Toolbox for fluorescent labelling of Pseudomonas aeruginosa across scales: from single cells to bacterial communities and host infection models
Abstract
Fluorescence microscopy is central to the study of bacterial cell biology, multicellular behaviours, and host–pathogen interactions. Bright, robust and photostable labelling is required for bacterial identification, sorting and quantitative analysis, driving continuous development of state-of-the-art labelling tools. Here, we developed a multi-color fluorescent cell labelling toolkit for Gram-negative bacteria carrying the attTn7 site, using the opportunistic human pathogen Pseudomonas aeruginosa as a model. Cell labelling is achieved by constitutive chromosomal expression of genes encoding a choice of four novel fluorescent proteins, mNeonGreen, mJuniper, mLychee and mScarlet-I3, codon-optimised for P. aeruginosa . These reporters provide bright, stable fluorescence with minimal photobleaching and excellent spectral separation during long-term imaging of single cells, macrocolonies and biofilms. Chromosomal expression of mNeonGreen yielded brighter and more homogeneous labelling than expression of the same construct from a plasmid. Interestingly, multi-color labelling of macrocolonies using our constructs allowed us to uncover and monitor a reversal of cell migration within motile colonies, thus highlighting the novel dynamics of motile communities. Finally, we demonstrate their applicability in biologically relevant host-pathogen contexts by imaging both live and fixed P. aeruginosa- infected human airway epithelial cells. This versatile cell labelling platform enables reliable bacterial identification, segmentation, tracking, and quantitative fluorescence imaging across spatial and temporal scales, and is readily adaptable to most other Gram-negative bacteria as the attTn7 integration site is well conserved.
Related
- Bioimaging With Fluorescent Nucleic‐Acid Aptamers for the Specific Detection and Quantification of Pseudomonas aeruginosa Alone and in Heterogeneous Bacterial Populations
- Corneal epithelial signals remodel bacterial physiology and extracellular vesicle cargo in Pseudomonas aeruginosa and Staphylococcus epidermidis
- Tag-stabilized fluorescent protein binders for endogenous cell type-specific protein labelling
- Host immune stress reveals a role for potassium homeostasis in Pseudomonas aeruginosa aggregate stability
- LytM family endopeptidase LmdC is controlled by regulated auto processing and proteolytic destruction in Pseudomonas aeruginosa
BibTeX
@misc{gerard2026toolbox,
title = {{Toolbox for fluorescent labelling of Pseudomonas aeruginosa across scales: from single cells to bacterial communities and host infection models}},
author = {Mattéo Gérard and Charlène Cornilleau and Vinciane Saint-Criq and Merve Nur Tunç and Maxime Deforet and Romain Briandet and Steven L Porter and Rut Carballido-López},
howpublished = {HAL},
year = {2026},
month = aug,
doi = {10.64898/2026.08.21.746161},
url = {https://hal.science/hal-05749934v1},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - UNPB
TI - Toolbox for fluorescent labelling of Pseudomonas aeruginosa across scales: from single cells to bacterial communities and host infection models
AU - Mattéo Gérard
AU - Charlène Cornilleau
AU - Vinciane Saint-Criq
AU - Merve Nur Tunç
AU - Maxime Deforet
AU - Romain Briandet
AU - Steven L Porter
AU - Rut Carballido-López
PY - 2026
DA - 2026/08/21
PB - HAL
DO - 10.64898/2026.08.21.746161
UR - https://hal.science/hal-05749934v1
AB - Fluorescence microscopy is central to the study of bacterial cell biology, multicellular behaviours, and host–pathogen interactions. Bright, robust and photostable labelling is required for bacterial identification, sorting and quantitative analysis, driving continuous development of state-of-the-art labelling tools. Here, we developed a multi-color fluorescent cell labelling toolkit for Gram-negative bacteria carrying the attTn7 site, using the opportunistic human pathogen Pseudomonas aeruginosa as a model. Cell labelling is achieved by constitutive chromosomal expression of genes encoding a choice of four novel fluorescent proteins, mNeonGreen, mJuniper, mLychee and mScarlet-I3, codon-optimised for P. aeruginosa . These reporters provide bright, stable fluorescence with minimal photobleaching and excellent spectral separation during long-term imaging of single cells, macrocolonies and biofilms. Chromosomal expression of mNeonGreen yielded brighter and more homogeneous labelling than expression of the same construct from a plasmid. Interestingly, multi-color labelling of macrocolonies using our constructs allowed us to uncover and monitor a reversal of cell migration within motile colonies, thus highlighting the novel dynamics of motile communities. Finally, we demonstrate their applicability in biologically relevant host-pathogen contexts by imaging both live and fixed P. aeruginosa- infected human airway epithelial cells. This versatile cell labelling platform enables reliable bacterial identification, segmentation, tracking, and quantitative fluorescence imaging across spatial and temporal scales, and is readily adaptable to most other Gram-negative bacteria as the attTn7 integration site is well conserved.
ER -