Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa Using Fluorescence-Based Biosensors

Rybtke, Morten; Chua, Song Lin; Yam, Joey Kuok Hoong; Givskov, Michael; Yang, Liang; Tolker‐Nielsen, Tim

doi:10.1007/978-1-4939-7240-1_8

Cited by 13 publications

(9 citation statements)

References 9 publications

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“…2A and B ). This indicates that P. aeruginosa bacteria in the planktonic state contain a low level of c-di-GMP, which is in agreement with previous work ( 35 ). The p lac -gfp -tagged strain showed a biofilm-like aggregation morphology at 8 h postinfection (hpi) in our mouse model of corneal infection ( Fig.…”

Section: Resultssupporting

confidence: 93%

“…To further address whether the intracellular c-di-GMP level in P. aeruginosa is elevated during corneal infection, we used P. aeruginosa carrying a p cdrA - gfp gene fusion, which is a well-established c-di-GMP reporter strain ( 34 – 36 ). In the planktonic state, the c-di-GMP reporter bacteria exhibited an extremely low expression level of the p cdrA - gfp fusion (below the detection limit) compared with a P. aeruginosa strain that carries a p lac -gfp fusion and constitutively expresses the green fluorescent protein (GFP) gene gfp ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Visualization of c-di-GMP level in Pseudomonas aeruginosa during infection. Procedures were carried out as mentioned previously (35). Briefly, the scratched corneas were infected with green fluorescent protein (GFP)-tagged P. aeruginosa PAO1/p cdrA -gfp or PAO1/p lac -gfp for observation of GFP expression during infection.…”

Section: Methodsmentioning

confidence: 99%

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Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa

et al. 2022

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Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a Δ wspF mutant strain (high c-di-GMP levels), and a p lac - yhjH -containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

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Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa

et al. 2022

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“…Intracellular levels of c-di-GMP were assessed with the fluorescence-based biosensor pCdrA-gfpC (63, 64), acquired as addgene plasmid #111614; http://n2t.net/addgene:111614 ; RRID:Addgene_111614. Purified plasmids were transformed by electroporation in evolved morphotypes obtained from static cultures (65).…”

Section: Methodsmentioning

confidence: 99%

Pseudomonas aeruginosa strains from both clinical and environmental origins readily adopt a stable small colony variant (SCV) phenotype resulting from single mutations in c-di-GMP pathways

Besse

Groleau

Trottier

et al. 2022

Preprint

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A subpopulation of Small Colony Variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolated from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread is the ability of P. aeruginosa to develop this phenotype and the genetic mechanism(s) behind SCVs emergence according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to, under laboratory culture conditions, spontaneously adopt a SCV-like smaller alternative morphotype distinguishable from the ancestral parent strain. We found that all the P. aeruginosa strains tested could adopt a SCV phenotype, regardless of their origin. Whole genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, Wsp and YfiBNR. We conclude that the ability to switch to a SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain.

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“…C-di-GMP levels were assessed with the fluorescence-based biosensor pCdrA-gfpC (53, 54). pCdrA-gfpC was constructed by Tim Tolker-Nielsen (addgene plasmid #111614; http://n2t.net/addgene:111614 ; RRID:Addgene_111614).…”

Section: Methodsmentioning

confidence: 99%

The ability ofPseudomonas aeruginosato adopt a Small Colony Variant (SCV) phenotype is conserved, and not restricted to clinical isolates

Besse

Trottier

Groleau

et al. 2021

Preprint

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A subpopulation of Small Colony Variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) lungs biofilms. SCVs have almost exclusively been reported from infected hosts, essentially CF individuals or, by extension, from laboratory cultivation of strains originated from infected hosts. We previously reported the identification of P. aeruginosa SCVs emerging from a non-clinical strain and displaying features shared with clinical SCVs. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to, under laboratory culture conditions, spontaneously adopt a SCV-like smaller alternative morphotype distinguishable from the ancestral parent strain. Unexpectedly, we found that all the P. aeruginosa strains tested have the ability to adopt a SCV morphotype, regardless of their origin. Based on the phenotypes already described for SCVs, the SCV-like morphotypes obtained were clustered in two groups displaying various phenotypic profiles, including one characteristic of already described SCVs. We conclude that the ability to switch to a SCV phenotype is a conserved feature in P. aeruginosa.

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Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa Using Fluorescence-Based Biosensors

Cited by 13 publications

References 9 publications

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa

Pseudomonas aeruginosa strains from both clinical and environmental origins readily adopt a stable small colony variant (SCV) phenotype resulting from single mutations in c-di-GMP pathways

The ability ofPseudomonas aeruginosato adopt a Small Colony Variant (SCV) phenotype is conserved, and not restricted to clinical isolates

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