Essential role for FtsL in activation of septal PG synthesis

Park, Kyung‐Tae; Du, Shishen; Lutkenhaus, Joe

doi:10.1101/2020.09.01.275982

Cited by 5 publications

(17 citation statements)

References 36 publications

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Section: Isolation Of Ftsw E289g and Ftsi K221i As Hyperactive Divisimentioning

confidence: 99%

“…In a recent study, we isolated dominant negative FtsL mutants that block cell division (21). These mutants support assembly of the divisome but are unable to divide because they are unable to interact with FtsI in response to FtsN (21). Such mutants are suppressed by the hyperactive ftsW M269I mutation which no longer requires the activation signal from FtsN (21).…”

Section: Isolation Of Ftsw E289g and Ftsi K221i As Hyperactive Divisimentioning

confidence: 99%

“…These mutants support assembly of the divisome but are unable to divide because they are unable to interact with FtsI in response to FtsN (21). Such mutants are suppressed by the hyperactive ftsW M269I mutation which no longer requires the activation signal from FtsN (21). To isolate additional active ftsW and ftsI mutations, we screened FtsW and FtsI mutant libraries for suppressors of one of these FtsL mutants (FtsL E87K ).…”

Section: Isolation Of Ftsw E289g and Ftsi K221i As Hyperactive Divisimentioning

confidence: 99%

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Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

Liu

Gong

et al. 2021

Preprint

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SEDS family peptidoglycan (PG) glycosyltransferases RodA and FtsW require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try and understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations and others affecting the activities of FtsWI on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.Author summaryBacterial cell division requires the synthesis of septal peptidoglycan by the widely conserved SEDS-bPBP protein complex FtsWI, but how the complex is activated during cell division is still poorly understood. Previous studies suggest that FtsN initiates a signaling cascade in the periplasm to activate FtsW. Here we isolated and characterized activated FtsW and FtsI mutants and confirmed that the signaling cascade for FtsW activation goes from FtsN to FtsQLB to FtsI and then to FtsW. The residues corresponding to mutations affecting FtsWI activation are clustered to a small region of the interaction interface between the pedestal domain of FtsI and the extracellular loop 4 of FtsW, suggesting that this interaction mediates activation of FtsW. This is strikingly similar to the proposed activation mechanism for the RodA-PBP2 complex, another SEDS-bPBP complex required for cell elongation. Thus, the two homologous SEDS-bPBP complexes are activated similarly by completely unrelated activators that modulate the interaction interface between the SEDS proteins and the bPBPs.

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Section: Isolation Of Ftsw E289g and Ftsi K221i As Hyperactive Divisimentioning

confidence: 99%

Section: Isolation Of Ftsw E289g and Ftsi K221i As Hyperactive Divisimentioning

confidence: 99%

Section: Isolation Of Ftsw E289g and Ftsi K221i As Hyperactive Divisimentioning

confidence: 99%

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Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

Liu

Gong

et al. 2021

Preprint

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“…We predict that other cell division proteins, such as ZipA or FtsEX may contribute to regulating the FtsA polymerization state [17,[43][44][45]. Once recruited by FtsA, FtsN would then activate FtsQBL and FtsWI for cell wall synthesis [18,45].…”

Section: Several Models Have Been Proposed To Resolve the Precise Indmentioning

confidence: 99%

“…FtsA and FtsZ act as the first of at least 12 proteins that localize to midcell during the early steps of division in E. coli [10][11][12][13]. FtsA interacts with many other cell division proteins directly including ZipA, FtsI, FtsEX, and, most notably, FtsN [14][15][16][17].The interaction between FtsA and FtsN has been suggested to serve as a trigger to engage FtsQBL, which activates FtsWI complex for peptidoglycan (PG) synthesis [18]. expressing mutant FtsA proteins defective for self-interaction bypass the requirement for ZipA, as well as FtsEX, but are dependent on FtsN, suggesting that these proteins may help promote monomerization of FtsA in vivo [17,[34][35][36][37].…”

Section: Introductionmentioning

confidence: 99%

Coregulated assembly of actin-like FtsA polymers with FtsZ during Z-ring formation and division inEscherichia coli

Morrison

Conti

Camberg

2021

Preprint

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In Escherichia coli, the actin homolog FtsA localizes the cell division machinery, beginning with the Z-ring, to the cytoplasmic membrane through direct interaction with FtsZ. FtsZ polymers are first to assemble at the Z-ring at midcell, where they direct constriction and septation. While FtsZ polymerization is critical for establishing a functional Z-ring that leads to constriction, the assembly state of FtsA and the role of FtsA ATP utilization during division in E. coli remain unclear. Here, we show that ATP hydrolysis, FtsZ interaction, and phospholipid vesicle remodeling by FtsA are impaired by a substitution mutation at the predicted active site for hydrolysis. This mutation, Glu 14 to Arg, also impairs Z-ring assembly and division in vivo. To further investigate the role of phospholipid engagement and ATP utilization in regulating FtsA function, we characterized a truncated E. coli FtsA variant, FtsA(ΔMTS), which lacks the region at the C-terminus important for engaging the membrane and is defective for ATP hydrolysis. We show that E. coli FtsA(ΔMTS) forms ATP-dependent actin-like filaments and assembly is antagonized by FtsZ. Polymerization of FtsZ with GTP, or a non-hydrolyzable analog, blocks inhibition of ATP-dependent FtsA assembly, and instead favors coassembly of stable FtsA/FtsZ polymers. In the cell, FtsA/FtsZ coassembly is favored at midcell, where FtsZ polymerizes, and inhibited at regions where FtsZ polymers are destabilized by regulators, such as MinC at the poles or SlmA at the nucleoid. We show that MinC prevents recruitment of FtsZ, via FtsA, to phospholipids, suggesting that local interactions of MinC with FtsZ block membrane tethering and uncouple the Z-ring from its major membrane contact. During Z-ring formation, the coassembly of FtsZ polymers with FtsA is coordinated and is a critical early step in division. This step also serves as a checkpoint by responding to the suite of FtsZ assembly regulators in the cell that modulate Z-ring position and dynamics prior to initiating cell wall synthesis.

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A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW

Yang¹,

McQuillen²,

Lyu³

et al. 2021

Nat Microbiol

156

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Synthesis of new septal peptidoglycan (sPG) is crucial for bacterial cell division. FtsW, an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a peptidoglycan glycosyltransferase (PGTase). Despite its importance, the septal PGTase activity of FtsW has not been demonstrated in vivo . How its activity is spatiotemporally regulated in vivo has also remained elusive. Here we confirmed FtsW as an essential septum-specific PGTase in vivo using an N -acetylmuramic acid analog incorporation assay. Next, using single-molecule tracking coupled with genetic manipulations, we identified two populations of processively moving FtsW molecules: a fast-moving population correlated with the treadmilling dynamics of the essential cytoskeletal FtsZ protein and a slow-moving population dependent on active sPG synthesis. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving population. Our results suggest a two-track model, in which inactive sPG synthases follow the “Z-track” to be distributed along the septum; FtsN promotes their release from the “Z-track” to become active in sPG synthesis on the slow “sPG-track”. This model provides a mechanistic framework for the spatiotemporal coordination of sPG synthesis in bacterial cell division.

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Essential role for FtsL in activation of septal PG synthesis

Cited by 5 publications

References 36 publications

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

Coregulated assembly of actin-like FtsA polymers with FtsZ during Z-ring formation and division inEscherichia coli

A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW

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