Dataset Information

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

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.

SUBMITTER: Parrello D

PROVIDER: S-EPMC4379052 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Parrello Damien D Mustin Christian C Brie David D Miron Sebastian S Billard Patrick P

PloS one 20150330 3

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 spectr ...[more]

PMID: 25822488

Similar Datasets

Project description:Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a key method for the detection of (uncultured) microorganisms in environmental and medical samples. A major limitation of standard FISH protocols, however, is the small number of phylogenetically distinct target organisms that can be detected simultaneously. In this study, we introduce a multicolor FISH approach that uses eight fluorophores with distinct spectral properties, which can unambiguously be distinguished by confocal laser scanning microscopy combined with white light laser technology. Hybridization of rRNA-targeted DNA oligonucleotide probes, which were mono-labeled with these fluorophores, to Escherichia coli cultures confirmed that the fluorophores did not affect probe melting behavior. Application of the new multicolor FISH method enabled the differentiation of seven (potentially up to eight) phylogenetically distinct microbial populations in an artificial community of mixed pure cultures (five bacteria, one archaeon, and one yeast strain) and in activated sludge from a full-scale wastewater treatment plant. In contrast to previously published multicolor FISH approaches, this method does not rely on combinatorial labeling of the same microorganisms with different fluorophores, which is prone to biases. Furthermore, images acquired by this method do not require elaborate post-processing prior to analysis. We also demonstrate that the newly developed multicolor FISH method is compatible with an improved cell fixation protocol for FISH targeting Gram-negative bacterial populations. This fixation approach uses agarose embedding during formaldehyde fixation to better preserve the three-dimensional structure of spatially complex samples such as biofilms and activated sludge flocs. The new multicolor FISH approach should be highly suitable for studying structural and functional aspects of microbial communities in virtually all types of samples that can be analyzed by conventional FISH methods.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure