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.
Highlights
Bacterial biofilms are confined, three-dimensional communities of adherent microbes
We present the quantitative investigation of the spatio-temporal fluorescence profiles of two fluorescent proteins, green fluorescent protein (GFP) and mCherry, constitutively expressed in biofilms of Escherichia coli grown under controlled fluid flow
By monitoring the spatio-temporal distribution of GFP and FAST fluorescence in the same biofilm growing under flow, we showed that the signal of the two proteins displays remarkably distinct spatial distributions and kinetics soon after biofilm starts to grow
Summary
Three-dimensional communities of adherent microbes. They display an outperforming specific lifestyle, which raises a number of fundamental and practical questions drawing interest from various disciplines[1,2,3,4,5,6]. We present the quantitative investigation of the spatio-temporal fluorescence profiles of two fluorescent proteins, GFP and mCherry, constitutively expressed in biofilms of Escherichia coli grown under controlled fluid flow. We show that both GFP and mCherry exhibit fluorescence kinetics quickly becoming inconsistent with the cell biomass development. The spatio-temporal fluorescence profiles appeared to closely match the oxygen shortage that arises as the biofilm develops To circumvent this problem, we tested the ability of a new small protein tag, FAST (Fluorescence-Activating and absorption-Shifting Tag), to report linearly on gene expression in oxygen-deficient biofilms. The recently developed small protein tag, FAST28, which fluoresces in the presence of an exogenously applied fluorogenic dye, offers an outstanding answer to this issue, enabling accurate measurement of gene expression in biofilms, irrespectively of the oxygen gradients
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