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Design and validation of a dual-fluorescence reporter system to monitor bacterial gene expression in the gut environment

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Abstract

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. However, available strategies to follow gene expression in bacteria within their natural ecosystem that can be typically rich and complex are scarce. In this work, we designed a plasmid-based tool ensuring both the identification of a strain of interest in complex environments and the monitoring of gene expression through the combination of two distinct fluorescent proteins as reporter genes. The tool was validated in Escherichia coli to monitor the expression of eut genes involved in the catabolism of ethanolamine. We demonstrated that the constructed reporter strain gradually responds with a bimodal output to increasing ethanolamine concentrations during in vitro cultures. The reporter strain was next inoculated to mice, and flow cytometry was used to detect the reporter strain among the dense microbiota of intestinal samples and to analyze specifically the expression of eut genes. This novel dual-fluorescent reporter system would be helpful to evaluate transcriptional processes in bacteria within complex environments.

Key points

• A reporter tool was developed to monitor bacterial gene expression in complex environments.

• Ethanolamine utilization (eut) genes are expressed by commensal E. coli in the mouse gut.

• Expression of eut genes follows a bimodal distribution.

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Acknowledgements

We thank the staff of the CLIC (Clermont Confocal Imaging) facility and especially Caroline VACHIAS for their excellent assistance in time-lapse microscopy experiments. This work was supported by funding from the National Research Institute for Agriculture, Food and Environment (INRAE). Maria Ines Moreira de Gouveia was a PhD Research Fellow funded by the French Ministry of Education and Research. Audrey Reuter was a Postdoctoral Research Fellow granted by the “Microbiology and Food Chain” Division of INRAE.

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Author notes

  1. Maria Ines Moreira de Gouveia and Audrey Reuter contributed equally to this work.

Authors and Affiliations

  1. Université Clermont Auvergne, INRAE, MEDIS UMR454, F-63000, Clermont-Ferrand, France

    Maria Ines Moreira de Gouveia, Audrey Reuter, Annie Garrivier, Julien Daniel, Annick Bernalier-Donadille & Gregory Jubelin

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  1. Maria Ines Moreira de Gouveia
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Contributions

GJ and ABD conceived and designed the research. MIMG, AR, AG, JD, and GJ conducted the experiments. MIMG and GJ analyzed the data. GJ wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Gregory Jubelin.

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Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The experiments performed herein were reviewed and approved by the Auvergne Committee for Animal Experimentation C2EA and received the agreement number #31333-2021042809157789v2. This article does not contain any studies with human participants performed by any of the authors.

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The authors declare no competing interests.

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Supplementary information

ESM 1

Figure S1. Sequence of pHL51 reporter plasmid

Figure S2. Sequence of the PCR product used to construct the HS eutR-T7pol strain. Homologous regions between the PCR and the genome of HS are indicated.

Figure S3. The BL21(DE3) strain carrying pHL51 was grown in LB + IPTG and the mid-log phase culture was washed once with PBS before quantification of BFP and GFP signals by fluorimetry. Data are presented as percentages of fluorescence relative to the signal recorded after the shift (T0). Results are presented as mean and standard error of the mean from 3 independent experiments.

Figure S4. The HS eutR-T7pol (pHL51) strain was grown in LB supplemented with indicated concentrations of vitamin B12 and/or EA during 6h and analysed by flow cytometry. Data are represented on histograms with GFP signal on the x-axis. GFP-negative population is colored in black and GFP-positive population is colored in green.

Figure S5. The HS eutR-T7pol strain carrying either pHL51 or pHL52 plasmid was grown in LB supplemented with vitamin B12 (150 nM), IPTG (1 mM) and/or EA at indicated concentrations during 6h and analysed by flow cytometry. Data are represented on histograms with GFP signal on the x-axis.

Figure S6. Influence of EutR on the bimodal expression of eut genes. The HS eutR-T7pol (pHL51) and HS eutR-T7pol Pcst-eutR (pHL51) strains were grown for 6 h in LB supplemented with indicated concentrations of EA and vitamin B12 and analysed by flow cytometry. Data are represented on two-dimensional dot plots with BFP and GFP signals on the y-axis and x-axis, respectively.

Figure S9. Time-lapse microscopy of HS eutR-T7pol (pHL51) strain grown on an agarose pad without EA and vitamin B12 supplementation. Time-lapse microscopy was performed during 200 minutes on several fields and a representative field is shown here. Images correspond to overlays of BFP, GFP and brightfield acquisitions.

Table S1. Bacterial strains and plasmids used in this study

Table S2. Primers used in this study

(PDF 2.95 mb)

Time-lapse movie of HS eutR-T7pol (pHL51) strain grown on an agarose pad supplemented with EA 30mM and vitamin B12 (AVI 60 mb)

Time-lapse movie of HS eutR-T7pol (pHL51) strain grown on an agarose pads without EA and vitamin B12 (AVI 51.3 mb)

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Moreira de Gouveia, M.I., Reuter, A., Garrivier, A. et al. Design and validation of a dual-fluorescence reporter system to monitor bacterial gene expression in the gut environment. Appl Microbiol Biotechnol 107, 7301–7312 (2023). https://doi.org/10.1007/s00253-023-12788-7

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