Discovery of a biofilm electrocline using real-time 3D metabolite analysis

Abstract

Bacteria are social organisms that possess multiple pathways for sensing and responding to small molecules produced by other microbes. Most bacteria in nature exist in sessile communities called biofilms, and the ability of biofilm bacteria to sense and respond to small molecule signals and cues produced by neighboring biofilm bacteria is particularly important. To understand microbial interactions between biofilms, it is necessary to perform rapid, real-time spatial quantification of small molecules in microenvironments immediately surrounding biofilms; however, such measurements have been elusive. In this study, scanning electrochemical microscopy was used to quantify small molecules surrounding a biofilm in 3D space. Measuring concentrations of the redox-active signaling molecule pyocyanin (PYO) produced by biofilms of the bacterium Pseudomonas aeruginosa revealed a high concentration of PYO that is actively maintained in the reduced state proximal to the biofilm. This gradient results in a reduced layer of PYO that we have termed the PYO "electrocline," a gradient of redox potential, which extends several hundred microns from the biofilm surface. We also demonstrate that the PYO electrocline is formed under electron acceptor-limiting conditions, and that growth conditions favoring formation of the PYO electrocline correlate to an increase in soluble iron. Additionally, we have taken a "reactive image" of a biofilm surface, demonstrating the rate of bacterial redox activity across a 2D surface. These studies establish methodology for spatially coordinated concentration and redox status measurements of microbe-produced small molecules and provide exciting insights into the roles these molecules play in microbial competition and nutrient acquisition.ultramicroelectrode | square wave voltammetry | local redox microenvironment | reduced pyocyanin layer

Review—Advances in Scanning Electrochemical Microscopy (SECM)

“…Several reviews have been dedicated to SECM investigations of such biological systems. [119][120][121][122][123] More recent applications have involved assessing multidrug resistance on cell co-culture patterns, 124 real-time mapping of local hydrogen peroxide production across a biofilm of oral bacteria and in observing its consumption in a polymicrobial biofilm through a mechanism involving one of the bacteria during spatial scanning, 125 in the discovery of a biofilm electrocline of oral bacteria using real-time 3D metabolite analysis, 126 and in the real-time monitoring of quorum sensing in bacterial aggregates. 127,128 Some examples are briefly discussed below.…”

BqsR/BqsS Constitute a Two-Component System That Senses Extracellular Fe(II) in Pseudomonas aeruginosa

“…Recently, it was shown that phenazine carboxylic acid can promote P. aeruginosa biofilm formation by stimulating Fe(II) acquisition in the presence of conalbumin (61). In addition, the presence of Fe(II) has been detected within a P. aeruginosa biofilm producing a gradient of the reduced phenazine pyocyanin (25).…”

Working Together for the Common Good: Cell-Cell Communication in Bacteria

“…In particular, Koley showed that a reduced layer of pyocyanin exists for 500 m above a Pseudomonas biofilm, even in the presence of oxygen. This may be critical to the organism's ability to scavenge iron (32). The SECM approach enables quantitative measurements of redox metabolite production in real time and in three dimensions (3D) that will undoubtedly facilitate a better understanding of how they aid bacterial survival and growth.…”