Interrogation of genes controlling biofilm formation using CRISPR interference in <i>Pseudomonas fluorescens</i>

Noirot‐Gros, Marie‐Françoise; Forrester, Sara; G, Malato; Pe, Larsen; Naveilhan, Philippe

doi:10.1101/476366

Cited by 3 publications

(3 citation statements)

References 81 publications

(135 reference statements)

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“…This ability to achieve tunable knockdown is particularly appealing for quantifying interstrain interactions, by contrast to the lethal phenotype of complete removal of essential genes. CRISPRi was recently used to identify genes that regulate biofilm formation in Pseudomonas fluorescens [39]; the efficacy of CRISPRi for B. subtilis in a colony/biofilm environment has yet to be ascertained.…”

Section: Introductionmentioning

confidence: 99%

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing

Arjes

Willis

Gui

et al. 2021

eLife

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Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential-gene CRISPRi knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm-colony co-culture via nutrient sharing. Rescue was enhanced in biofilm-colony co-culture with a matrix-deficient parent, due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.

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Section: Introductionmentioning

confidence: 99%

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing

Arjes

Willis

Gui

et al. 2021

eLife

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“…Identification of genes and pathways specifically related to biofilm formation or the interaction with surfaces has recently been boosted by the potential of CRISPR for high-throughput functional genomics. Systematic disruption of gene expression levels using CRISPR interference has been used to interrogate control of biofilm formation in multiple strains of Pseudomonas fluorescens (Noirot-Gros et al, 2019). These can be complemented with computational tools.…”

Section: Computational Genomicsmentioning

confidence: 99%

Advances in experimental and computational methodologies for the study of microbial-surface interactions at different omics levels

et al. 2022

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The study of the biological response of microbial cells interacting with natural and synthetic interfaces has acquired a new dimension with the development and constant progress of advanced omics technologies. New methods allow the isolation and analysis of nucleic acids, proteins and metabolites from complex samples, of interest in diverse research areas, such as materials sciences, biomedical sciences, forensic sciences, biotechnology and archeology, among others. The study of the bacterial recognition and response to surface contact or the diagnosis and evolution of ancient pathogens contained in archeological tissues require, in many cases, the availability of specialized methods and tools. The current review describes advances in in vitro and in silico approaches to tackle existing challenges (e.g., low-quality sample, low amount, presence of inhibitors, chelators, etc.) in the isolation of high-quality samples and in the analysis of microbial cells at genomic, transcriptomic, proteomic and metabolomic levels, when present in complex interfaces. From the experimental point of view, tailored manual and automatized methodologies, commercial and in-house developed protocols, are described. The computational level focuses on the discussion of novel tools and approaches designed to solve associated issues, such as sample contamination, low quality reads, low coverage, etc. Finally, approaches to obtain a systems level understanding of these complex interactions by integrating multi omics datasets are presented.

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“…This ability to achieve tunable knockdown is particularly appealing for quantifying interstrain interactions, by contrast to the lethal phenotype of complete removal of essential genes. CRISPRi was recently used to identify genes that regulate biofilm formation in Pseudomonas fluorescens [38]; the efficacy of CRISPRi for B. subtilis in a colony/biofilm environment has yet to be ascertained.…”

Section: Introductionmentioning

confidence: 99%

Three-dimensional biofilm growth supports a mutualism involving matrix and nutrient sharing

Arjes

Willis

Gui

et al. 2020

Preprint

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Life in a three-dimensional structure such as a biofilm is typical for many bacteria, yet little is known about how strains with different genotypes interact in this context. Here, to systematically explore gene knockdowns across various three-dimensional contexts, we created arrayed libraries of essential-gene CRISPRi knockdowns in the model biofilm-forming bacterium Bacillus subtilis and measured competitive fitness during colony co-culture with a wild type-like parent on different media and at different knockdown levels. Partial knockdown led to a wide range of fitness phenotypes, with targeting of translation-related genes often leading to lower growth rates and rapid out-competition by the parent. Several knockdowns competed differentially in biofilms versus non-biofilm colonies, in some cases due to lack of a particular nutrient in one medium. Cells depleted for the alanine racemase AlrA died in monoculture, but co-cultures survived via nutrient sharing in a biofilm but not in liquid. This rescue was enhanced in biofilm co-culture with a parent unable to produce extracellular matrix, due to a mutualism involving nutrient and matrix sharing. Including alrA, we identified several examples of mutualism involving matrix sharing that occurred in a three-dimensional biofilm colony but not when growth was constrained to two dimensions. These findings demonstrate that growth in a three-dimensional biofilm can promote genetic diversity through sharing of secreted factors, and illustrate the role of matrix production in determining trajectories for biofilm evolution that may be relevant to pathogens and other environmental bacteria.

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

Cited by 3 publications

References 81 publications

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing

Advances in experimental and computational methodologies for the study of microbial-surface interactions at different omics levels

Three-dimensional biofilm growth supports a mutualism involving matrix and nutrient sharing

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