Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants
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Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants. / Nair, Harikrishnan A.S.; Subramoni, Sujatha; Poh, Wee Han; Hasnuddin, Nabilah Taqiah Binte; Tay, Martin; Givskov, Michael; Tolker-Nielsen, Tim; Kjelleberg, Staffan; McDougald, Diane; Rice, Scott A.
In: BMC Microbiology, Vol. 21, 255, 2021.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants
AU - Nair, Harikrishnan A.S.
AU - Subramoni, Sujatha
AU - Poh, Wee Han
AU - Hasnuddin, Nabilah Taqiah Binte
AU - Tay, Martin
AU - Givskov, Michael
AU - Tolker-Nielsen, Tim
AU - Kjelleberg, Staffan
AU - McDougald, Diane
AU - Rice, Scott A.
N1 - Publisher Copyright: © 2021, The Author(s).
PY - 2021
Y1 - 2021
N2 - Background: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. Results: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. Conclusions: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.
AB - Background: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. Results: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. Conclusions: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.
KW - Biofilm development
KW - Bioreporter
KW - C-di-GMP
KW - Dispersal
KW - Image-based quantification
KW - Morphotypic variants
KW - Pseudomonas aeruginosa
KW - Starvation
U2 - 10.1186/s12866-021-02318-8
DO - 10.1186/s12866-021-02318-8
M3 - Journal article
C2 - 34551714
AN - SCOPUS:85115311723
VL - 21
JO - BMC Microbiology
JF - BMC Microbiology
SN - 1471-2180
M1 - 255
ER -
ID: 280725198