Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy

Abstract

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein–protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell

“…The fluorescence molecules currently used in biological research can be divided into two categories: genetically encoded fluorescence proteins (Zhang et al, 1996) and fluorogenic molecules. The latter can be further distinguished into two subcategories: molecules that do not require a binding partner to emit detectable fluorescence upon excitation (Strack, 2021), and molecules that greatly increase their emission intensity after binding to a target molecule (Iyer et al, 2021;Plamont et al, 2016). Both fluorescent proteins and fluorogenic molecules have advantages and disadvantages, and they greatly differ in their mechanism of action.…”

“…Some of the main advantages of these molecules over FPs are that they do not have to mature, have greater photostability, emit more photons and are much smaller. Some fluorogenic probes can be used as standalone molecules (Braut-Boucher et al, 1995;Liu et al, 2020) due to their natural high brightness and quantum yield, while others need to be paired with macromolecules (Iyer et al, 2021;Plamont et al, 2016;Pothoulakis et al, 2014), since only after interacting with a specific binding pocket they become (highly) fluorescent. Possible limitations of fluorogenic molecules, especially for in vivo research, are (i) their potential cytotoxicity and impact on the growth of cells, (ii) the non-trivial specific labeling of macromolecules or supramolecular structures and (iii) the targeting to specific compartments.…”

“…binding motifs have been developed (Iyer et al, 2021;Plamont et al, 2016). These proteins can be used for genetic tagging of targets in the same way that FPs are used.…”

“…These dyes need to be virtually non-fluorescent when unbound, and increase their fluorescence and change their lifetime upon interaction with the macromolecular partner. This system has been applied to create both hybrid protein-dye systems (Iyer et al, 2021;Plamont et al, 2016), and RNA-dye systems (Autour et al, 2018;Pothoulakis, Ceroni, Reeve, & Ellis, 2014), which can be used for imaging purposes (Gautier et al, 2008;Ouellet, 2016) or to develop biosensors ( Jepsen et al, 2018;Tebo et al, 2018;Wang, Wilson, & Hammond, 2016).…”

Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging

“…The introduced linkers or reactive groups for chemical labelling at specific sites can cause potential cytotoxicity to bacteria [ 29 , 30 ]. Genetic engineering, which endows bacteria with inductively or spontaneously expressed imageable proteins, such as fluorescence proteins and gas vesicle proteins, is an elegant approach to solve these limitations [ [31] , [32] , [33] , [34] ]. Representatively, as one of fluorescent proteins, green fluorescent protein (GFP) provides a simple yet robust way to genetically tag proteins of interest in bacteria [ 35 , 36 ].…”