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The Tol-Pal proteins stabilize the outer membrane during cell division in many Gram-negative bacteria, including Escherichia coli . Pal is an outer membrane lipoprotein that can bind peptidoglycan. It accumulates at the septum during division by a mobilization-and-capture mechanism. This work further substantiates and extends knowledge of Pal's localization in E. coli using immunolabelling; this method enables the detection of endogenous proteins. The midcell localization of Pal and TolB, as seen with fluorescent protein fusions, during cell division, was confirmed. The retention of Pal in newly formed cell poles seemed to persist longer than observed with fluorescent Pal fusions. The concentration of endogenous Pal during the cell division cycle fluctuated: it decreased initially (to half the fluorescence concentration (32.1 au µm -3 ) of the maximum (64.1 au µm -3 ) reached during the cell cycle) and then increased during the second half of the cell division cycle. We probed for possible regulators and proposed two new putative regulators of Pal. By deleting the periplasmic protease, Prc decreased the total Pal abundance (to ~65% of the fluorescence concentration in WT cells) and affected its concentration fluctuation during the cell cycle. This suggests that Prc controls a cell division stage-specific regulator of Pal. Immunolabelling also supported the prediction that the small RNA MicA suppresses Pal expression (the fluorescence concentration of Pal in cells without MicA is double that of Pal in WT cells). However, the regulation by MicA occurred in a cell cycle-independent manner. All these findings urge further research on the tight regulation of the dividing cell envelope stability., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors.)

The fluorescent proteins superfolder mTurquoise2 ox (sfTq2 ox ) and mNeonGreen function excellently in mammalian cells, but are not well expressed in E. coli when forming the N-terminus of constructs. Expression was increased by decreasing structures at the start of their coding sequences in the mRNA. Unfortunately, the expression of mNeonGreen started from methionine at position ten as optimisation introduced an alternative RBS in the GFP N-terminus of mNeonGreen. The original start-codon was not deleted, which caused the expression of isomers starting at the original start-codon and at the start-codon at the beginning of the GFP N-terminus. By omitting the GFP N-terminus of mNeonGreen and optimising the structure of its mRNA, the expression of a mixture of isomers was avoided, and up to ~ 50-fold higher expression rates were achieved for mNeonGreen. Both fluorescent proteins can now be expressed at readily detectable levels in E. coli and can be used for various purposes. One explored application is the detection of in-cell protein interactions by FRET. mNeonGreen and sfTq2 ox form a FRET pair with a larger dynamic range than commonly used donor-acceptor pairs, allowing for an excellent signal-to-noise ratio, which supports the detection of conformational changes that affect the distance between the interacting proteins., (© 2022. The Author(s).)

This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in all compartments of Escherichia coli by Förster Resonance Energy Transfer (FRET) using the Superfolder mTurquoise2 ox-mNeonGreen FRET pair (sfTq2 ox -mNG). This FRET pair has more than twice the detection range for FRET interaction studies in the cytoplasm or periplasm of E. coli compared to other pairs to date. These protein-interaction studies can be performed in vivo because fluorescent proteins can be genetically encoded as fusions to proteins of interest and expressed in the cell. sfTq2 ox and mNG fluorescent protein fusions are co-expressed in bacterial cells and the fluorescence emission spectra are measured. By also measuring reference spectra for the background, sfTq2 ox -only and mNG-only samples, expected emission spectra can be calculated. Sensitized emission for mNG above the expected spectrum can be attributed to FRET and quantified by spectral unmixing. This bio-protocol discusses the sfTq2 ox -mNG FRET pair and provides a practical guide in preparing the protein fusions, setting up and running the FRET experiments, measuring fluorescence spectra and gives the tools to analyze the collected data., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)

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 (sfTq2 ox ) and is able to withstand challenging, oxidizing environments. sfTq2 ox 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 sfTq2 ox has the same spectroscopic properties as mTq2, that is, high fluorescence lifetime and quantum yield. The sfTq2 ox -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 (sfTq2 ox ), 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 sfTq2 ox as FRET donor, we show the direct in vivo interaction and disruption of unique periplasmic antibiotic targets FtsB and FtsL., (© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)