Multicolor whole-cell bacterial sensing using a synchronous fluorescence spectroscopy-based approach.

Damien Parrello,Sebastian Miron,Christian Mustin,Patrick Billard,David Brie

doi:10.1371/journal.pone.0122848

Abstract

The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems.

Highlights

Fluorescent proteins (FPs) have become valuable tools for investigating biological processes in living cells
The pPB-lac plasmids are derivatives of the broad host range promoter-probe vector pPROBE’-GFP[LVA][20] utilizing different fluorescent reporter genes controlled by the strong lac promoter PA1/O4/O3, which is constitutive in E. coli TOP10
This spectral simplification and bandwidth narrowing holds for TurboYFP and DsRed-Express2 as indicated by the systematic reduction of full width at half maximum (FWHM) of spectra acquired synchronously compared to emission peaks (Table 2)

Summary

Introduction

Fluorescent proteins (FPs) have become valuable tools for investigating biological processes in living cells. FPs are regularly used, for instance, as reporters for non-invasive monitoring of gene expression as well as for studying subcellular localization and dynamics of proteins [1] In ecological studies, they can serve as tags to track bacteria in complex environmental matrices such as biofilms or soils, and to survey mutualistic or pathogenic associations that bacteria form with plants or animals [2]. They can serve as tags to track bacteria in complex environmental matrices such as biofilms or soils, and to survey mutualistic or pathogenic associations that bacteria form with plants or animals [2] Another broad application area is the development of whole cell bacterial sensors, which typically relies on gene fusions between a stress.

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Conclusion