scholarly journals An oscillating MinD protein determines the cellular positioning of the motility machinery in archaea

2020 ◽  
Author(s):  
Phillip Nußbaum ◽  
Solenne Ithurbide ◽  
James C. Walsh ◽  
Megha Patro ◽  
Floriane Delpech ◽  
...  
Keyword(s):  
Cell Division ◽  
Point Mutations ◽  
Sister Group ◽  
Atpase Domain ◽  
Homologous Proteins ◽  
Cell Axis ◽  
E Coli ◽  
Self Organized ◽  
Z Ring ◽  
The Mind

MinD proteins are well studied in rod-shaped bacteria such as E. coli, where they display self-organized pole-to-pole oscillations that are important for correct positioning of the Z-ring at mid-cell for cell division. Archaea also encode proteins belonging to the MinD family, but their functions are unknown. MinD homologous proteins were found to be widespread in Euryarchaeota and form a sister group to the bacterial MinD family, distinct from the ParA and other related ATPase families. We aimed to identify the function of four archaeal MinD proteins in the model archaeon Haloferax volcanii. Deletion of the minD genes did not cause cell division or size defects, and the Z-ring was still correctly positioned. Instead, one of the mutations (ΔminD4) reduced swimming motility, and hampered the correct formation of motility machinery at the cell poles. In ΔminD4 cells, there is reduced formation of the motility structure and chemosensory arrays, which are essential for signal transduction. In bacteria, several members of the ParA family can position the motility structure and chemosensory arrays via binding to a landmark protein, and consequently these proteins do not oscillate along the cell axis. However, GFP-MinD4 displayed pole-to-pole oscillation and formed polar patches or foci in H. volcanii. The MinD4 membrane targeting sequence (MTS), homologous to the bacterial MinD MTS, was essential for the oscillation. Surprisingly, MinD4 ATPase domain point-mutations did not block oscillation, but they failed to form pole-patches. Thus, MinD4 from H. volcanii combines traits of different bacterial ParA/MinD proteins.

MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

2017 ◽  
Vol 474 (18) ◽  
pp. 3189-3205 ◽  
Author(s):  
Ashoka Chary Taviti ◽  
Tushar Kant Beuria
Keyword(s):  
Cell Division ◽  
Single Point ◽  
Point Mutations ◽  
Direct Interaction ◽  
Ring Formation ◽  
Z Ring ◽  
Single Point Mutations ◽  
Biochemical Analyses ◽  
Ftsz Assembly ◽  
The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

scholarly journals FtsZ from Divergent Foreign Bacteria Can Function for Cell Division in Escherichia coli

2006 ◽  
Vol 188 (20) ◽  
pp. 7132-7140 ◽  
Author(s):  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Point Mutations ◽  
Loss Of Function ◽  
Mycoplasma Pulmonis ◽  
E Coli ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT FtsZs from Mycoplasma pulmonis (MpuFtsZ) and Bacillus subtilis (BsFtsZ) are only 46% and 53% identical in amino acid sequence to FtsZ from Escherichia coli (EcFtsZ). In the present study we show that MpuFtsZ and BsFtsZ can function for cell division in E. coli provided we make two modifications. First, we replaced their C-terminal tails with that from E. coli, giving the foreign FtsZ the binding site for E. coli FtsA and ZipA. Second, we selected for mutations in the E. coli genome that facilitated division by the foreign FtsZs. These suppressor strains arose at a relatively high frequency of 10−3 to 10−5, suggesting that they involve loss-of-function mutations in multigene pathways. These pathways may be negative regulators of FtsZ or structural pathways that facilitate division by slightly defective FtsZ. Related suppressor strains were obtained for EcFtsZ containing certain point mutations or insertions of yellow fluorescent protein. The ability of highly divergent FtsZs to function for division in E. coli is consistent with a two-part mechanism. FtsZ assembles the Z ring, and perhaps generates the constriction force, through self interactions; the downstream division proteins remodel the peptidoglycan wall by interacting with each other and the wall. The C-terminal peptide of FtsZ, which binds FtsA, provides the link between FtsZ assembly and peptidoglycan remodeling.

scholarly journals High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP

2020 ◽  
Vol 76 (2) ◽  
pp. 94-102 ◽  
Author(s):  
Maria A. Schumacher ◽  
Tomoo Ohashi ◽  
Lauren Corbin ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Cell Division ◽  
High Resolution ◽  
Crystal Structures ◽  
Bacterial Cell ◽  
Model System ◽  
E Coli ◽  
Z Ring ◽  
Main Model

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.

scholarly journals A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2021 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Multiple Alternative ◽  
Z Ring ◽  
Cell Division Inhibitor

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism. Importance: Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

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 A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2020 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Z Ring ◽  
Response To Stress ◽  
Cell Division Inhibitor

AbstractRod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC+ phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple division inhibition pathways exist during the stress-induced SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism.ImportanceFilamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

scholarly journals Interaction between Cell Division Proteins FtsE and FtsZ

2007 ◽  
Vol 189 (8) ◽  
pp. 3026-3035 ◽  
Author(s):  
Brian D. Corbin ◽  
Yipeng Wang ◽  
Tushar K. Beuria ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
Abc Transporters ◽  
Fluorescent Protein ◽  
Direct Interaction ◽  
Bacterial Cell Division ◽  
E Coli ◽  
Cell Extracts ◽  
Z Ring ◽  
Green Fluorescent ◽  
Binding Component

ABSTRACT FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.

scholarly journals The Escherichia coli Outer Membrane β-Barrel Assembly Machinery (BAM) Crosstalks with the Divisome

2021 ◽  
Vol 22 (22) ◽  
pp. 12101
Author(s):  
Elisa Consoli ◽  
Joen Luirink ◽  
Tanneke den Blaauwen
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Outer Membrane ◽  
De Novo ◽  
Lateral Wall ◽  
Specific Cell ◽  
E Coli ◽  
Z Ring ◽  
Integral Proteins ◽  
Membrane Lipoproteins

The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli, it consists of a transmembrane β-barrel subunit, BamA, and four outer membrane lipoproteins (BamB-E). Using BAM-specific antibodies, in E. coli cells, the complex is shown to localize in the lateral wall in foci. The machinery was shown to be enriched at midcell with specific cell cycle timing. The inhibition of septation by aztreonam did not alter the BAM midcell localization substantially. Furthermore, the absence of late cell division proteins at midcell did not impact BAM timing or localization. These results imply that the BAM enrichment at the site of constriction does not require an active cell division machinery. Expression of the Tre1 toxin, which impairs the FtsZ filamentation and therefore midcell localization, resulted in the complete loss of BAM midcell enrichment. A similar effect was observed for YidC, which is involved in the membrane insertion of cell division proteins in the inner membrane. The presence of the Z-ring is needed for preseptal peptidoglycan (PG) synthesis. As BAM was shown to be embedded in the PG layer, it is possible that BAM is inserted preferentially simultaneously with de novo PG synthesis to facilitate the insertion of OMPs in the newly synthesized outer membrane.

scholarly journals Sublethal Concentrations of the Aminoglycoside Amikacin Interfere with Cell Division without Affecting Chromosome Dynamics

2006 ◽  
Vol 51 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Christophe Possoz ◽  
Jason Newmark ◽  
Nohemy Sorto ◽  
David J. Sherratt ◽  
Marcelo E. Tolmasky
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Acceptor Site ◽  
Protein Fusion ◽  
E Coli ◽  
Z Ring ◽  
A Site ◽  
Derivatives Of ◽  
Sublethal Concentrations

ABSTRACT Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated Escherichia coli cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome ori and ter loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in E. coli strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells.

scholarly journals The bacterial DNA binding protein MatP involved in linking the nucleoid terminal domain to the divisome at midcell interacts with lipid membranes

2018 ◽  
Author(s):  
Begoña Monterroso ◽  
Silvia Zorrilla ◽  
Marta Sobrinos-Sanguino ◽  
Miguel Ángel Robles-Ramos ◽  
Carlos Alfonso ◽  
...  
Keyword(s):  
Cell Division ◽  
Dna Binding ◽  
Dna Sequences ◽  
Lipid Membranes ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
E Coli ◽  
Daughter Cells ◽  
Z Ring

ABSTRACTDivision ring formation at midcell is controlled by various mechanisms inEscherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipidsin vitro. We found that MatP, when encapsulated inside microdroplets generated by microfluidics and giant vesicles, accumulates at phospholipid bilayers and monolayers matching the lipid composition in theE. coliinner membrane. MatP binding to lipids was independently confirmed using lipid coated microbeads and bio-layer interferometry assays. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the interaction of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.IMPORTANCEThe division of anE. colicell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integratedin vivoandin vitroanalysis provides evidence that MatP can interact with lipid membranes comprising the phospholipid mixture in theE. coliinner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

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