Pleiotropic Effects of GacA on Pseudomonas fluorescens Pf0-1
            <i>In Vitro</i>
            and in Soil

Seaton, Sarah C.; Silby, Mark W.; Levy, Stuart B.

doi:10.1128/aem.00819-13

Cited by 30 publications

(41 citation statements)

References 40 publications

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“…These sRNAs bind to the global regulator RsmA, releasing its repression of genes necessary for biofilm formation 20,40 . The decrease in biofilm formation observed upon CRISPRimediated silencing of gacS is in agreement with the regulatory model and with phenotypes observed in gacS mutants in various Pseudomonas 17,18,25,40,68 . In SBW25, GacS is also regulating genes involved in oxidative stress response, and the gacS::Tn5 mutant has a reduced capacity to survive exposure to sub-lethal doses of H2O2 25 .…”

Section: Discussionsupporting

confidence: 88%

“…fluorescens phenotypes related to mobility and biofilm are typically measured after 48 hours, we investigated CRISPRi-mediated silencing of the pleiotropic gacS (PFLU3777) gene. We found that upon induction of dCas9 expression, the strain expressing the gacSNT guide was totally impaired in swarming motility after 48 hours ( Figure 3), in keeping with the tight control of motility by gacS in Pseudomonas 17,24,25,67,68 . Expression of the gacST guide also led to a clear defect in swarming (supplementary Fig.…”

Section: Silencing Of the Two-component Sensor Kinase Gacs Impairs Mosupporting

confidence: 69%

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Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Noirot‐Gros

Forrester

et al. 2018

Preprint

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Bacterial biofilm formation involves multigenic signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm complex 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Genetic approaches based on gene inactivation are powerful for mechanistic studies but often are labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in P. fluorescens. We found that CRISPRi is applicable to three genetically and physiologically diverse species, SBW25, WH6 and Pf0-1 and affords extended periods of time to study complex phenotypes such as cell morphology, motility and biofilm formation. In SBW25, CRISPRi-mediated silencing of the GacA/S two-component system and genes regulated by cylic-di-GMP produced phenotypes similar to those previously described after gene inactivation in various Pseudomonas. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with biofilm architecture and extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 protein. Thus, CRISPRi is a reliable and scalable approach to interrogate gene networks in the diverse P. fluorescens group.

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Section: Discussionsupporting

confidence: 88%

Section: Silencing Of the Two-component Sensor Kinase Gacs Impairs Mosupporting

confidence: 69%

Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Noirot‐Gros

Forrester

et al. 2018

Preprint

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“…; Seaton et al. ). These mutations can confer significant fitness benefits (Duffy and Défago ; Driscoll et al.…”

Section: Discussionmentioning

confidence: 98%

“…For example, loss-of-function mutations of global regulators gacA and gacS arise at high rates in the laboratory and in natural populations (Duffy and Défago 2000;van den Broek et al 2005;Driscoll et al 2011;Seaton et al 2013). These mutations can confer significant fitness benefits (Duffy and Défago 2000;Driscoll et al 2011), and lead to loss of public goods, including extracellular enzymes, antibiotics, and biofilm formation (Bull et al 2001;Seaton et al 2013), but also private goods involved in primary metabolism (Wei et al 2013). Similarly, mutations in a LysR-type regulator in Pseudomonas aureofaciens lead to an inability to produce antifungal compounds (Silby et al 2005).…”

Section: Discussionmentioning

confidence: 99%

Pleiotropy and the low cost of individual traits promote cooperation

Mitri

Foster

2016

Evolution

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The evolution of cooperation is thought to be promoted by pleiotropy, whereby cooperative traits are coregulated with traits that are important for personal fitness. However, this hypothesis faces a key challenge: what happens if mutation targets a cooperative trait specifically rather than the pleiotropic regulator? Here, we explore this question with the bacterium Pseudomonas aeruginosa, which cooperatively digests complex proteins using elastase. We empirically measure and theoretically model the fate of two mutants--one missing the whole regulatory circuit behind elastase production and the other with only the elastase gene mutated--relative to the wild-type (WT). We first show that, when elastase is needed, neither of the mutants can grow if the WT is absent. And, consistent with previous findings, we show that regulatory gene mutants can grow faster than the WT when there are no pleiotropic costs. However, we find that mutants only lacking elastase production do not outcompete the WT, because the individual cooperative trait has a low cost. We argue that the intrinsic architecture of molecular networks makes pleiotropy an effective way to stabilize cooperative evolution. Although individual cooperative traits experience loss-of-function mutations, these mutations may result in weak benefits, and need not undermine the protection from pleiotropy.

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“…As in P. aeruginosa, this system stimulates biofilm formation (Barahona et al, 2010;Cheng et al, 2013;Duque et al, 2013). Recent transcriptional profiling studies using a gacS mutant of P. fluorescens have shown that the system positively affects the expression of genes involved in exopolysaccharide synthesis, cyclic lipopeptide synthesis and c-di-GMP signalling (Cheng et al, 2013;Seaton et al, 2013). A role of the GacAS system in the regulation of motility appears to be strain-dependent (Martinez-Granero et al, 2006;Barahona et al, 2010;.…”

Section: Involvement Of Srnas In P Putida and P Fluorescens Biofilmmentioning

confidence: 97%

Regulation of biofilm formation in Pseudomonas and Burkholderia species

Fazli

Almblad

Rybtke

et al. 2014

Environmental Microbiology

258

185

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SummaryIn the present review, we describe and compare the molecular mechanisms that are involved in the regulation of biofilm formation by Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Burkholderia cenocepacia. Our current knowledge suggests that biofilm formation is regulated by cyclic diguanosine-5′-monophosphate (c-di-GMP), small RNAs (sRNA) and quorum sensing (QS) in all these bacterial species. The systems that employ c-di-GMP as a second messenger regulate the production of exopolysaccharides and surface proteins which function as extracellular matrix components in the biofilms formed by the bacteria. The systems that make use of sRNAs appear to regulate the production of exopolysaccharide biofilm matrix material in all these species. In the pseudomonads, QS regulates the production of extracellular DNA, lectins and biosurfactants which all play a role in biofilm formation. In B. cenocepacia QS regulates the expression of a large surface protein, lectins and extracellular DNA that all function as biofilm matrix components. Although the three regulatory systems all regulate the production of factors used for biofilm formation, the molecular mechanisms involved in transducing the signals into expression of the biofilm matrix components differ between the species. Under the conditions tested, exopolysaccharides appears to be the most important biofilm matrix components for P. aeruginosa, whereas large surface proteins appear to be the most important biofilm matrix components for P. putida, P. fluorescens, and B. cenocepacia.

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Pleiotropic Effects of GacA on Pseudomonas fluorescens Pf0-1 In Vitro and in Soil

Cited by 30 publications

References 40 publications

Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Pleiotropy and the low cost of individual traits promote cooperation

Regulation of biofilm formation in Pseudomonas and Burkholderia species

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