Regulation of the Peptidoglycan Polymerase Activity of PBP1b by Antagonist Actions of the Core Divisome Proteins FtsBLQ and FtsN

Boes, Adrien; Olatunji, Samir; Breukink, Eefjan; Terrak, Mohammed

doi:10.1128/mbio.01912-18

Cited by 78 publications

(128 citation statements)

References 73 publications

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“…Importantly, overexpression of other late division proteins has not been associated with reductions in cell size. Simultaneous overexpression of the ftsQ , ftsL , and ftsB causes cell filamentation [48], and in our hands overexpression of ftsI also modestly increases cell length (SI Appendix, Figure S10).…”

Section: Resultsmentioning

confidence: 60%

“…S. aureus and S. pneumoniae still undergo pH-dependent changes in size but lack identifiable homologs of FtsN (SI Appendix, Figure S3) [70], 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 [48,71].…”

Section: Discussionmentioning

confidence: 99%

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Environmental pH impacts division assembly and cell size inEscherichia coli

Mueller

Westfall

Levin

2019

Preprint

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Cell size is a complex trait, derived from both genetic and environmental factors. Environmental determinants of bacterial cell size identified to date primarily target assembly of cytosolic components of the cell division machinery. Whether certain environmental cues also impact cell size through changes in the assembly or activity of extracytoplasmic division proteins remains an open question. Here, we identify extracellular pH as a modulator of cell division and a key determinant of cell size across evolutionarily distant bacterial species. In the Gram-negative model organism Escherichia coli, our data indicate environmental pH impacts the length at which cells divide by altering the ability of the terminal cell division protein FtsN to localize to the cytokinetic machinery and activate division. Acidic environments lead to enrichment of FtsN at the septum and activation of division at a reduced cell length, while alkaline pH inhibits FtsN localization and suppress division activation. Altogether, our work reveals a previously unappreciated role for pH in bacterial cell size control.

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Section: Resultsmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Environmental pH impacts division assembly and cell size inEscherichia coli

Mueller

Westfall

Levin

2019

Preprint

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“…The periplasm contains many relevant potential antibiotic targets including the cell division subcomplex of FtsB, FtsL and FtsQ (FtsBLQ) (den Blaauwen et al , ). All three components are essential and are thought to form the regulating link between the cytoplasmic and periplasmic machineries of cell division (Liu et al , ; Boes et al , ). Assaying the interactions in this complex could allow for the screening of novel antibiotics in vivo , determining the efficacy and specificity at the same time.…”

Section: Resultsmentioning

confidence: 99%

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

Meiresonne

Consoli

Mertens

et al. 2019

Molecular Microbiology

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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.

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“…, FtsW, FtsW-PBP3, MurJ13 , and FtsN12 . Stock proteins (1 mg/ml) were stored at −20 °C in the following buffers: PBP1b(25 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.7% CHAPS).…”

mentioning

confidence: 99%

“…1). The functions of these proteins are coordinated with those of interacting proteins in the elongasome and divisome complexes to ensure optimal growth and division of the bacterial cell [10][11][12][13] .…”

mentioning

confidence: 99%

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

Boes

Olatunji

Mohammadi

et al. 2020

Sci Rep

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Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid ii-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. in addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

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Regulation of the Peptidoglycan Polymerase Activity of PBP1b by Antagonist Actions of the Core Divisome Proteins FtsBLQ and FtsN

Cited by 78 publications

References 73 publications

Environmental pH impacts division assembly and cell size inEscherichia coli

Environmental pH impacts division assembly and cell size inEscherichia coli

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

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

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Regulation of the Peptidoglycan Polymerase Activity of PBP1b by Antagonist Actions of the Core Divisome Proteins FtsBLQ and FtsN

Cited by 78 publications

References 73 publications

Environmental pH impacts division assembly and cell size inEscherichia coli

Environmental pH impacts division assembly and cell size inEscherichia coli

Superfolder mTurquoise2ox optimized for the bacterial periplasm allows high efficiency in vivo FRET of cell division antibiotic targets

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

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