis of PesA on 10 μg of total RNA extracted at the end of the exponential growth phase, treated (+) or untreated (−) with terminator 5′-phosphate-dependent exonuclease (T-ex).
Fig 2.
PesA gene dissemination and expression levels among environmental, CF and COPD clinical isolates.
Assays on environmental, CF and COPD isolates are shown in three panels, A, B and C, respectively. The strain-collection was plated on BHI-agar plates. After overnight growth at 37°C, culture samples were taken and processed for total RNA extraction and analysis by Northern blot, and for genomic DNA extraction. Positive or negative PCR-amplification outcomes are indicated as “+” or “-” in the “pesA gene amplification” row, below each Northern Blot. PAO1 and PA14 were used as controls of Northern Blot analysis and for negative or positive gene amplification, respectively.
Fig 3.
PesA expression is induced by temperature and low availability of oxygen.
Levels of PesA RNA in: A) Wild-type PA14 grown in BHI at 20°C (lanes 1 and 4), 37°C (lanes 3 and 6) or following 20 min of acclimation (AC) from 20 to 37°C (lanes 2 and 5). Culture samples were taken at middle (OD600 of 0.8) and late (OD600 of 1.8) exponential growth phase. B) Wild-type PA14 grown in BHI anaerobically (NO3−; lanes 1 and 2), aerobically (O2, lane 6) and aerobically until an OD600 of 0.8 and then shifted to anaerobic conditions (O2 → NO3−; lanes 3, 4 and 5). Samples were taken 20 and 150 min after the shift to anaerobic conditions (t20 and t150). After sampling, cell cultures were processed for total RNA extraction and analysis by Northern blot. Intensities of the bands of PesA were quantified and normalized to those of 5S RNA in the same lane. Values are expressed as arbitrary units (AU) in the histograms below each Northern blot and represent the mean ± Standard Deviation (SD) of three independent experiments.
Fig 4.
PesA deletion enhances sensitivity to ciprofloxacin.
Antibiotic disk diffusion was performed on LB-agar plates spread with 106 CFU bacterial cells of wild-type PA14 and ΔpesA mutant strains. The diameters of the inhibitory zones were measured after overnight incubation at 37°C. Data derive from three independent experiments. Values represent the mean ± SD. Statistical significance by Student’s t-Test is indicated: **p< 0.01.
Fig 5.
PesA deletion enhances UV sensitivity similarly to pyoS3 operon deletion.
3 μl of cultures of PA14 wt, ΔpesA and ΔpyoS3, serially diluted 10-fold, were spotted onto LB-agar plates, and treated with UV light at the indicated doses. Surviving CFUs were observed after overnight incubation at 37°C.
Fig 6.
Time course of cell viability of IB3-1 cells following bacterial infection with P. aeruginosa PA14 wild-type and ΔpesA.
Cell viability, assessed as a reduction of MTT salt, was quantified by the optical density (OD) at 490 nm. IB3-1 cells were seeded at a density of 5 × 104 cells/well into 96-well microplates, and infected with 5 × 106 bacterial cells (MOI 1:100). At every time point, data are shown as the difference in OD490 between the PA14 wild-type strain and the sRNA-deleted mutant ΔpesA. Uninfected cells were used as positive control of cell viability. Data derive from three independent experiments. Results are shown as the difference in the OD490 reached at the different time points by IB3-1 cells infected by the mutant strain or non-infected, subtracted of the OD490 reached by IB3-cells infected with the wild-type strain. Values represent the mean ± standard error of the mean (SEM). Statistical significance between wild-type and ΔpesA strains by Student’s t-Test is indicated: *p< 0.05.
Fig 7.
Interaction of PesA with pyoS3A-I mRNA.
A) Prediction by TargetRNA software of the base-pairing interactions between PesA and pyoS3A-I mRNA. B) In vitro interaction between PesA RNA and pyoS3A-I mRNA by an electrophoretic mobility shift assay. Increasing amounts of PesA RNA (0, 0.08, 0.15, and 0.25 pmol; lanes 1–4) or, as a negative control, E. coli RseX RNA (0.25 and 2.5 pmol; lanes 5 and 6) were incubated at 37°C for 20 min with 0.15 pmol radiolabeled pyoS3A-I mRNA and loaded onto a native 6% polyacrylamide gel.
Fig 8.
PesA deletion impairs the production of pyocin S3.
Plate showing the effects of pyocin S3 present in the filtered supernatants of PA14 wild-type, ΔpesA and ΔpyoS3A-I on the killing of the indicator strain P. aeruginosa ATCC 27853. Drops of 5 μl of filtered supernatants from PA14 wild-type, ΔpesA and ΔpyoS3A-I cultures at OD600 = 1 were deposited on Luria-Bertani agar plates. A layer of the indicator strain P. aeruginosa ATCC 27853 was plated over the dried drops by inclusion in 0.7% agar. Plates were incubated overnight at 37°C. Clearer haloes represent inhibition (killing) of the indicator strain by pyocin S3 present in the sedimented supernatants.
Fig 9.
PesA positively regulates the expression of both the pyoS3A and pyoS3I translational fusions in PA14.
Comparison of the sfGFP and mCherry activities expressed in arbitrary units (AU) resulting from the translational fusion of (A) lacZ::pyos3A-I::sfGFP, (B) pyoS3I::sfGFP and (C) Cherry::pyoS3A-I::sfGFP combined with the control vector (pGM931) or the plasmid overexpressing PesA (pGM-pesA), in PA14 wild-type and PA14 ΔpesA. The strains were grown to an OD600 of 1.8 in LB medium supplemented with gentamicin and carbenicillin, to maintain pBBR1- and pGM- plasmids, respectively, and arabinose, to induce PesA overexpression. Cells were harvested and treated for sfGFP and mCherry activity determination by measuring fluorescence polarization FP485/535 and fluorescence intensity FI590/635, respectively. sfGFP and mCherry activities are expressed as ratio FP485/535/Abs595 and FI590/635/Abs595, respectively. Data derive from three independent experiments. Values represent the mean ± SD. Statistical significance by Student’s t-Test is indicated: *p<0.05; **p< 0.01.