The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity

Glas, Marjolein; Saparoea, H. van den Berg van Bart; McLaughlin, Stephen H.; Roseboom, Winfried; Liu, Fan; Koningstein, Gregory M.; Fish, Alexander; Blaauwen, Tanneke den; Heck, Albert J. R.; Jong, Luitzen de; Bitter, Wilbert; Esch, Iwan J. P. de; Luirink, Joen

doi:10.1074/jbc.m115.654756

Cited by 37 publications

(58 citation statements)

References 48 publications

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“…5). These interactions are in agreement with the interactions reported for the periplasmic domains of these proteins (16). In addition, as has been shown in two-hybrid assays for FtsQ (28,29), our results show that FtsQ from C. crescentus interacts with itself; however, we also detected self-interaction of FtsB.…”

Section: Resultssupporting

confidence: 93%

“…After a time delay, the FtsQ, FtsL, FtsB, FtsW, FtsI, and FtsN proteins are rapidly recruited (14). These proteins probably do not accumulate at the division site simultaneously, since FtsQLB forms a complex that is required for divisome stabilization and the recruitment of other division proteins (2,(15)(16)(17)(18). The FtsQLB complex has also been proposed to activate the synthesis of septal peptidoglycan in response to the state of the divisome (19,20).…”

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A New Essential Cell Division Protein in Caulobacter crescentus

et al. 2017

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Bacterial cell division is a complex process that relies on a multiprotein complex composed of a core of widely conserved and generally essential proteins and on accessory proteins that vary in number and identity in different bacteria. The assembly of this complex and, particularly, the initiation of constriction are regulated processes that have come under intensive study. In this work, we characterize the function of DipI, a protein conserved in Alphaproteobacteria and Betaproteobacteria that is essential in Caulobacter crescentus. Our results show that DipI is a periplasmic protein that is recruited late to the division site and that it is required for the initiation of constriction. The recruitment of the conserved cell division proteins is not affected by the absence of DipI, but localization of DipI to the division site occurs only after a mature divisome has formed. Yeast two-hybrid analysis showed that DipI strongly interacts with the FtsQLB complex, which has been recently implicated in regulating constriction initiation. A possible role of DipI in this process is discussed.IMPORTANCE Bacterial cell division is a complex process for which most bacterial cells assemble a multiprotein complex that consists of conserved proteins and of accessory proteins that differ among bacterial groups. In this work, we describe a new cell division protein (DipI) present only in a group of bacteria but essential in Caulobacter crescentus. Cells devoid of DipI cannot constrict. Although a mature divisome is required for DipI recruitment, DipI is not needed for recruiting other division proteins. These results, together with the interaction of DipI with a protein complex that has been suggested to regulate cell wall synthesis during division, suggest that DipI may be part of the regulatory mechanism that controls constriction initiation.KEYWORDS Caulobacter crescentus, SH3 domain, bacterial cell division, constriction initiation, divisome B acterial cell division is a complex process involving the action of a multiprotein complex known as the divisome (1, 2). In Escherichia coli, the divisome is composed of approximately 30 proteins that assemble into a complex spanning from the cytoplasm to the outer membrane (OM). Of these proteins, only 12 are essential in E. coli and 11 are widely conserved in other bacteria, suggesting that they constitute the core of the divisome (3, 4). The divisome complex starts assembling when a ring formed by the FtsZ protein is stabilized at the site where division will occur (5, 6). FtsZ is a tubulin-related cytoplasmic protein that is brought near the cytoplasmic membrane through its interactions with the membrane-associated protein FtsA and, in E. coli, the transmembrane protein ZipA (7,8). The FtsZ ring generates a force sufficient to deform lipid membranes and probably drives the constriction of the inner membrane (9, 10). After establishment of the FtsZ ring, the FtsEX complex is recruited through the interaction of FtsE with FtsZ (11). The divisome then expands to the peripla...

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Section: Resultssupporting

confidence: 93%

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A New Essential Cell Division Protein in Caulobacter crescentus

et al. 2017

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“…Exactly how and in what stoichiometry the complex forms and functions is still a matter of debate (Karimova et al, 2005;Villanelo et al, 2011;Robichon et al, 2011;Glas et al, 2015;Condon et al, 2018). Interactions between FtsB and FtsL seem to rely on their coiled-coil transmembrane helices (Robichon et al, 2011;Condon et al, 2018) while the interaction of FtsQ and FtsB requires their periplasmic soluble domains and forms independently of FtsL (van den Berg van Saparoea et al, 2013;Glas et al, 2015;Kureisaite-Ciziene et al, 2018;Choi et al, 2018;Boes et al, 2019). Our findings confirm the former interaction in vivo and provide a tool for the further dissection of FtsBLQ interactions.…”

Section: Discussionsupporting

confidence: 69%

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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“…FtsB and FtsL are by themselves unstable in both E. coli and B. subtilis (FtsB is called DivIC in the latter species), but together they associate into a stable complex 83–85 associated with FtsQ (DivIB in B. subtilis and other Gram positives ). Interestingly, the extracellular domain of S. aureus DivIB can bind peptidoglycan 86 despite lacking a binding domain typical of other divisome proteins such as FtsN (see next section).…”

Section: Divisome Maturation and Cytokinesismentioning

confidence: 99%

Splitsville: structural and functional insights into the dynamic bacterial Z ring

Haeusser

Margolin²

2016

Nat Rev Microbiol

299

283

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Preface Bacteria must divide in order to increase in number and colonize their niche. Binary fission is the most widespread means of bacterial cell division, but even this relatively simple mechanism displays many variations on a theme. In most bacteria, the tubulin homolog FtsZ assembles into a ring structure (Z ring) at the site of cytokinesis and recruits additional proteins to form a large protein machine (divisome) that spans the membrane. Here we discuss current insights into the regulation of Z ring assembly and how the divisome drives membrane invagination and septal cell wall growth while still flexibly responding to various cellular inputs.

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The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity

Cited by 37 publications

References 48 publications

A New Essential Cell Division Protein in Caulobacter crescentus

A New Essential Cell Division Protein in Caulobacter crescentus

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

Splitsville: structural and functional insights into the dynamic bacterial Z ring

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The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity

Cited by 37 publications

References 48 publications

A New Essential Cell Division Protein in Caulobacter crescentus

A New Essential Cell Division Protein in Caulobacter crescentus

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

Splitsville: structural and functional insights into the dynamic bacterial Z ring

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Product

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