scholarly journals MinC N- and C-Domain Interactions Modulate FtsZ Assembly, Division Site Selection, and MinD-Dependent Oscillation inEscherichia coli

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Christopher J. LaBreck ◽  
Joseph Conti ◽  
Marissa G. Viola ◽  
Jodi L. Camberg
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Site Selection ◽  
Fluorescent Protein ◽  
Site Mutation ◽  
Content Type ◽  
Ftsz Ring ◽  
Division Site ◽  
Z Ring

ABSTRACTThe Min system inEscherichia coli, consisting of MinC, MinD, and MinE proteins, regulates division site selection by preventing assembly of the FtsZ-ring (Z-ring) and exhibits polar oscillationin vivo. MinC antagonizes FtsZ polymerization, andin vivo, the cellular location of MinC is controlled by a direct association with MinD at the membrane. To further understand the interactions of MinC with FtsZ and MinD, we performed a mutagenesis screen to identify substitutions inminCthat are associated with defects in cell division. We identified amino acids in both the N- and C-domains of MinC that are important for direct interactions with FtsZ and MinDin vitro, as well as mutations that modify the observedin vivooscillation of green fluorescent protein (GFP)-MinC. Our results indicate that there are two distinct surface-exposed sites on MinC that are important for direct interactions with FtsZ, one at a cleft on the surface of the N-domain and a second on the C-domain that is adjacent to the MinD interaction site. Mutation of either of these sites leads to slower oscillation of GFP-MinCin vivo, although the MinC mutant proteins are still capable of a direct interaction with MinD in phospholipid recruitment assays. Furthermore, we demonstrate that interactions between FtsZ and both sites of MinC identified here are important for assembly of FtsZ-MinC-MinD complexes and that the conserved C-terminal end of FtsZ is not required for MinC-MinD complex formation with GTP-dependent FtsZ polymers.IMPORTANCEBacterial cell division proceeds through the coordinated assembly of the FtsZ-ring, or Z-ring, at the site of division. Assembly of the Z-ring requires polymerization of FtsZ, which is regulated by several proteins in the cell. InEscherichia coli, the Min system, which contains MinC, MinD, and MinE proteins, exhibits polar oscillation and inhibits the assembly of FtsZ at nonseptal locations. Here, we identify regions on the surface of MinC that are important for contacting FtsZ and destabilizing FtsZ polymers.

scholarly journals Assembly Dynamics of FtsZ Rings in Bacillus subtilis and Escherichia coli and Effects of FtsZ-Regulating Proteins

2004 ◽  
Vol 186 (17) ◽  
pp. 5775-5781 ◽  
Author(s):  
David E. Anderson ◽  
Frederico J. Gueiros-Filho ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Fluorescence Recovery ◽  
Half Time ◽  
Experimental Conditions ◽  
Z Ring

ABSTRACT FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (∼9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an ∼3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, ∼8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

scholarly journals Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

2020 ◽  
Vol 203 (2) ◽  
pp. e00463-20
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Gene Product ◽  
Fluorescent Protein ◽  
Lytic Bacteriophage ◽  
Cell Wall Synthesis ◽  
Content Type ◽  
Ftsz Ring ◽  
Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

scholarly journals The C-Terminal Domain of MinC Inhibits Assembly of the Z Ring in Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 236-243 ◽  
Author(s):  
Daisuke Shiomi ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Additional Function ◽  
Ring Assembly ◽  
C Terminus ◽  
Min Proteins ◽  
Z Ring ◽  
Ftsz Assembly ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC122-231) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC122-231, the C terminus of full-length MinC, or the C terminus of MinC122-231 perturbed MinC function, which may explain why cell division inhibition by MinC122-231 was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

scholarly journals Structural Determinants Required To Target Penicillin-Binding Protein 3 to the Septum of Escherichia coli

2004 ◽  
Vol 186 (18) ◽  
pp. 6110-6117 ◽  
Author(s):  
André Piette ◽  
Claudine Fraipont ◽  
Tanneke den Blaauwen ◽  
Mirjam E. G. Aarsman ◽  
Soumya Pastoret ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Structural Determinants ◽  
Membrane Anchor ◽  
Interaction Sites ◽  
Division Site ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.

scholarly journals Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Desmond A. Moore ◽  
Zakiya N. Whatley ◽  
Chandra P. Joshi ◽  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Cell Division ◽  
Fluorescent Proteins ◽  
Sole Source ◽  
Ring Structure ◽  
Content Type ◽  
Z Ring ◽  
Complete Cell ◽  
The Right

ABSTRACT FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

scholarly journals MinC, MinD, and MinE Drive Counter-oscillation of Early-Cell-Division Proteins Prior to Escherichia coli Septum Formation

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Paola Bisicchia ◽  
Senthil Arumugam ◽  
Petra Schwille ◽  
David Sherratt
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Lipid Bilayers ◽  
Epifluorescence Microscopy ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Septum Formation ◽  
Early Stages ◽  
Z Ring ◽  
High Concentrations

ABSTRACTBacterial cell division initiates with the formation of a ring-like structure at the cell center composed of the tubulin homolog FtsZ (the Z-ring), which acts as a scaffold for the assembly of the cell division complex, the divisome. Previous studies have suggested that the divisome is initially composed of FtsZ polymers stabilized by membrane anchors FtsA and ZipA, which then recruit the remaining division proteins. The MinCDE proteins prevent the formation of the Z-ring at poles by oscillating from pole to pole, thereby ensuring that the concentration of the Z-ring inhibitor, MinC, is lowest at the cell center. We show that prior to septum formation, the early-division proteins ZipA, ZapA, and ZapB, along with FtsZ, assemble into complexes that counter-oscillate with respect to MinC, and with the same period. We propose that FtsZ molecules distal from high concentrations of MinC form relatively slowly diffusing filaments that are bound by ZapAB and targeted to the inner membrane by ZipA or FtsA. These complexes may facilitate the early stages of divisome assembly at midcell. As MinC oscillates toward these complexes, FtsZ oligomerization and bundling are inhibited, leading to shorter or monomeric FtsZ complexes, which become less visible by epifluorescence microscopy because of their rapid diffusion. Reconstitution of FtsZ-Min waves on lipid bilayers shows that FtsZ bundles partition away from high concentrations of MinC and that ZapA appears to protect FtsZ from MinC by inhibiting FtsZ turnover.IMPORTANCEA big issue in biology for the past 100 years has been that of how a cell finds its middle. InEscherichia coli, over 20 proteins assemble at the cell center at the time of division. We show that the MinCDE proteins, which prevent the formation of septa at the cell pole by inhibiting FtsZ, drive the counter-oscillation of early-cell-division proteins ZapA, ZapB, and ZipA, along with FtsZ. We propose that FtsZ forms filaments at the pole where the MinC concentration is the lowest and acts as a scaffold for binding of ZapA, ZapB, and ZipA: such complexes are disassembled by MinC and reform within the MinC oscillation period before accumulating at the cell center at the time of division. The ability of FtsZ to be targeted to the cell center in the form of oligomers bound by ZipA and ZapAB may facilitate the early stages of divisome assembly.

scholarly journals Genetic Dissection of DivIVA Functions in Listeria monocytogenes

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
Karan Gautam Kaval ◽  
Samuel Hauf ◽  
Jeanine Rismondo ◽  
Birgitt Hahn ◽  
Sven Halbedel
Keyword(s):  
Cell Division ◽  
Listeria Monocytogenes ◽  
Protein Interactions ◽  
Site Selection ◽  
Genetic Dissection ◽  
Flagellar Motility ◽  
Content Type ◽  
Division Site ◽  
Interaction Partners ◽  
Species Specific

ABSTRACT DivIVA is a membrane binding protein that clusters at curved membrane regions, such as the cell poles and the membrane invaginations occurring during cell division. DivIVA proteins recruit many other proteins to these subcellular sites through direct protein-protein interactions. DivIVA-dependent functions are typically associated with cell growth and division, even though species-specific differences in the spectrum of DivIVA functions and their causative interaction partners exist. DivIVA from the Gram-positive human pathogen Listeria monocytogenes has at least three different functions. In this bacterium, DivIVA is required for precise positioning of the septum at midcell, it contributes to the secretion of autolysins required for the breakdown of peptidoglycan at the septum after the completion of cell division, and it is essential for flagellar motility. While the DivIVA interaction partners for control of division site selection are well established, the proteins connecting DivIVA with autolysin secretion or swarming motility are completely unknown. We set out to identify divIVA alleles in which these three DivIVA functions could be separated, since the question of the degree to which the three functions of L. monocytogenes DivIVA are interlinked could not be answered before. Here, we identify such alleles, and our results show that division site selection, autolysin secretion, and swarming represent three discrete pathways that are independently influenced by DivIVA. These findings provide the required basis for the identification of DivIVA interaction partners controlling autolysin secretion and swarming in the future. IMPORTANCE DivIVA of the pathogenic bacterium Listeria monocytogenes is a central scaffold protein that influences at least three different cellular processes, namely, cell division, protein secretion, and bacterial motility. How DivIVA coordinates these rather unrelated processes is not known. We here identify variants of L. monocytogenes DivIVA, in which these functions are separated from each other. These results have important implications for the models explaining how DivIVA interacts with other proteins.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Piotr Szwedziak ◽  
Qing Wang ◽  
Tanmay A M Bharat ◽  
Matthew Tsim ◽  
Jan Löwe
Keyword(s):  
Cell Division ◽  
Molecular Model ◽  
Three Dimensional ◽  
Bacterial Cell Division ◽  
Electron Cryomicroscopy ◽  
E Coli ◽  
Ftsz Ring ◽  
Z Ring

Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding.

scholarly journals LocZ Is a New Cell Division Protein Involved in Proper Septum Placement in Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 6 (1) ◽  
Author(s):  
Nela Holečková ◽  
Linda Doubravová ◽  
Orietta Massidda ◽  
Virginie Molle ◽  
Karolína Buriánková ◽  
...  
Keyword(s):  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Protein Kinase ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Daughter Cells ◽  
Division Site ◽  
Z Ring ◽  
Specific Shape ◽  
Cell Division Protein

ABSTRACTHow bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement inStreptococcus pneumoniae. We show thatlocZis not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division.IMPORTANCEBacterial cell division is a highly ordered process regulated in time and space. Recently, we reported that the Ser/Thr protein kinase StkP regulates cell division in Streptococcus pneumoniae, through phosphorylation of several key proteins. Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ. We show that LocZ is a new cell division protein important for proper septum placement and likely functions as a marker of the cell division site. Consistently, LocZ supports proper Z-ring positioning at midcell. LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.

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