# Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

## TL;DR

A drop in temperature helps Pseudomonas aeruginosa form biofilms by activating a specific signaling module that increases sticky substance production.

## Contribution

The study identifies the SiaABCD module as a novel temperature-sensing pathway that promotes biofilm formation through c-di-GMP elevation.

## Key findings

- Temperature downshift from 37°C to 21°C promotes biofilm formation in 63% of P. aeruginosa strains.
- The SiaABCD module, especially SiaD and SiaA, drives c-di-GMP elevation and biofilm promotion at lower temperatures.
- Membrane perturbations triggered by temperature changes activate the SiaABCD module to increase Psl production.

## Abstract

Pseudomonas aeruginosa is a highly adaptable Gram-negative pathogen known for its remarkable ability of forming biofilms. Understanding the environmental cues and regulatory mechanisms that drive biofilm formation is essential for developing effective control strategies. In this study, we screened 57 clinical and environmental P. aeruginosa isolates and discovered that a universal environmental cue, temperature downshift from host-associated 37 °C to room temperature (21 °C), significantly promotes biofilm formation in 63% of the strains. Using the ATCC 27853 strain as a model, we demonstrate that this enhancement results from increased production of the Psl exopolysaccharides at lower temperature. LC-MS/MS analysis revealed elevated levels of the secondary messenger c-di-GMP, a key regulator of the motile-to-sessile transition, at room temperature. Through screening a mutant library targeting 18 c-di-GMP metabolic enzymes, we identified the diguanylate cyclase SiaD within the SiaABCD signaling and functional module as a principal driver of c-di-GMP elevation and biofilm promotion. Further investigation showed that the entire SiaABCD module, especially the signal-sensing domain of SiaA, mediates the temperature-dependent response. Integrating lipidomics with genetics and physiological assays, we show that a temperature downshift triggers rapid membrane perturbations that activate the SiaABCD signaling module, thereby increasing Psl production to strengthen surface adhesion and drive robust biofilm formation. These findings establish temperature downshift as a previously unrecognized physiological cue that promotes biofilm formation in P. aeruginosa and define an adaptive regulatory pathway linking specific environmental stresses of membrane perturbation to dedicated c-di-GMP signaling module, paving the way for new strategies to disrupt biofilm-associated infections and transmission.