Release of extracellular DNA byPseudomonasspecies as a major determinant for biofilm switching and an early indicator for cell population control

Abstract

Microbial populations undergo phenotypic switching as a response to environmental perturbations. For instance, some bacteria switch from a planktonic lifestyle to biofilm, resulting in altered physiological properties such as increased robustness depending on the conditions. However, the precise detection of phenotypic switching events during the bacterial life cycle is still a technical challenge. Propidium iodide (PI) is one of the most frequently used fluorescence indicators for assessing cell viability based on membrane permeability, yet PI-stained cells sometimes display a red-but-not-dead phenotype. In Escherichia coli, this phenomenon is connected to modulation of porins in the outer membrane (OM) to adapt OM permeability according to nutrient availability. In this study, we explored PI staining to assess phenotypic changes in Pseudomonas sp. during biofilm development. We show that this switch is linked to excretion of extracellular DNA (eDNA), rather than modification of OM permeability. Confocal laser scanning microscopy (CLSM) enabled direct visualization of red fluorescent clusters outside intact membranes of viable cells, suggesting that PI binds eDNA. Besides, the occurrence of PI-positive sub-populations was correlated with biofilm formation in the model bacterium Pseudomonas putida KT2440. Engineered derivatives thereof with altered biofilm-forming capabilities exposed a whole continuum of phenotypic states involving planktonic cells and aggregates, and were identified according to the dynamic change of PI-positive cells with flow cytometry analysis. Our results demonstrate that PI is a fast, convenient and versatile staining for eDNA to rapidly monitor the phenotypic switching of Pseudomonas sp. during transitions in the bacterial lifestyle.