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.

CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5

“…Previously, CRISPRi of quorum sensing gene luxS was used to inhibit biofilm formation in E. coli (Zuberi et al 2017). Later, biofilm formation and swarming motility were strongly inhibited by CRISPRi of environmental sensor histidine kinase gene gacS in Pseudomonas fluorescens (Noirot-Gros et al 2019). In B. subtilis, mutations in spo0A caused a defect in biofilm formation (Hamon and Lazazzera 2001).…”

Current State and Promising Opportunities on Pharmaceutical Approaches in the Treatment of Polymicrobial Diseases

“…Another novel approach that can be harnessed to tackle the biofilm-related issues is the use of a clustered, regularly interspaced short palindromic repeats (CRISPR)-mediated method. Studies have revealed the potential use of the CRISPR interference (CRISPRi) system as a strategy to inhibit bacterial biofilm [ 242 , 243 ]. Using the CRISPRi method, Zuberi et al demonstrated that inhibition of genes that are directly involved in quorum sensing could lead to the inhibition of biofilm formation in E. coli [ 242 ].…”

“…Using the CRISPRi method, Zuberi et al demonstrated that inhibition of genes that are directly involved in quorum sensing could lead to the inhibition of biofilm formation in E. coli [ 242 ]. Taking a similar step using P. fluorescens as a model, Noirot-Gros et al (2019) investigated the gene network that leads to the disruption in the biofilm formation [ 243 ]. It seems that the silencing of bacterial genes responsible in the formation of biofilm by CRISPRi may hold a strategic advantage in the management of biofilm-associated polymicrobial diseases.…”

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

“…Both systems were demonstrated to be functional for repression of genes encoding fluorescent proteins, and Kim et al (2020) also employed CRISPRi for metabolic engineering via gene repression by depleting the GlpR regulator to enhance the glycerol-dependent synthesis of mevalonate. Other examples on the development of CRISPRi systems have been reported for P. aeruginosa (Peters et al, 2019;Qu et al, 2019) and P. fluorescens (Noirot-Gros et al, 2019). While the CRISPRi toolbox for Pseudomonas species offers alternatives depending on the intended application (ranging from fundamental studies to simple metabolic engineering manipulations), the techniques applied so far are afflicted by either leaky expression of the components or limited ability to titrate repression levelsthus restricting the applicability of the tool in complex engineering approaches.…”

“…aeruginosa (Peters et al , ; Qu et al , ) and P . fluorescens (Noirot‐Gros et al , ). While the CRISPRi toolbox for Pseudomonas species offers alternatives depending on the intended application (ranging from fundamental studies to simple metabolic engineering manipulations), the techniques applied so far are afflicted by either leaky expression of the components or limited ability to titrate repression levels – thus restricting the applicability of the tool in complex engineering approaches.…”