A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST).

Visualizing protein localization and characterizing gene expression activity in live cells is limited for lack of a real-time, highly fluorescent, oxygen-independent reporter system. Enzymatic reporter systems have been used successfully for many years with spp.; however, these assays do not allow for real-time analysis of gene expression activity with flow cytometry or for visualizing protein localization through fusion proteins. Commonly used fluorescent reporter proteins require oxygen for chromophore maturation and cannot be used for most strictly anaerobic organisms. Here we show that the fluorescence-activating and absorption-shifting tag protein (FAST), when associated with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR; now commercially available) and other commercially available ligands, is highly fluorescent in under anaerobic conditions. Using flow cytometry and a fluorescence microplate reader, we demonstrated FAST as a reporter system by employing the promoters of the thiolase (), acetoacetate decarboxylase (), and phosphotransbutyrylase () metabolic genes, as well as a mutant P and modified ribosome binding site (RBS) versions of P and P Flow cytometry-based sorting was efficient and fast in sorting FAST-expressing cells, and positively and negatively sorted cells could be effectively recultured. FAST was also used to tag and examine protein localization of the predicted cell division FtsZ partner protein, ZapA, to visualize the divisome localization in live cells. Our findings suggest that FAST can be used to further investigate divisomes and more broadly the localization and expression levels of other proteins in organisms, thus enabling cell biology studies with these organisms. FAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system in FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that FAST is highly fluorescent under anaerobic conditions, it can be used in several applications of this and likely many organisms and other strict anaerobes, including studies involving cell sorting, sporulation dynamics, and population characterization in pure as well as mixed cultures, such as those in various native or synthetic microbiomes and syntrophic cultures.