Pseudomonas aeruginosa is joining the family! A team of Ottawa University led by Prof. Campbell-Valois have just reported multicolor imaging of P. aeruginosa with FAST. Check their findings in Front. Microbiol. 2023.
P. aeruginosa thrives in the airways of individuals with cystic fibrosis. Forming robust biofilms, they often are resistant to immune clearance or antibiotic treatment. In the cystic fibrosis lung, the thickened mucus layers create an oxygen gradient, often culminating into anoxic pockets.
Current fluorescent reporters for studying P. aeruginosa are limited to the GFP and related analogs. However, these reporters require oxygen for the maturation of their chromophore. Hence, making them unsuitable for the study of anaerobically grown P. aeruginosa.
That’s why the authors have evaluated seven alternative fluorescent proteins, including iLOV, phiLOV2.1, evoglow-Bs2, LucY, UnaG, FAST, and iRFP670. Those are indeed reported to emit light under oxygen-limiting conditions. So, the authors generated a series of plasmids encoding these proteins. They further verified their fluorescence using both plate reader assays and confocal microscopy. They found in particular, phiLOV2.1 and FAST to provide superior fluorescence stability. Most interestingly, the couple enabled dual-color imaging of both planktonic and biofilm cultures with a normalized response.
Due to the broad host range of their expression vector, the phiLOV2.1 and FAST-based reporters may be applicable to other Gram-negative bacteria in similar low-oxygen niches. Furthermore, one may retrieve plasmids from Addgene (e.g., plasmid #208808).
The Twinkle Factory offers commercial fluorogens for FAST, pFAST, greenFAST & redFAST, frFAST, and their related split versions, e.g., splitFAST or tripartite-splitFAST. tfFox-NP, a bright non-permeant fluorogen, and tfDarth, a quencher, have recently completed the product range.
Read more about FAST for multicolor imaging in anaerobes
Front. Microbiol. 2023 – Tchagang, C. F., Mah, T. F., & Campbell-Valois, F. X. Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa.
Appl. Environ. Microbiol. 2020 – Charubin, K., Streett, H., & Papoutsakis, E. T. Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins