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William Margolin - One of the best experts on this subject based on the ideXlab platform.
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peptide linkers within the essential ftsz membrane tethers zipa and FtsA are nonessential for cell division
Journal of Bacteriology, 2020Co-Authors: Kara M Schoenemann, Daniel E Vega, William MargolinAbstract:ABSTRACT Bacteria such as Escherichia coli divide by organizing filaments of FtsZ, a tubulin homolog that assembles into dynamic treadmilling membrane-associated protein filaments at the cell midpoint. FtsA and ZipA proteins are required to tether these filaments to the inner face of the cytoplasmic membrane, and loss of either tether is lethal. ZipA from E. coli and other closely related species harbors a long linker region that connects the essential N-terminal transmembrane domain to the C-terminal globular FtsZ-binding domain, and part of this linker includes a P/Q-rich peptide that is predicted to be intrinsically disordered. We found unexpectedly that several large deletions of the ZipA linker region, including the entire P/Q rich peptide, had no effect on cell division under normal conditions. However, we found that the loss of the P/Q region made cells more resistant to excess levels of FtsA and more sensitive to conditions that displaced FtsA from FtsZ. FtsA also harbors a short ∼20-residue peptide linker that connects the main globular domain with the C-terminal amphipathic helix that is important for membrane binding. In analogy with ZipA, deletion of 11 of the central residues in the FtsA linker had little effect on FtsA function in cell division. IMPORTANCEEscherichia coli cells divide using a cytokinetic ring composed of polymers of the tubulin-like FtsZ. To function properly, these polymers must attach to the inner surface of the cytoplasmic membrane via two essential membrane-associated tethers, FtsA and ZipA. Both FtsA and ZipA contain peptide linkers that connect their membrane-binding domains with their FtsZ-binding domains. Although they are presumed to be crucial for cell division activity, the importance of these linkers has not yet been rigorously tested. Here, we show that large segments of these linkers can be removed with few consequences for cell division, although several subtle defects were uncovered. Our results suggest that ZipA, in particular, can function in cell division without an extended linker.
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direct interaction between the two z ring membrane anchors FtsA and zipa
Journal of Bacteriology, 2019Co-Authors: Daniel E Vega, William MargolinAbstract:The initiation of Escherichia coli cell division requires three proteins, FtsZ, FtsA, and ZipA, which assemble in a dynamic ring-like structure at midcell. Along with the transmembrane protein ZipA, the actin-like FtsA helps to tether treadmilling polymers of tubulin-like FtsZ to the membrane. In addition to forming homo-oligomers, FtsA and ZipA interact directly with the C-terminal conserved domain of FtsZ. Gain-of-function mutants of FtsA are deficient in forming oligomers and can bypass the need for ZipA, suggesting that ZipA may normally function to disrupt FtsA oligomers, although no direct interaction between FtsA and ZipA has been reported. Here, we use in vivo cross-linking to show that FtsA and ZipA indeed interact directly. We identify the exposed surface of FtsA helix 7, which also participates in binding to ATP through its internal surface, as a key interface needed for the interaction with ZipA. This interaction suggests that FtsZ's membrane tethers may regulate each other's activities.IMPORTANCE To divide, most bacteria first construct a protein machine at the plane of division and then recruit the machinery that will synthesize the division septum. In Escherichia coli, this first stage involves the assembly of FtsZ polymers at midcell, which directly bind to membrane-associated proteins FtsA and ZipA to form a discontinuous ring structure. Although FtsZ directly binds both FtsA and ZipA, it is unclear why FtsZ requires two separate membrane tethers. Here, we uncover a new direct interaction between the tethers, which involves a helix within FtsA that is adjacent to its ATP binding pocket. Our findings imply that in addition to their known roles as FtsZ membrane anchors, FtsA and ZipA may regulate each other's structure and function.
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gain of function variants of FtsA form diverse oligomeric structures on lipids and enhance ftsz protofilament bundling
Molecular Microbiology, 2018Co-Authors: Kara M Schoenemann, Marcin Krupka, Veronica W Rowlett, Steven L Distelhorst, William MargolinAbstract:Escherichia coli requires FtsZ, FtsA and ZipA proteins for early stages of cell division, the latter two tethering FtsZ polymers to the cytoplasmic membrane. Hypermorphic mutants of FtsA such as FtsA* (R286W) map to the FtsA self-interaction interface and can bypass the need for ZipA. Purified FtsA forms closed minirings on lipid monolayers that antagonize bundling of FtsZ protofilaments, whereas FtsA* forms smaller oligomeric arcs that enable bundling. Here, we examined three additional FtsA*-like mutant proteins for their ability to form oligomers on lipid monolayers and bundle FtsZ. Surprisingly, all three formed distinct structures ranging from mostly arcs (T249M), a mixture of minirings, arcs and straight filaments (Y139D) or short straight double filaments (G50E). All three could form filament sheets at higher concentrations with added ATP. Despite forming these diverse structures, all three mutant proteins acted like FtsA* to enable FtsZ protofilament bundling on lipid monolayers. Synthesis of the FtsA*-like proteins in vivo suppressed the toxic effects of a bundling-defective FtsZ, exacerbated effects of a hyper-bundled FtsZ, and rescued some thermosensitive cell division alleles. Together, the data suggest that conversion of FtsA minirings into any type of non-miniring oligomer can promote progression of cytokinesis through FtsZ bundling and other mechanisms.
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escherichia coli FtsA forms lipid bound minirings that antagonize lateral interactions between ftsz protofilaments
Nature Communications, 2017Co-Authors: Marcin Krupka, Kara M Schoenemann, Veronica W Rowlett, Dustin R Morado, Heidi Vitrac, Jun Liu, William MargolinAbstract:The actin-like protein FtsA and the tubulin-like protein FtsZ play crucial roles during cell division in most bacteria. Here, the authors show that FtsA forms minirings on lipid monolayers, and present evidence supporting that its oligomeric state modulates the bundling o…
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a mutation in escherichia coli ftsz bypasses the requirement for the essential division gene zipa and confers resistance to ftsz assembly inhibitors by stabilizing protofilament bundling
Molecular Microbiology, 2015Co-Authors: Daniel P Haeusser, Veronica W Rowlett, William MargolinAbstract:Summary The earliest step in Escherichia coli cell division consists of the assembly of FtsZ protein into a proto-ring structure, tethered to the cytoplasmic membrane by FtsA and ZipA. The proto-ring then recruits additional cell division proteins to form the divisome. Previously we described an ftsZ allele, ftsZL169R, which maps to the side of the FtsZ subunit and confers resistance to FtsZ assembly inhibitory factors including Kil of bacteriophage λ. Here we further characterize this allele and its mechanism of resistance. We found that FtsZL169R permits the bypass of the normally essential ZipA, a property previously observed for FtsA gain-of-function mutants such as FtsA* or increased levels of the FtsA-interacting protein FtsN. Similar to FtsA*, FtsZL169R also can partially suppress thermosensitive mutants of ftsQ or ftsK, which encode additional divisome proteins, and confers strong resistance to excess levels of FtsA, which normally inhibit FtsZ ring function. Additional genetic and biochemical assays provide further evidence that FtsZL169R enhances FtsZ protofilament bundling, thereby conferring resistance to assembly inhibitors and bypassing the normal requirement for ZipA. This work highlights the importance of FtsZ protofilament bundling during cell division and its likely role in regulating additional divisome activities.
Joe Lutkenhaus - One of the best experts on this subject based on the ideXlab platform.
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FtsA acts through ftsw to promote cell wall synthesis during cell division in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2021Co-Authors: Kyungtae Park, Sebastien Pichoff, Joe LutkenhausAbstract:In Escherichia coli, FtsQLB is required to recruit the essential septal peptidoglycan (sPG) synthase FtsWI to FtsA, which tethers FtsZ filaments to the membrane. The arrival of FtsN switches FtsQLB in the periplasm and FtsA in the cytoplasm from a recruitment role to active forms that synergize to activate FtsWI. Genetic evidence indicates that the active form of FtsQLB has an altered conformation with an exposed domain of FtsL that acts on FtsI to activate FtsW. However, how FtsA contributes to the activation of FtsW is not clear, as it could promote the conformational change in FtsQLB or act directly on FtsW. Here, we show that the overexpression of an activated FtsA (FtsA*) bypasses FtsQ, indicating it can compensate for FtsQ's recruitment function. Consistent with this, FtsA* also rescued FtsL and FtsB mutants deficient in FtsW recruitment. FtsA* also rescued an FtsL mutant unable to deliver the periplasmic signal from FtsN, consistent with FtsA* acting on FtsW. In support of this, an FtsW mutant was isolated that was rescued by an activated FtsQLB but not by FtsA*, indicating it was specifically defective in activation by FtsA. Our results suggest that in response to FtsN, the active form of FtsA acts on FtsW in the cytoplasm and synergizes with the active form of FtsQLB acting on FtsI in the periplasm to activate FtsWI to carry out sPG synthesis.
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how ftsex localizes to the z ring and interacts with FtsA to regulate cell division
Molecular Microbiology, 2019Co-Authors: Wyatt Henke, Sebastien Pichoff, Joe LutkenhausAbstract:In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N-terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.
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disruption of divisome assembly rescued by ftsn FtsA interaction in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Sebastien Pichoff, Joe LutkenhausAbstract:Cell division requires the assembly of a protein complex called the divisome. The divisome assembles in a hierarchical manner, with FtsA functioning as a hub to connect the Z-ring with the rest of the divisome and FtsN arriving last to activate the machine to synthesize peptidoglycan. FtsEX arrives as the Z-ring forms and acts on FtsA to initiate recruitment of the other divisome components. In the absence of FtsEX, recruitment is blocked; however, a multitude of conditions allow FtsEX to be bypassed. Here, we find that all such FtsEX bypass conditions, as well as the bypass of FtsK, depend upon the interaction of FtsN with FtsA, which promotes the back-recruitment of the late components of the divisome. Furthermore, our results suggest that these bypass conditions enhance the weak interaction of FtsN with FtsA and its periplasmic partners so that the divisome proteins are brought to the Z-ring when the normal hierarchical pathway is disrupted.
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ftsex acts on FtsA to regulate divisome assembly and activity
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Sebastien Pichoff, Joe LutkenhausAbstract:Bacterial cell division is driven by the divisome, a ring-shaped protein complex organized by the bacterial tubulin homolog FtsZ. Although most of the division proteins in Escherichia coli have been identified, how they assemble into the divisome and synthesize the septum remains poorly understood. Recent studies suggest that the bacterial actin homolog FtsA plays a critical role in divisome assembly and acts synergistically with the FtsQLB complex to regulate the activity of the divisome. FtsEX, an ATP-binding cassette transporter-like complex, is also necessary for divisome assembly and inhibits division when its ATPase activity is inactivated. However, its role in division is not clear. Here, we find that FtsEX acts on FtsA to regulate both divisome assembly and activity. FtsX interacts with FtsA and this interaction is required for divisome assembly and inhibition of divisome function by ATPase mutants of FtsEX. Our results suggest that FtsEX antagonizes FtsA polymerization to promote divisome assembly and the ATPase mutants of FtsEX block divisome activity by locking FtsA in the inactive form or preventing FtsA from communicating with other divisome proteins. Because FtsEX is known to govern cell wall hydrolysis at the septum, our findings indicate that FtsEX acts on FtsA to promote divisome assembly and to coordinate cell wall synthesis and hydrolysis at the septum. Furthermore, our study provides evidence that FtsA mutants impaired for self-interaction are favored for division, and FtsW plays a critical role in divisome activation in addition to the FtsQLB complex.
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the bypass of zipa by overexpression of ftsn requires a previously unknown conserved ftsn motif essential for FtsA ftsn interaction supporting a model in which FtsA monomers recruit late cell division proteins to the z ring
Molecular Microbiology, 2015Co-Authors: Sebastien Pichoff, Joe LutkenhausAbstract:Assembly of the divisome in Escherichia coli occurs in two temporally distinct steps. First, FtsZ filaments attached to the membrane through interaction with FtsA and ZipA coalesce into a Z ring at midcell. Then, additional proteins are recruited to the Z ring in a hierarchical manner to form a complete divisome, activated by the arrival of FtsN. Recently, we proposed that the interaction of FtsA with itself competes with its ability to recruit downstream division proteins (both require the IC domain of FtsA) and ZipA's essential function is to promote the formation of FtsA monomers. Here, we tested whether overexpression of a downstream division protein could make ZipA dispensable, presumably by shifting the FtsA equilibrium to monomers. Only overexpression of FtsN bypassed ZipA and a conserved motif in the cytoplasmic domain of FtsN was required for both the bypass and interaction with FtsA. Also, this cytoplasmic motif had to be linked to the periplasmic E domain of FtsN to bypass ZipA, indicating that linkage of FtsA to periplasmic components of the divisome through FtsN was essential under these conditions. These results are used to further elaborate our model for the role of FtsA in recruiting downstream division proteins.
Ane Urtiaga - One of the best experts on this subject based on the ideXlab platform.
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Electrochemical oxidation of 6:2 fluorotelomer sulfonic acid (6:2 FtsA) on BDD: electrode characterization and mechanistic investigation
Journal of Applied Electrochemistry, 2018Co-Authors: Jordi Carrillo-abad, Valentín Pérez-herranz, Ane UrtiagaAbstract:6:2 Fluorotelomer sulfonic acid (6:2 FtsA) is used as surfactant and foam stabilizer in the formulation of air firefighting foams (AFFFs). 6:2 FtsA is produced as an alternative to persistent and bioaccumulative long-chain perfluoroalkyl compounds. This study investigates the electrochemical degradation of 6:2 FtsA on a boron-doped diamond (BDD) anode. First, the BDD anode was characterized by cyclic voltammetry, revealing that the direct oxidation of 6:2 FtsA occurred at an anodic potential of 2.72 V versus Ag/AgCl (saturated KCl) electrode. Increasing the scan rate resulted in an increased current intensity of the direct oxidation peak, and this relationship was analyzed using the Randles–Sevcik equation to calculate the diffusion coefficient of 6:2 FtsA in aqueous media ( D = 4.16 × 10^−6 cm^2 s^−1 at room temperature). This value is in close agreement to the predicted value obtained by the Wilke–Chang correlation. In electrolysis experiments under potentiostatic control, increasing the anode potential over 2.72 V greatly enhanced the 6:2 FtsA removal, and the simultaneous formation of short-chain perfluorocarboxylic acids (perfluorohexanoic acid, perfluorpentanoic acid and perfluorobutanoic acid) and fluoride release were observed. Based on these observations, the 6:2 FtsA degradation pathway was predicted to start by the attack of hydroxyl radicals to the non-fluorinated carbons to form a perfluorocarboxylate, followed by a single electron transfer to the anode to yield a reactive radical C_6F_13COO‧. The latter species decarboxylated and finally combined with hydroxyl radicals to allow defluorination to form shorter-chain perfluorocarboxylic acids.Graphical Abstract
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bdd anodic treatment of 6 2 fluorotelomer sulfonate 6 2 FtsA evaluation of operating variables and by product formation
Chemosphere, 2018Co-Authors: Ane Urtiaga, Alvaro Soriano, J CarrilloabadAbstract:The concerns about the undesired impacts on human health and the environment of long chain perfluorinated alkyl substances (PFASs) have driven industrial initiatives to replace PFASs by shorter chain fluorinated homologues. 6:2 fluorotelomer sulfonic acid (6:2 FtsA) is applied as alternative to PFOS in metal plating and fluoropolymer manufacture. This study reports the electrochemical treatment of aqueous 6:2 FtsA solutions on microcrystalline BDD anodes. Bench scale batch experiments were performed, focused on assessing the effect of the electrolyte and the applied current density (5-600 A m-2) on the removal of 6:2 FtsA, the reduction of total organic carbon (TOC) and the fluoride release. Results showed that at the low range of applied current density (J = 50 A m-2), using NaCl, Na2SO4 and NaClO4, the electrolyte exerted a minimal effect on removal rates. The formation of toxic inorganic chlorine species such as ClO4- was not observed. When using Na2SO4 electrolyte, increasing the applied current density to 350-600 A m-2 promoted a notable enhancement of the 6:2 FtsA removal and defluorination rates, pointing to the positive contribution of electrogenerated secondary oxidants to the overall removal rate. 6:2 FtsA was transformed into shorter-chain PFCAs, and eventually into CO2 and fluoride, as TOC reduction was >90%. Finally, it was demonstrated that diffusion in the liquid phase was controlling the overall kinetic rate, although with moderate improvements due to secondary oxidants at very high current densities.
Sebastien Pichoff - One of the best experts on this subject based on the ideXlab platform.
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FtsA acts through ftsw to promote cell wall synthesis during cell division in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2021Co-Authors: Kyungtae Park, Sebastien Pichoff, Joe LutkenhausAbstract:In Escherichia coli, FtsQLB is required to recruit the essential septal peptidoglycan (sPG) synthase FtsWI to FtsA, which tethers FtsZ filaments to the membrane. The arrival of FtsN switches FtsQLB in the periplasm and FtsA in the cytoplasm from a recruitment role to active forms that synergize to activate FtsWI. Genetic evidence indicates that the active form of FtsQLB has an altered conformation with an exposed domain of FtsL that acts on FtsI to activate FtsW. However, how FtsA contributes to the activation of FtsW is not clear, as it could promote the conformational change in FtsQLB or act directly on FtsW. Here, we show that the overexpression of an activated FtsA (FtsA*) bypasses FtsQ, indicating it can compensate for FtsQ's recruitment function. Consistent with this, FtsA* also rescued FtsL and FtsB mutants deficient in FtsW recruitment. FtsA* also rescued an FtsL mutant unable to deliver the periplasmic signal from FtsN, consistent with FtsA* acting on FtsW. In support of this, an FtsW mutant was isolated that was rescued by an activated FtsQLB but not by FtsA*, indicating it was specifically defective in activation by FtsA. Our results suggest that in response to FtsN, the active form of FtsA acts on FtsW in the cytoplasm and synergizes with the active form of FtsQLB acting on FtsI in the periplasm to activate FtsWI to carry out sPG synthesis.
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how ftsex localizes to the z ring and interacts with FtsA to regulate cell division
Molecular Microbiology, 2019Co-Authors: Wyatt Henke, Sebastien Pichoff, Joe LutkenhausAbstract:In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N-terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.
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disruption of divisome assembly rescued by ftsn FtsA interaction in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Sebastien Pichoff, Joe LutkenhausAbstract:Cell division requires the assembly of a protein complex called the divisome. The divisome assembles in a hierarchical manner, with FtsA functioning as a hub to connect the Z-ring with the rest of the divisome and FtsN arriving last to activate the machine to synthesize peptidoglycan. FtsEX arrives as the Z-ring forms and acts on FtsA to initiate recruitment of the other divisome components. In the absence of FtsEX, recruitment is blocked; however, a multitude of conditions allow FtsEX to be bypassed. Here, we find that all such FtsEX bypass conditions, as well as the bypass of FtsK, depend upon the interaction of FtsN with FtsA, which promotes the back-recruitment of the late components of the divisome. Furthermore, our results suggest that these bypass conditions enhance the weak interaction of FtsN with FtsA and its periplasmic partners so that the divisome proteins are brought to the Z-ring when the normal hierarchical pathway is disrupted.
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ftsex acts on FtsA to regulate divisome assembly and activity
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Sebastien Pichoff, Joe LutkenhausAbstract:Bacterial cell division is driven by the divisome, a ring-shaped protein complex organized by the bacterial tubulin homolog FtsZ. Although most of the division proteins in Escherichia coli have been identified, how they assemble into the divisome and synthesize the septum remains poorly understood. Recent studies suggest that the bacterial actin homolog FtsA plays a critical role in divisome assembly and acts synergistically with the FtsQLB complex to regulate the activity of the divisome. FtsEX, an ATP-binding cassette transporter-like complex, is also necessary for divisome assembly and inhibits division when its ATPase activity is inactivated. However, its role in division is not clear. Here, we find that FtsEX acts on FtsA to regulate both divisome assembly and activity. FtsX interacts with FtsA and this interaction is required for divisome assembly and inhibition of divisome function by ATPase mutants of FtsEX. Our results suggest that FtsEX antagonizes FtsA polymerization to promote divisome assembly and the ATPase mutants of FtsEX block divisome activity by locking FtsA in the inactive form or preventing FtsA from communicating with other divisome proteins. Because FtsEX is known to govern cell wall hydrolysis at the septum, our findings indicate that FtsEX acts on FtsA to promote divisome assembly and to coordinate cell wall synthesis and hydrolysis at the septum. Furthermore, our study provides evidence that FtsA mutants impaired for self-interaction are favored for division, and FtsW plays a critical role in divisome activation in addition to the FtsQLB complex.
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the bypass of zipa by overexpression of ftsn requires a previously unknown conserved ftsn motif essential for FtsA ftsn interaction supporting a model in which FtsA monomers recruit late cell division proteins to the z ring
Molecular Microbiology, 2015Co-Authors: Sebastien Pichoff, Joe LutkenhausAbstract:Assembly of the divisome in Escherichia coli occurs in two temporally distinct steps. First, FtsZ filaments attached to the membrane through interaction with FtsA and ZipA coalesce into a Z ring at midcell. Then, additional proteins are recruited to the Z ring in a hierarchical manner to form a complete divisome, activated by the arrival of FtsN. Recently, we proposed that the interaction of FtsA with itself competes with its ability to recruit downstream division proteins (both require the IC domain of FtsA) and ZipA's essential function is to promote the formation of FtsA monomers. Here, we tested whether overexpression of a downstream division protein could make ZipA dispensable, presumably by shifting the FtsA equilibrium to monomers. Only overexpression of FtsN bypassed ZipA and a conserved motif in the cytoplasmic domain of FtsN was required for both the bypass and interaction with FtsA. Also, this cytoplasmic motif had to be linked to the periplasmic E domain of FtsN to bypass ZipA, indicating that linkage of FtsA to periplasmic components of the divisome through FtsN was essential under these conditions. These results are used to further elaborate our model for the role of FtsA in recruiting downstream division proteins.
M A Vicente - One of the best experts on this subject based on the ideXlab platform.
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the hypermorph FtsA protein has an in vivo role in relieving the escherichia coli proto ring block caused by excess zapc
PLOS ONE, 2017Co-Authors: Cristina Ortiz, Mercedes Casanova, Pilar Palacios, M A VicenteAbstract:Assembly of the proto-ring, formed by the essential FtsZ, FtsA and ZipA proteins, and its progression into a divisome, are essential events for Escherichia coli division. ZapC is a cytoplasmic protein that belongs to a group of non-essential components that assist FtsZ during proto-ring assembly. Any overproduction of these proteins leads to faulty FtsZ-rings, resulting in a cell division block. We show that ZapC overproduction can be counteracted by an excess of the ZipA-independent hypermorph FtsA* mutant, but not by similar amounts of wild type FtsA+. An excess of FtsA+ allowed regular spacing of the ZapC-blocked FtsZ-rings, but failed to promote recruitment of the late-assembling proteins FtsQ, FtsK and FtsN and therefore, to activate constriction. In contrast, overproduction of FtsA*, besides allowing correct FtsZ-ring localization at midcell, restored the ability of FtsQ, FtsK and FtsN to be incorporated into active divisomes.
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key role of two terminal domains in the bidirectional polymerization of FtsA protein
Journal of Biological Chemistry, 2012Co-Authors: Marcin Krupka, Ana Isabel Rico, German Rivas, M A VicenteAbstract:The effect of two different truncations involving either the 1C domain or the simultaneous absence of the S12–13 β-strands of the FtsA protein from Streptococcus pneumoniae, located at opposite terminal sides in the molecular structure, suggests that they are essential for ATP-dependent polymerization. These two truncated proteins are not able to polymerize themselves but can be incorporated to some extent into the FtsA+ polymers during the assembling process. Consequently, they block the growth of the FtsA+ polymers and slow down the polymerization rate. The combined action of the two truncated proteins produces an additive effect on the inhibition of FtsA+ polymerization, indicating that each truncation affects a different interaction site within the FtsA molecule.
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reconstitution and organization of escherichia coli proto ring elements ftsz and FtsA inside giant unilamellar vesicles obtained from bacterial inner membranes
Journal of Biological Chemistry, 2011Co-Authors: Mercedes Jimenez, Ariadna Martos, M A Vicente, German RivasAbstract:We have incorporated, for the first time, FtsZ and FtsA (the soluble proto-ring proteins from Escherichia coli) into bacterial giant unilamellar inner membrane vesicles (GUIMVs). Inside the vesicles, the structural organization and spatial distribution of fluorescently labeled FtsZ and FtsA were determined by confocal microscopy. We found that, in the presence of GDP, FtsZ was homogeneously distributed in the lumen of the vesicle. In the presence of GTP analogs, FtsZ assembled inside the GUIMVs, forming a web of dense spots and fibers. Whereas isolated FtsA was found adsorbed to the inner face of GUIMVs, the addition of FtsZ together with GTP analogs resulted in its dislodgement and its association with the FtsZ fibers in the lumen, suggesting that the FtsA-membrane interaction can be modulated by FtsZ polymers. The use of this novel in vitro system to probe interactions between divisome components will help to determine the biological implications of these findings.
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phage display and correlated mutations identify an essential region of subdomain 1c involved in homodimerization of escherichia coli FtsA
Proteins, 2002Co-Authors: Daniele Carettoni, Jesus Mingorance, M A Vicente, Paulino Gomezpuertas, Lucia Yim, Orietta Massidda, Alfonso Valencia, Enrico Domenici, Daniela AnderluzziAbstract:FtsA plays an essential role in Escherichia coli cell division and is nearly ubiquitous in eubacteria. Several evidences postulated the ability of FtsA to interact with other septation proteins and with itself. To investigate these binding properties, we screened a phage-display library with FtsA. The isolated peptides defined a degenerate consensus sequence, which in turn displayed a striking similarity with residues 126-133 of FtsA itself. This result suggested that residues 126-133 were involved in homodimerization of FtsA. The hypothesis was supported by the analysis of correlated mutations, which identified a mutual relationship between a group of amino acids encompassing the ATP-binding site and a set of residues immediately downstream to amino acids 126-133. This information was used to assemble a model of a FtsA homodimer, whose accuracy was confirmed by probing multiple alternative docking solutions. Moreover, a prediction of residues responsible for protein-protein interaction validated the proposed model and confirmed once more the importance of residues 126-133 for homodimerization. To functionally characterize this region, we introduced a deletion in FtsA, where residues 126-133 were skipped. This mutant failed to complement conditional lethal alleles of FtsA, demonstrating that amino acids 126-133 play an essential role in E. coli.
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role of the carboxy terminus of escherichia coli FtsA in self interaction and cell division
Journal of Bacteriology, 2000Co-Authors: Lucia Yim, Jesus Mingorance, Mercedes Casanova, Guy Vandenbussche, Sonsoles Rueda, Jean Marie Ruysschaert, M A VicenteAbstract:The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of the FtsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362-5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsADelta1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsADelta27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsADelta27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.
