# Nitric Oxide Donor Spermine-NONOate Elicits Endogenous Dispersal-Associated Transcriptional Responses to Promote Biofilm Dispersal in Pseudomonas aeruginosa

## TL;DR

This study shows that the NO donor Spermine-NONOate promotes biofilm dispersal in Pseudomonas aeruginosa by activating key dispersal-related genes.

## Contribution

The study identifies specific transcriptional responses linked to NO-induced biofilm dispersal and validates known dispersal biomarkers.

## Key findings

- SP-NONO activates canonical NO-regulated responses and disrupts energy metabolism pathways.
- Shared transcriptional shifts in SP-NONO-treated and spontaneously dispersed biofilms include branched-chain amino acid catabolism genes.
- Nine of 14 known dispersal biomarkers were confirmed in SP-NONO-treated biofilms.

## Abstract

Background/Objectives: Bacterial biofilms are structured communities of sessile cells embedded in a self-produced extracellular matrix. Within biofilms, bacteria become highly tolerant toenvironmental stressors such as host immune responses and antimicrobial treatments. In response to specific cues, however, biofilm cells can revert to a planktonic free-swimming lifestyle through a process termed biofilm dispersal. When dispersed cells escape the biofilm matrix, they lose biofilm-associated antibiotic tolerance, a major barrier to treating medical biofilms. As such, dispersal-inducing compounds like nitric oxide (NO) are actively investigated as adjuvants to potentiate the biofilm-eradicating activity of existing antibiotics. We recently characterised the transcriptomic responses elicited during spontaneous biofilm dispersal in closed culture-grown Pseudomonas aeruginosa biofilms. Here, we evaluated the transcriptional profiles of P. aeruginosa biofilms treated with the NO donor Spermine-NONOate (SP-NONO) and the nitroxide C-TEMPO, an NO analogue, to determine potential pathways involved in NO-mediated dispersal. Methods: Dispersal activity on P. aeruginosa PAO1 biofilms by SP-NONOate and C-TEMPO was quantified by crystal violet staining. Cellular responses to each compound were profiled by RNA-seq on treated and untreated cells. Results: While both compounds disrupted the transcription of ANR-regulated energy metabolism pathways, only SP-NONO activated canonical NO-regulated responses. Considering that only SP-NONO showed biofilm dispersal activity in this culture system, we investigated shared transcriptional shifts in SP-NONO-treated and spontaneously dispersed biofilms to identify pathways likely involved in central dispersal responses. These mostly included genes involved in the catabolism of branched-chain amino acids (leucine, valine, isoleucine) and lysine, as well as 9 of 14 genes previously defined as transcriptional biomarkers of spontaneous biofilm dispersal. Conclusions: This study suggests that NO disrupts biofilm maturation by prematurely stimulating central pathways of spontaneous biofilm dispersal and highlights this set of biomarkers as robust indicators of dispersal responses.