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Title: CRISPR interference to interrogate genes that control biofilm formation in Pseudomonas fluorescens

Abstract

Bacterial biofilm formation involves signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Gene inactivation is a powerful approach for functional studies but it is often labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in diverse strain isolates of P. fluorescens, SBW25, WH6 and Pf0-1. We found that CRISPRi is applicable to study complex phenotypes such as cell morphology, motility and biofilm formation over extended periods of time. In SBW25, CRISPRi-mediated silencing of genes encoding the GacA/S two-component system and regulatory proteins associated with the cylic di-GMP signaling messenger produced swarming and biofilm phenotypes similar to those obtained after gene inactivation. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 operon. We conclude that CRISPRi is a reliable and scalable approach to investigate gene networks in the diverse P. fluorescens group.

Authors:
ORCiD logo [1];  [1];  [1];  [2];  [1]
  1. Argonne National Lab. (ANL), Argonne, IL (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Illinois, Chicago, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1578041
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Noirot-Gros, Marie-Francoise, Forrester, Sara, Malato, Grace, Larsen, Peter E., and Noirot, Philippe. CRISPR interference to interrogate genes that control biofilm formation in Pseudomonas fluorescens. United States: N. p., 2019. Web. doi:10.1038/s41598-019-52400-5.
Noirot-Gros, Marie-Francoise, Forrester, Sara, Malato, Grace, Larsen, Peter E., & Noirot, Philippe. CRISPR interference to interrogate genes that control biofilm formation in Pseudomonas fluorescens. United States. doi:10.1038/s41598-019-52400-5.
Noirot-Gros, Marie-Francoise, Forrester, Sara, Malato, Grace, Larsen, Peter E., and Noirot, Philippe. Mon . "CRISPR interference to interrogate genes that control biofilm formation in Pseudomonas fluorescens". United States. doi:10.1038/s41598-019-52400-5. https://www.osti.gov/servlets/purl/1578041.
@article{osti_1578041,
title = {CRISPR interference to interrogate genes that control biofilm formation in Pseudomonas fluorescens},
author = {Noirot-Gros, Marie-Francoise and Forrester, Sara and Malato, Grace and Larsen, Peter E. and Noirot, Philippe},
abstractNote = {Bacterial biofilm formation involves signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Gene inactivation is a powerful approach for functional studies but it is often labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in diverse strain isolates of P. fluorescens, SBW25, WH6 and Pf0-1. We found that CRISPRi is applicable to study complex phenotypes such as cell morphology, motility and biofilm formation over extended periods of time. In SBW25, CRISPRi-mediated silencing of genes encoding the GacA/S two-component system and regulatory proteins associated with the cylic di-GMP signaling messenger produced swarming and biofilm phenotypes similar to those obtained after gene inactivation. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 operon. We conclude that CRISPRi is a reliable and scalable approach to investigate gene networks in the diverse P. fluorescens group.},
doi = {10.1038/s41598-019-52400-5},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United States},
year = {2019},
month = {11}
}

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