Tracking and controlling phenotypic switching of Pseudomonas sp. associated with biofilm formation

Abstract
Phenotypic switching to a biofilm lifestyle is a significant survival strategy employed by bacteria under stressful conditions, necessitating a transition to a biofilm-specific phenotype. Pseudomonas sp. is renowned for its remarkable ability to thrive in extreme conditions, both in natural environments and within the human body, owing to its capacity to form biofilms. An example of this is Pseudomonas putida KT2440, a model plant-beneficial bacterium commonly found in soil, which can form biofilms depending on the surrounding environmental conditions. P. putida is widespread in the natural environment, colonizing the rhizosphere of various crop plants and persisting in the ecosystem.
The newly proposed biofilm developmental cycle in Pseudomonas sp. progresses through distinct stages, including aggregation and attachment, growth and accumulation, and finally disaggregation and detachment. Our research focuses on investigating the early initiation of biofilm formation, specifically the transition of individual cells from free-floating bacteria to the initial stages of biofilm formation, where aggregates float in the liquid phase. However, characterizing this process is challenging due to the lack of suitable fluorescent reporters and the formation of cell aggregates during the transition, which hinders single-cell analysis. To address this, we identified PI staining as a rapid, convenient, and reliable single-cell proxy that effectively monitors the phenotypic switching occurring in the early stages of biofilm development. By utilizing PI staining, we validated the significance of extracellular DNA (eDNA) in the cell-decision-making process, particularly in the initial stage of biofilm formation, shedding light on its impact on phenotypic diversification.
To further advance our research, we leveraged the association between PI-eDNA-associated cells and implemented reactive flow cytometry, coupled with a well-designed control strategy and an appropriate single-cell biomarker, in an advanced system known as Segrogostat. This approach allowed us to effectively assess and control the initial biofilm-phenotype state of bacteria with high temporal resolution.
Furthermore, our findings demonstrated that the early stages of biofilm development can be detected in planktonic cultures by evaluating the level of auto-aggregation and co-aggregation using flow cytometry as a high-throughput method, along with the identification of PI-eDNA-associated/PI-positive cells. We established a strong relationship between PI staining and cell auto-aggregation, reinforcing the link between these factors and biofilm formation.
This study opens novel avenues to possible applications of PI as a single-cell proxy, allowing us to capture the "key" subpopulations of cells involved in biofilm formation.In this sense, this work paves the way to further understanding the early phenotypic switching involved in biofilm formation.