Allosteric activation of cell wall synthesis during bacterial growth

Shlosman, Irina; Fivenson, Elayne M.; Gilman, M.S.A.; Sisley, Tyler A.; Walker, Suzanne; Bernhardt, Thomas G.; Kruse, Andrew C.; Loparo, Joseph J.

doi:10.1101/2022.11.07.515454

Cited by 6 publications

(5 citation statements)

References 66 publications

(87 reference statements)

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“…We conclude that MreC is required for proper midcell localization and for elongasome PG synthesis by the bPBP2b:RodA synthase. This result is consistent with the previous conclusion that MreC allosterically activates the GT/TP activity of RodA:PBP2 in the PG elongasome of E. coli (16,67,105).…”

Section: Bpbp2b Localization Becomes Diffuse and Circumferential Move...supporting

confidence: 93%

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell ofStreptococcus pneumoniae

Perez,

Lamanna,

Bruce

et al. 2024

Preprint

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Ovoid-shaped bacteria, such asStreptococcus pneumoniae(pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed non-processive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

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Section: Bpbp2b Localization Becomes Diffuse and Circumferential Move...supporting

confidence: 93%

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell ofStreptococcus pneumoniae

Perez,

Lamanna,

Bruce

et al. 2024

Preprint

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“…Since our sample is active in vitro, we assume that substrate binding by Lipid II might play a key role in the interconversion of the two states. Recently, a study on RodA-PBP2 reported a similar upswinging mechanism 22 , which indicates that the concept of regulating the TP activity via restricting its access to the PG layer might be conserved between the divisome and the elongasome. However, significant differences exist between divisome and elongasome regarding the conformation before the upswinging motion and most likely also between the signals required to initiate this conformational change.…”

Section: Comparison With Roda-pbp2 Structures and Structure Predictionsmentioning

confidence: 87%

“…Lipid II used to monitor glycosyltransferase activity of the protein complex was dried using a nitrogen stream and dissolved in an equal volume of dimethyl sulfoxide (DMSO). The reaction and detection of glycan strands was adopted from previously published protocols 5,[20][21][22] . Briefly, PaFtsWIQLB and EcFtsWIQLB were mixed at a final protein concentration of 1 µM in 10µL with 1 µL 10x Reaction Buffer (500 mM Tris pH 7.5, 200 mM MnCl2), 1 µL DMSO and 1 µL Lipid II and incubated for 30 min at 25°C.…”

Section: Transglycosylase Activity Assaymentioning

confidence: 99%

Divisome core complex in bacterial cell division revealed by cryo-EM

Käshammer

Ent

Jeffery

et al. 2022

Preprint

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Cell division, or cytokinesis is a fundamental process of life and, in most bacteria, is driven by peptidoglycan synthesis at the septum. It is catalysed by the divisome, a multi-protein complex with more than 20 components that spans the cell envelope in bacteria harbouring a cell wall. Central to the divisome is the peptidoglycan-synthesising protein complex FtsWI, with the transglycosylase (TG) FtsW polymerising glycan strands from its substrate Lipid II, and the transpeptidase (TP) FtsI crosslinking peptide stems, thus forming a covalent mesh between glycan strands (Fig. 1a). Septal peptidoglycan synthesis occurs after activation of the divisome glycosyltransferase-transpeptidase pair FtsWI, in particular through an interaction with the heterotrimer FtsQBL. Here, we present the cryo-EM structure of the catalytic divisome core complex FtsWIQBL from Pseudomonas aeruginosa at 3.7 Angstrom resolution. The structure reveals the intricate details of the periplasmic interfaces within FtsWIQBL, including the positioning of FtsI by the coiled coil of FtsBL, as well as a transmembrane domain containing FtsWIBL but not FtsQ. With our structure we are able to provide molecular mechanisms of a multitude of known mutations that interfere with divisome activation and regulation. Finally, we reveal a large conformational switch between presumably inactive and active states of the FtsWI core enzymes. Our work is foundational for further structural, biochemical and genetic studies elucidating the molecular mechanisms of bacterial cell division. Since the divisome peptidoglycan synthase is essential for cell division in most bacteria, and is absent in eukaryotic cells entirely, it is a key target of important antibiotics and antibiotic development, and we suggest that our structure will help to accelerate these efforts.

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“…One intriguing question that remains unanswered is how E FtsN's binding to the AWI FtsL-FtsI complex is transmitted to the active site of FtsW located in the periplasm proximal to the membrane. Recently, Shlosman et al 34 proposed that an extended open conformation of PBP2 (promoted by MreC) activates RodA. Jan Lowe's group used cryo-EM and found that PaFtsI complexed with PaFtsQLB exists in a partially open form with its TP domain heading away from PaFtsW but short of reaching the PG layer 35 .…”

Section: Discussionmentioning

confidence: 99%

The essential domain of FtsN triggers cell division by promoting interaction between FtsL and FtsI

Park

Johnson

Pichoff

et al. 2023

Preprint

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Cell division in bacteria requires the activation of FtsWI at the division site to synthesize septal peptidoglycan. In E. coli FtsN activates FtsWI and a previous model posited that the essential domain of FtsN (EFtsN) acts on FtsQLB causing conformational changes so that a domain of FtsL, called AWI (AWIFtsL), contacts FtsI resulting in activation of FtsW. In this study we use genetic analysis along with an AlphaFold2 model to test this activation model. Based on our findings we propose an updated model wherein theAWIFtsL and FtsI interaction is stabilized byEFtsN to activate FtsW and that this interaction is enhanced by theCytoFtsN-FtsA interaction. Thus, FtsN acts as both a sensor for divisome assembly and an activator. In addition, we elucidate the role played by two critical FtsL residues in FtsW activation.

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Allosteric activation of cell wall synthesis during bacterial growth

Cited by 6 publications

References 66 publications

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell ofStreptococcus pneumoniae

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell ofStreptococcus pneumoniae

Divisome core complex in bacterial cell division revealed by cryo-EM

The essential domain of FtsN triggers cell division by promoting interaction between FtsL and FtsI

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