Abstract
To increase our understanding of bacterial biofilm complexity, real- time quantitative analyses of the living community functions are required. To reach this goal, accurate fluorescent reporters are needed. In this paper, we used the classical fluorescent genetic reporters of the GFP family and demonstrated their limits in the context of a living biofilm. We showed that fluorescence signal saturated after only a few hours of growth and related this saturation to the reduction of oxygen concentration induced by bacterial consumption. This behaviour prevents the use of GFP-like fluorescent proteins for quantitative measurement in living biofilms. To overcome this limitation, we propose the use of a recently introduced small protein tag, FAST, which is fluorescent in the presence of an exogenously applied fluorogenic dye, enabling to avoid the oxygen sensitivity issue. We compared the ability of FAST to report on biofilm growth with that of GFP and mCherry, and demonstrated the superiority of the FAST:fluorogen probes for investigating dynamics in the complex environment of a living biofilm.