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

Marie-Francoise Noirot-Gros,Grace Malato,Philippe Noirot,Peter E Larsen,Sara Forrester

doi:10.1038/s41598-019-52400-5

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.

Highlights

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
The amplitude of the inhibition depended on the DNA site and strand targeted by the gRNA and the concentration of aTc inducer, demonstrating that these features can be used to modulate the level of inhibition
Silencing by CRISPR interference (CRISPRi) was used in SBW25 to investigate genes involved in cell division and cell morphology for which phenotypes are observed after 3–18 hours of growth, and genes involved in the regulation of motility and biofilm formation for which phenotypes are scored after 48 hours on plate and liquid culture assays

Summary

Introduction

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. EPS production is regulated by the Gac/Rsm signaling cascade, involving GacA/S TCS and the non-coding small regulatory RNAs RsmZ and RsmY17,18,22–24 This signaling cascade regulates about 700 genes involved in a wide range of biological functions, including biofilm formation and oxidative stress response[17,24,25]. Additional levels of regulation involve the PDE RimA which modulates the activity of RimK, an enzyme that modifies the ribosomal protein RpsF by adding glutamate residues to its C terminus, altering ribosome abundance and function[40,41] These studies reveal that c-di-GMP-binding enzymes and proteins act in a coordinated manner to control the various stages of the planktonic-to-biofilm transition, altering motility, promoting cell adhesion, producing EPS and shaping biofilm architecture. While this assay is rapid and reliable, it cannot report on the full range of phenotypes (e.g., cell abundance, EPS architecture) that characterize biofilm development

Methods

Results

Conclusion