Toolbox for fluorescent labelling of Pseudomonas aeruginosa across scales: from single cells to bacterial communities and host infection models

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.