Spatiotemporal analysis of quorum-sensing responses reveals diffusion constraints in Vibrio cholerae biofilms

Abstract

Bacteria frequently form multicellular, surface-associated communities called biofilms. Biofilms provide advantages to residing cells, protecting them from environmental stresses such as antibiotics and mechanical perturbation. It has been proposed that these advantages stem from limited diffusion of small molecules into and out of the biofilm. However, due to the challenge of probing events occurring inside the biofilm structure, direct measurements of diffusion through the biofilm are limited. Here, I use single-molecule fluorescent in situ hybridization (smFISH) to measure spatiotemporal transcriptional responses of quorum-sensing (QS)-regulated genes in Vibrio cholerae biofilms following perturbations in levels of the QS signaling molecule called autoinducer-2 (AI-2), allowing gene expression to be used as a proxy to infer the spatial distribution of AI-2 over time within biofilms. Spatial position within the biofilm influenced QS response kinetics, with peripheral cells responding more rapidly than interior cells, particularly during the early response to AI-2 addition. Transitions between QS states were also asymmetric, occurring more rapidly following AI-2 addition than removal. Increased extracellular matrix production also slowed these responses following AI-2 addition. Collectively, these results suggest that diffusion limitations within the biofilm matrix restrict autoinducer movement and modulate QS responses. This study establishes a framework for linking gene expression dynamics to spatial transport processes and demonstrates how physical constraints within biofilms shape collective bacterial behavior.