scholarly journals Escherichia coliZipA organizes FtsZ polymers into dynamic ring-like protofilament structures

2018 ◽  
Author(s):  
Marcin Krupka ◽  
Marta Sobrinos-Sanguino ◽  
Mercedes Jiménez ◽  
Germán Rivas ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
High Surface ◽  
Bacterial Cells ◽  
E Coli ◽  
Daughter Cells ◽  
Membrane Attachment ◽  
Lipid Surface ◽  
Confocal Fluorescence Imaging

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nm in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at mid-cell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are only observed when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.

scholarly journals Escherichia coliZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Marcin Krupka ◽  
Marta Sobrinos-Sanguino ◽  
Mercedes Jiménez ◽  
Germán Rivas ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
High Surface ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Membrane Attachment ◽  
Lipid Surface ◽  
Confocal Fluorescence Imaging

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.

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 FtsW exhibits distinct processive motions driven by either septal cell wall synthesis or FtsZ treadmilling in E. coli

2019 ◽  
Author(s):  
Xinxing Yang ◽  
Ryan McQuillen ◽  
Zhixin Lyu ◽  
Polly Phillips-Mason ◽  
Ana De La Cruz ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Single Molecule ◽  
Bacterial Cell ◽  
Spatial Information ◽  
Bacterial Species ◽  
E Coli ◽  
Slow Moving

AbstractDuring bacterial cell division, synthesis of new septal peptidoglycan (sPG) is crucial for successful cytokinesis and cell pole morphogenesis. FtsW, a SEDS (Shape, Elongation, Division and Sporulation) family protein and an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a new monofunctional peptidoglycan glycosyltransferase (PGTase). FtsW and its cognate monofunctional transpeptidase (TPase) class B penicillin binding protein (PBP3 or FtsI in E. coli) may constitute the essential, bifunctional sPG synthase specific for new sPG synthesis. Despite its importance, the septal PGTase activity of FtsW has not been documented in vivo. How its activity is spatiotemporally regulated in vivo has also remained unknown. Here we investigated the septal PGTase activity and dynamics of FtsW in E. coli cells using a combination of single-molecule imaging and genetic manipulations. We show that FtsW exhibits robust activity to incorporate an N-acetylmuramic acid analog at septa in the absence of other known PGTases, confirming FtsW as the essential septum-specific PGTase in vivo. Notably, we identified two populations of processive moving FtsW molecules at septa. A fast-moving population is driven by the treadmilling dynamics of FtsZ and independent of sPG synthesis. A slow-moving population is driven by active sPG synthesis and independent of FtsZ’s treadmilling dynamics. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving, sPG synthesis-dependent population. Our results support a two-track model, in which inactive sPG synthase molecules follow the fast treadmilling “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 explains how the spatial information is integrated into the regulation of sPG synthesis activity and suggests a new mechanistic framework for the spatiotemporal coordination of bacterial cell wall constriction.

scholarly journals Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator inPseudomonas aeruginosa

2018 ◽  
Vol 115 (12) ◽  
pp. 3150-3155 ◽  
Author(s):  
Neil G. Greene ◽  
Coralie Fumeaux ◽  
Thomas G. Bernhardt
Keyword(s):  
Cell Wall ◽  
Drug Targets ◽  
Bacterial Cells ◽  
Acinetobacter Baylyi ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membrane Lipoprotein ◽  
Synthase Regulation

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoproteinEcLpoB was identified as a critical activator ofEscherichia coliPBP1b (EcPBP1b), one of the major PG synthases of this organism. Activation ofEcPBP1b is mediated through the association ofEcLpoB with a regulatory domain onEcPBP1b called UB2H. Notably,Pseudomonas aeruginosaalso encodes PBP1b (PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potentialPaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity ofPaPBP1b. We named this protein LpoP and found that it interacts directly withPaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated thatPaLpoP-PaPBP1b as well as an equivalent protein pair fromAcinetobacter baylyican fully substitute forEcLpoB-EcPBP1b inE. colifor PG synthesis. Furthermore, we show that amino acid changes inPaPBP1b that bypass thePaLpoP requirement map to similar locations in the protein as changes promotingEcLpoB bypass inEcPBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

Effects of high hydrostatic pressure on bacterial cytoskeleton FtsZ polymers in vivo and in vitro

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1965-1972 ◽  
Author(s):  
Akihiro Ishii ◽  
Takako Sato ◽  
Masaaki Wachi ◽  
Kazuo Nagai ◽  
Chiaki Kato
Keyword(s):  
Cell Division ◽  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Elevated Pressure ◽  
Immunofluorescent Staining ◽  
E Coli ◽  
Division Process ◽  
Indirect Immunofluorescent

Some rod-shaped bacteria, including Escherichia coli, exhibit cell filamentation without septum formation under high-hydrostatic-pressure conditions, indicating that the cell-division process is affected by hydrostatic pressure. The effects of elevated pressure on FtsZ-ring formation in E. coli cells were examined using indirect immunofluorescence microscopy. Elevated pressure of 40 MPa completely inhibited colony formation of E. coli cells under the cultivation conditions used, and the cells exhibited obviously filamentous shapes. In the elongated cells, normal cell-division processes appeared to be inhibited, because no FtsZ rings were observed by indirect immunofluorescent staining. In addition, it was observed that hydrostatic pressure dissociated the E. coli FtsZ polymers in vitro. These results suggest that high hydrostatic pressure directly affects cell survival and morphology through the dissociation of the cytoskeletal frameworks.

scholarly journals Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

2022 ◽  
Author(s):  
James A Sawitzke ◽  
Nina C Costantino ◽  
Ellen Hutchinson ◽  
Lynn Thomason ◽  
Donald L Court
Keyword(s):  
Dna Assembly ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Engineering Technology ◽  
E Coli ◽  
Linear Dna ◽  
Recombination System ◽  
Genetic Engineering Technology

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

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.

scholarly journals ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces

2021 ◽  
Author(s):  
Yamal Al-ramahi ◽  
Akos Nyerges ◽  
Yago Margolles ◽  
Lidia Cerdán ◽  
Gyorgyi Ferenc ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Industrial Biotechnology ◽  
Bacterial Cells ◽  
E Coli ◽  
Immunomagnetic Capture ◽  
Bacterial Surfaces ◽  
Potential Applications ◽  
Novel Target

SUMMARYIn vivo evolution of antibodies facilitates emergence of novel target specificities from pre-existing clones. In this work we show how mutagenic ssDNA recombineering of camel-derived nanobodies encoded in a bacterial genome enables clonal hyper-diversification and the rise of new properties. As a proof-of-principle we used a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) and evolved it towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying on their surface this nanobody fused to the intimin outer membrane anchor domain were subjected to multiple rounds of mutagenic ssDNA recombineering targeted to the CDR1, CDR2 and CDR3 regions of its genomically encoded VHH sequence. Binders to the new antigen (EPEC TirM) were then selected upon immunomagnetic capture of bacteria bearing the corresponding nanobody variants. As a result, several modified nanobodies were identified which maintained recognition of EHEC TirM but acquired the ability to bind the new antigen with high affinity (Kd ~20 nM). The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest. Our experimental workflow empowers the evolution of nanobodies displayed on the surface of bacterial cells for a large number of potential applications in medical and industrial biotechnology.

scholarly journals Reversible inhibition of bacterial growth after specific inhibition of spermidine synthase by dicyclohexylamine

1984 ◽  
Vol 223 (3) ◽  
pp. 823-830 ◽  
Author(s):  
T Mattila ◽  
T Honkanen-Buzalski ◽  
H Pösö
Keyword(s):  
Final Concentration ◽  
Spermidine Synthase ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
E Coli ◽  
Inhibition Of Growth ◽  
Streptococcus Uberis ◽  
Mastitis Pathogens

The effect of dicyclohexylamine on seven freshly isolated bacterial strains of mastitis pathogens was studied. Streptococcus uberis was the most sensitive strain investigated, since 5 mM-dicyclohexylamine totally arrested its growth and 1.25 mM of the drug caused 60% growth inhibition. The Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains were also sensitive to the drug, but less so than Strep. uberis, since 5 mM drug caused only partial inhibition of growth. Micrococcus sp. and Klebsiella sp. grew in the presence of 10.0 mM-dicyclohexylamine, and, finally the growth of Streptococcus agalactiae was not at all affected by dicyclohexylamine. These different sensitivities towards dicyclohexylamine in vivo were paralleled by different sensitivities of the bacteria's spermidine synthase to the drug in vitro, and also by the ability of the drug to lower spermidine concentration in bacterial cells. Spermidine synthase from sensitive bacteria was inhibited by more than 90% by 50 microM-dicyclohexylamine in vitro, and the concentration of spermidine was decreased in E. coli and Ps. aeruginosa by 70% and in Strep. uberis by 95%, whereas in Strep. agalactiae 5 mM-dicyclohexylamine did not affect the concentration of spermidine at all. Dicyclohexylamine treatment led to the accumulation of putrescine in Strep. uberis. Spermidine synthesis catalysed by the extracts of Micrococcus sp. required 500 microM-dicyclohexylamine for 90% inhibition, and Strep. agalactiae contained a spermidine synthase that was still active at 1000 microM-dicyclohexylamine, The observed inhibition of growth was totally reversed by adding 50 microM-spermidine (final concentration) to the medium. Putrescine reversed the inhibition only when bacteria had a spermidine synthase activity insensitive to dicyclohexylamine. Spermine did not overcome the inhibition of growth caused by dicyclohexylamine, probably because it was not taken up by the bacterial cells used in this study. The inhibition of the growth by dicyclohexylamine (even in the case of Strep. uberis) was reversible in the sense that addition of 50 microM-spermidine 18 h after dicyclohexylamine still restored the growth rate of untreated controls.

scholarly journals In Vitro and In Vivo Functional Activity of Chlamydia MurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance

2003 ◽  
Vol 185 (4) ◽  
pp. 1218-1228 ◽  
Author(s):  
Andrea J. McCoy ◽  
Robin C. Sandlin ◽  
Anthony T. Maurelli
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Enzymatic Reaction ◽  
Developmental Cycle ◽  
E Coli ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Fosfomycin Resistance

ABSTRACT Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal ΔmurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

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