Imaging living obligate anaerobic bacteria with bilin-binding fluorescent proteins

Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a

“…(Detailed plasmid construction is provided in the supplemental material.) UnaG fluoresces only after the addition of cell-permeable bilirubin and, unlike many fluorophores, does not require oxygen, making it helpful in studying members of the gut microbiota ( 18 ). UnaG is maximally excited at 495 nm when bound to bilirubin, with a maximal emission at 525 nm.…”

Ruminococcus bromii enables the growth of proximal Bacteroides thetaiotaomicron by releasing glucose during starch degradation

“…Δsus UnaG Bt was generated by counter-selectable allelic exchange in a ΔsusA-G Bt ( Δtdk ) thymidine kinase deletion mutant with codon optimized UnaG (45) and grown as previously described (46). Bt ATCC 29148 (VPI-5482) and its derivative Δsus UnaG Bt were grown in TYG medium (47) or minimal medium (48).…”

“…Rb grows on potato amylopectin similar to growth on fructose and glycogen as carbon source The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.07.479236 doi: bioRxiv preprint (Figure S3a). Since Bt can also grow on amylopectin, we used a ∆sus Bt strain in which the sus operon (susA-G) is deleted, and tagged it with the anaerobic fluorescent protein UnaG, which fluoresces in the presence of a bilirubin cofactor when excited at 488 nm (45). The constructed ∆sus UnaG Bt strain cannot grow on potato amylopectin but can grow on glucose and fluoresces in the presence of a bilirubin cofactor when excited at 488 nm (Figure S3b and c).…”

Characterization of flavin binding in oxygen‐independent fluorescent reporters

“…[5][6][7][8][9] Unlike GFP, chemogenetic reporters function by pairing proteins with synthetic dyes or biogenic fluorophores such as bilins or flavin mononucleotide (FMN), thereby retaining the ability to fluoresce even in oxygen-free conditions. [10][11][12][13][14][15] Compared to bilins and synthetic dyes, FMN is an essential metabolite found in all living systems, 16 which makes it possible to use FMN based fluorescent proteins to label cells without requiring external agents to be delivered across the largely impenetrable bacterial and fungal cell walls. FMN-based reporters are derived from light, oxygen, and voltage sensing (LOV) domains that are found in certain photoactive proteins, which use FMN to initiate cell signaling by absorbing light and forming a covalent bond with a cysteine residue located in the FMN binding cavity.…”

“…However, flagship reporters based on the green fluorescent protein (GFP) depend on environmental oxygen to emit light, 4 which renders them unusable in gut microbes, archaea, anaerobic communities, marine bacteria, and other biological systems that thrive in oxygen‐free environments 5‐9 . Unlike GFP, chemogenetic reporters function by pairing proteins with synthetic dyes or biogenic fluorophores such as bilins or flavin mononucleotide (FMN), thereby retaining the ability to fluoresce even in oxygen‐free conditions 10‐15 . Compared to bilins and synthetic dyes, FMN is an essential metabolite found in all living systems, 16 which makes it possible to use FMN based fluorescent proteins to label cells without requiring external agents to be delivered across the largely impenetrable bacterial and fungal cell walls.…”