doi: 10.1128/JB.01889-14. Epub 2014 Jul 14.
Biological cost of pyocin production during the SOS response in Pseudomonas aeruginosa
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- PMID:25022851
- PMCID: PMC4135695
- DOI: 10.1128/JB.01889-14
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Biological cost of pyocin production during the SOS response in Pseudomonas aeruginosa
Jon Penterman et al. J Bacteriol.2014 Sep.
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Abstract
LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Figures
Autoproteolytic activity of PrtR is required for pyocin production. (A) Expression of thePA0614p::lacZ translational reporter gene in theprtRS162A mutant and the parent strain treated with 1 μg/ml ciprofloxacin. Error bars indicate standard deviations. (B) Production of pyocin by theprtRS162A mutant and the parent strain. Filtered supernatant from cultures treated with MMC (3 μg/ml) and from liquid cultures incubated overnight were spotted onto lawns of the indicator strains 13s, PML1516d, and NIH5. Clearing of the bacterial lawn is indicative of pyocin-mediated death (see Materials and Methods; see Fig. S1A in the supplemental material for information on the specificity of indicator strains for different pyocin types). (C) Expression of therecAp::GFP transcriptional reporter gene in theprtRS162A mutant, thelexAG86V mutant, and the parent strain treated with 1 μg/ml ciprofloxacin. LexAG86V protein is resistant to autoproteolytic cleavage (37). Error bars indicate standard deviations.
Autoproteolytic activity of PrtR reduces survival during genotoxic stress. (A) Total CFU counts in cultures of wild-type PAO1 and theprtRS162A mutant treated with a bactericidal concentration of ciprofloxacin (2 μg/ml). Error bars indicate standard deviations. (B) Total CFU counts of wild-type PAO1 and theprtRS162A mutant on agar plates containing 0 and 15 μg/ml of MMC after overnight incubation. Error bars indicate standard deviations. (C) Surviving CFU counts of wild-type PAO1 and theprtRS162A mutant after UV treatment. Cultures were serially diluted 10-fold, spotted onto agar, and treated with UV light at the indicated doses. Surviving CFU counts were determined after overnight incubation.
PrtR-regulated lysis and R2 pyocin genes are determinants of UV irradiation survival. (A) Production of extracellular pyocin in cultures treated with 3 μg/ml of MMC for 2 h. Clearing of the bacterial lawn of the indicator strains is indicative of pyocin-mediated death. (B) Surviving CFU counts of the lysis-defective Δ0614 Δ0629 mutant strain and pyocin mutant strains after UV treatment (30 J/m2). Cultures were serially diluted 10-fold, spotted onto agar, and treated with UV light. Surviving CFU counts were determined after overnight incubation. S2-, PA1150-H12::ISlacZ/hah allele; S4-, PA3866-F01::ISlacZ/hah allele; F2-, PA0633-E12::ISlacZ/hah allele; R1-, PA0625-H05::ISlacZ/hah allele. (C) Total CFU counts in cultures of wild-type PAO1 andprtRS162A, Δ0614 Δ0629, and ΔR2 mutant cells treated with a bactericidal concentration of ciprofloxacin (2 μg/ml). Error bars indicate standard deviations.
Extracellular R2 pyocin is lethal to cells that initially survive UV treatment. (A) Survival of the wild type, the ΔR2 mutant, and a 1:1 mixture of the wild type and the ΔR2 mutant after UV treatment (30 J/m2). (B) Survival of UV-irradiated ΔR2 mutant cells with or without purified R2 pyocin. Cultures were serially diluted 10-fold, mixed 1:1 with saline or saline with purified R2 pyocin, and spotted onto agar prior to UV treatment. Surviving CFU counts were determined after overnight incubation. Note that ΔR2 mutant cells not treated with UV light were resistant to purified R2 pyocin.
Loss of R2 pyocin resistance after UV treatment is not regulated by RecA-stimulated cleavage of PrtR, LexA, and PA0906 during the SOS response. Cultures were serially diluted 10-fold, mixed 1:1 with saline or saline with purified R2 pyocin, and spotted onto agar prior to treatment with UV (10 J/m2). Surviving CFU counts were determined after overnight incubation.
Relative expression of B-band and A-band O-antigen synthesis genes after treatment of wild-type cells with UV (15 J/m2). (A) Relative expression of A-band synthesis genes (wbpZ,rmd) and B-band synthesis genes (wbpJ,wbpA,wzx) in wild-type cells on agar. Nontreated control cells were incubated on agar for 15 min. Error bars indicate 95% confidence intervals. (B) Relative expression oflpxA andwaaP in wild-type cells on agar. Error bars indicate 95% confidence intervals. (C) Relative expression of A-band synthesis genes (wbpZ,rmd) and B-band synthesis genes (wbpJ,wbpA) inPA0906S153AlexAG86VprtRS162A triple mutant cells on agar. Nontreated control cells were incubated on agar for 15 min. Error bars indicate 95% confidence intervals.
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References
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