Superfolder mTurquoise2^ox optimized for the bacterial periplasm allows high efficiency in vivo FRET of cell division antibiotic targets

Abstract

Summary Fluorescent proteins (FPs) are of vital importance to biomedical research. Many of the currently available fluorescent proteins do not fluoresce when expressed in non‐native environments, such as the bacterial periplasm. This strongly limits the options for applications that employ multiple FPs, such as multiplex imaging and Förster resonance energy transfer (FRET). To address this issue, we have engineered a new cyan fluorescent protein based on mTurquoise2 (mTq2). The new variant is dubbed superfolder turquoise2ox (sfTq2ox) and is able to withstand challenging, oxidizing environments. sfTq2ox has improved folding capabilities and can be expressed in the periplasm at higher concentrations without toxicity. This was tied to the replacement of native cysteines that may otherwise form promiscuous disulfide bonds. The improved sfTq2ox has the same spectroscopic properties as mTq2, that is, high fluorescence lifetime and quantum yield. The sfTq2ox‐mNeongreen FRET pair allows the detection of periplasmic protein‐protein interactions with energy transfer rates exceeding 40%. Employing the new FRET pair, we show the direct interaction of two essential periplasmic cell division proteins FtsL and FtsB and disrupt it by mutations, paving the way for in vivo antibiotic screening. Significance The periplasmic space of Gram‐negative bacteria contains many regulatory, transport and cell wall‐maintaining proteins. A preferred method to investigate these proteins in vivo is by the detection of fluorescent protein fusions. This is challenging since most fluorescent proteins do not fluoresce in the oxidative environment of the periplasm. We assayed popular fluorescent proteins for periplasmic functionality and describe key factors responsible for periplasmic fluorescence. Using this knowledge, we engineered superfolder mTurquoise2ox (sfTq2ox), a new cyan fluorescent protein, capable of bright fluorescence in the periplasm. We show that our improvements come without a trade‐off from its parent mTurquoise2. Employing sfTq2ox as FRET donor, we show the direct in vivo interaction and disruption of unique periplasmic antibiotic targets FtsB and FtsL.

pH-dependent activation of cytokinesis modulates Escherichia coli cell size

“…S. aureus and S. pneumoniae still undergo pH-dependent changes in size but lack identifiable homologs of FtsN (S3 Fig) [25], indicating the existence of additional pH-responsive divisome components at least in these organisms. Recent technological advancements, including the use of FRET biosensors to probe interactions between division proteins and new methods to assay activity and interactions between the membrane-associated divisome components, offer promising avenues to dissect the impact of pH specific divisome interactions in future studies [64,82].…”

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

“…This would argue in favor of a more complex mechanism that also requires proper folding for periplasmic fluorescence. Based on mTurquoise2 [30], a superfolder variant named sfTq2 (Figure 1), which incorporates all six mutations of sfGFP, was created [31]. The advantage with bright cyan FPs donors is that they can accommodate a large palette of acceptors, either green, yellow, or orange, with high Förster resonance energy transfer (FRET) efficiencies.…”

“…sfTq2 is now used in FRET experiments with acceptor mNeonGreen [32] for the detection of protein–protein interactions in cytoplasmic and periplasmic compartments. sfTq2 mutant C70V named sfTq2 ox , when paired with mNeonGreen shows even brighter fluorescence signal in the periplasm [31].…”

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins

“…Labeling OM proteins, however, is challenging. Many fluorescent reporter proteins fail to mature in the periplasm [57]; thus, genetic fusions with fluorescent proteins are limited to a handful of options [58,59,60,61]. An alternative approach is non-covalent affinity-based labeling by using antibody–reporter protein fusions, affinity tags, or other high-affinity interactions, for example, colicins ColE9 and ColIa for labeling of the vitamin B12 transporter BtuB [62].…”