scholarly journals RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape

2017 ◽  
Author(s):  
Alexandre Colavin ◽  
Handuo Shi ◽  
Kerwyn Casey Huang
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Spatial Organization ◽  
Dependent Manner ◽  
Cell Width ◽  
E Coli ◽  
Wall Growth ◽  
The Rich ◽  
Dynamics Simulations ◽  
Geometric Localization

AbstractIn the rod-shaped bacteriumEscherichia coli, the actin-like protein MreB localizes in a curvature-dependent manner and spatially coordinates cell-wall insertion to maintain cell shape across changing environments, although the molecular mechanism by which cell width is regulated remains unknown. Here, we demonstrate that the bitopic membrane protein RodZ regulates the biophysical properties of MreB and alters the spatial organization ofE. colicell-wall growth. The relative expression levels of MreB and RodZ changed in a manner commensurate with variations in growth rate and cell width. We carried out single-cell analyses to determine that RodZ systematically alters the curvature-based localization of MreB and cell width in a manner dependent on the concentration of RodZ. Finally, we identified MreB mutants that we predict using molecular dynamics simulations to alter the bending properties of MreB filaments at the molecular scale similar to RodZ binding, and showed that these mutants rescued rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Together, our results show thatE. colicontrols its shape and dimensions by differentially regulating RodZ and MreB to alter the patterning of cell-wall insertion, highlighting the rich regulatory landscape of cytoskeletal molecular biophysics.

scholarly journals Probing bacterial cell wall growth by tracing wall-anchored protein complexes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yi-Jen Sun ◽  
Fan Bai ◽  
An-Chi Luo ◽  
Xiang-Yu Zhuang ◽  
Tsai-Shun Lin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Bernoulli Shift ◽  
Protein Complexes ◽  
Axial Direction ◽  
Cell Wall Growth ◽  
Flagellar Motors ◽  
Shift Map ◽  
Dynamic Assembly ◽  
Wall Growth

AbstractThe dynamic assembly of the cell wall is key to the maintenance of cell shape during bacterial growth. Here, we present a method for the analysis of Escherichia coli cell wall growth at high spatial and temporal resolution, which is achieved by tracing the movement of fluorescently labeled cell wall-anchored flagellar motors. Using this method, we clearly identify the active and inert zones of cell wall growth during bacterial elongation. Within the active zone, the insertion of newly synthesized peptidoglycan occurs homogeneously in the axial direction without twisting of the cell body. Based on the measured parameters, we formulate a Bernoulli shift map model to predict the partitioning of cell wall-anchored proteins following cell division.

scholarly journals FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

2021 ◽  
Author(s):  
Zhixin Lyu ◽  
Atsushi Yahashiri ◽  
Xinxing Yang ◽  
Joshua W McCausland ◽  
Gabriela M Kaus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Single Molecule ◽  
Spatial Organization ◽  
Genetic Manipulation ◽  
Cell Wall Synthesis ◽  
Single Population ◽  
E Coli ◽  
Ftsz Ring ◽  
Synthesis Activity

The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of ~ 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the ~ 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZ's treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.

scholarly journals RodZ promotes MreB polymer formation and curvature localization to determine the cylindrical uniformity of E. coli shape

2017 ◽  
Author(s):  
Randy M. Morgenstein ◽  
Benjamin P. Bratton ◽  
Joshua W. Shaevitz ◽  
Zemer Gitai
Keyword(s):  
Cell Wall ◽  
Single Cell ◽  
Cell Body ◽  
Cell Shape ◽  
E Coli ◽  
Total Length ◽  
Polymer Formation ◽  
Mutant Strains ◽  
Geometric Cues ◽  
Cell Changes

AbstractCell shape in bacteria is determined by the cell wall, which is synthesized by a variety of proteins whose actions are coordinated by the actin-like MreB protein. MreB uses local geometric cues of envelope curvature to avoid the cell poles and localize to specific regions of the cell body. However, it remains unclear whether MreB’s curvature preference is regulated by additional factors, and which features of MreB are essential for specific aspects of rod shape growth, such as cylindrical uniformity. Here we show that in addition to its previously-described role in mediating MreB motion, RodZ also modulates MreB polymer number and curvature preference. MreB polymer number and curvature localization can be regulated independently. Quantitative 3D measurements and a series of mutant strains show that among various properties of MreB, polymer number, total length of MreB polymers, and MreB curvature preference are the key determinants of cylindrical uniformity, a measure of the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2=0.93). Our data suggest a model for rod shape in which RodZ promotes the assembly of multiple long MreB polymers that ensure the growth of a uniform cylinder.

scholarly journals RNA polymerase organizes into distinct spatial clusters independent of ribosomal RNA transcription in E. coli

2018 ◽  
Author(s):  
Xiaoli Weng ◽  
Christopher H. Bohrer ◽  
Kelsey Bettridge ◽  
Arvin Cesar Lagda ◽  
Cedric Cagliero ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Growth Condition ◽  
Ribosomal Rna ◽  
Spatial Organization ◽  
Large Fraction ◽  
Rrna Synthesis ◽  
Rich Medium ◽  
E Coli ◽  
The Rich ◽  
Molecular Components

AbstractRecent studies have shown that RNA polymerase (RNAP) is spatially organized into distinct clusters in E. coli and B. subtilis cells. Spatially organized molecular components in prokaryotic systems imply compartmentalization without the use of membranes, which may offer new insights into pertinent functions and regulations. However, the function of RNAP clusters and whether its formation is driven by active ribosomal RNA (rRNA) transcription remain elusive. In this work, we investigated the spatial organization of RNAP in E. coli cells using quantitative superresolution imaging. We observed that RNAP formed large, distinct clusters under a rich medium growth condition and preferentially located in the center of the nucleoid. Two-color superresolution colocalization imaging showed that under the rich medium growth condition, nearly all RNAP clusters were active in synthesizing rRNA, suggesting that rRNA synthesis may be spatially separated from mRNA synthesis that most likely occurs at the nucleoid periphery. Surprisingly, a large fraction of RNAP clusters persisted under conditions in which rRNA synthesis was reduced or abolished, or when only one out of the seven rRNA operons (rrn) remained. Furthermore, when gyrase activity was inhibited, we observed a similar rRNA synthesis level, but multiple dispersed, smaller rRNA and RNAP clusters occupying not only the center but also the periphery of the nucleoid, comparable to an expanded nucleoid. These results suggested that RNAP was organized into active transcription centers for rRNA synthesis under the rich medium growth condition; their presence and spatial organization, however, were independent of rRNA synthesis activity under the conditions used but were instead influenced by the structure and characteristics of the underlying nucleoid. Our work opens the door for further investigations of the function and molecular nature of RNAP clusters and points to a potentially new mechanism of transcription regulation by the spatial organization of individual molecular components.

scholarly journals AimB is a small protein regulator of cell size and MreB assembly

2019 ◽  
Author(s):  
John N. Werner ◽  
Handuo Shi ◽  
Jen Hsin ◽  
Kerwyn Casey Huang ◽  
Zemer Gitai ◽  
...  
Keyword(s):  
Unnatural Amino Acids ◽  
Cell Shape ◽  
Caulobacter Crescentus ◽  
Specific Activity ◽  
Interacting Proteins ◽  
Cell Width ◽  
Site Specific ◽  
Small Protein ◽  
Dynamics Simulations ◽  
Species Specific

AbstractThe MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeletons, few MreB interacting proteins have been well characterized. Here we identify a small protein fromCaulobacter crescentus, AimB, as anAssemblyInhibitor ofMreB. AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft. We validate this model through functional analysis of point mutants in both AimB and MreB, photo-crosslinking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings indicate that AimB promotes MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor.

scholarly journals Tyrosine phosphorylation-dependent localization of TmaR, a novel E. coli polar protein that controls activity of the major sugar regulator by polar sequestration

2020 ◽  
Author(s):  
Tamar Szoke ◽  
Nitsan Albocher ◽  
Sutharsan Govindarajan ◽  
Anat Nussbaum-Shochat ◽  
Orna Amster-Choder
Keyword(s):  
Tyrosine Phosphorylation ◽  
Spatial Organization ◽  
Sugar Metabolism ◽  
Sugar Uptake ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Sugar Consumption ◽  
E Coli ◽  
Underlying Mechanisms ◽  
Novel Protein

ABSTRACTThe poles of E. coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a novel protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization of the general PTS protein Enzyme I (EI) by preventing it from exerting its activity by binding and polar sequestration, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results argue that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.SIGNIFICANCEIn recent years, we have learnt that bacterial cells have intricate spatial organization despite the lack of membrane-bounded organelles. The endcaps of rod-shaped bacteria, termed poles, are emerging as hubs for sensing and responding, but the underlying mechanisms for positioning macromolecules there are largely unknown. We discovered a novel protein, TmaR, whose polar localization depends on a phospho-tyrosine modification. We show that TmaR controls the activity of EI, the major regulator of sugar metabolism in most bacteria, by polar sequestration and release. Notably, TmaR is essential for survival in conditions that E. coli often encounters in nature. Hence, TmaR is a key regulator that connects tyrosine phosphorylation, spatial regulation, sugar metabolism and survival in bacteria and the first protein reported to recruit proteins to the E. coli cell poles.

scholarly journals Cell wall synthesis and remodeling dynamics determine bacterial division site architecture and cell shape

2021 ◽  
Author(s):  
Paula P. Navarro ◽  
Andrea Vettiger ◽  
Virly Y Ananda ◽  
Paula Montero Llopis ◽  
Christoph Allolio ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Electron Tomography ◽  
Three Dimensional ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Division Site ◽  
Bacterial Division ◽  
Mutant Cells ◽  
Bacterial Domain

The bacterial division apparatus builds daughter cell poles by catalyzing the synthesis and remodeling of the septal peptidoglycan (sPG) cell wall. Understanding of this essential process has been limited by the lack of native three-dimensional visualization of developing septa. Here, we used state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to understand the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. Our results with mutant cells altered in the regulation of sPG biogenesis revealed a striking and unexpected similarity between the architecture of E. coli septa with those from Gram-positive bacteria, suggesting a conserved morphogenic mechanism. Furthermore, we found that the cell elongation and division machineries are in competition and that their relative activities determine the shape of cell constrictions and the poles they form. Overall, our results highlight how the activity of the division system can be modulated to generate the diverse array of morphologies observed in the bacterial domain.

scholarly journals Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth

mSystems ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Valerie J. Carabetta ◽  
Todd M. Greco ◽  
Andrew W. Tanner ◽  
Ileana M. Cristea ◽  
David Dubnau
Keyword(s):  
Cell Wall ◽  
Cell Growth ◽  
Posttranslational Modification ◽  
Lysine Acetylation ◽  
Growth Phases ◽  
Cell Width ◽  
Cell Wall Growth ◽  
Content Type ◽  
Temporal Regulation ◽  
Wall Growth

ABSTRACT The past decade highlighted N ε-lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell shape-determining protein. Our findings led us to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth. N ε-Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. IMPORTANCE The past decade highlighted N ε-lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell shape-determining protein. Our findings led us to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth.

scholarly journals Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

Open Biology ◽  
2017 ◽  
Vol 7 (2) ◽  
pp. 160274 ◽  
Author(s):  
Deborah A. Goldspink ◽  
Chris Rookyard ◽  
Benjamin J. Tyrrell ◽  
Jonathan Gadsby ◽  
James Perkins ◽  
...  
Keyword(s):  
Cell Shape ◽  
Spatial Organization ◽  
Dependent Manner ◽  
Microtubule Organizing Centres ◽  
Functional Inhibition ◽  
Genes Encoding ◽  
Anchoring Protein ◽  
Microtubule Formation ◽  
And Function

Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150 Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs.

CELL WALL REPLICATION: II. CELL WALL GROWTH AND CROSS WALL FORMATION OF ESCHERICHIA COLI AND STREPTOCOCCUS FAECALIS

1964 ◽  
Vol 10 (3) ◽  
pp. 473-482 ◽  
Author(s):  
K. L. Chung ◽  
R. Z. Hawirko ◽  
P. K. Isaac
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Initial Step ◽  
Wall Material ◽  
Cross Wall ◽  
Cell Wall Growth ◽  
E Coli ◽  
Rapid Enlargement ◽  
Wall Growth ◽  
Polar Type

Cell wall replication in E. coli and S. faecalis was studied by differential labelling of living cells with fluorescent and non-fluorescent antibody.In E. coli the initial step in cell division was the formation of a cross wall at the cell equator, followed by the appearance of new cell wall on either side of the cross wall. The process was repeated in sequence at subsequent sites in the polar, the subcentral, and the subpolar areas. Constriction occurred at random so that the divided parent cells were composed of several daughter cells.A polar type of unidirectional cell wall growth and elongation was also observed in E. coli. It was initiated by the synthesis of a ring of new cell wall material around the polar tip. A second ring was then formed at the subpolar area during the rapid enlargement of the first ring in a single direction.Evidence shows that cell wall synthesis is independent of cell division and that in E. coli, it is initiated at multiple but specific sites within the cell and not by diffuse intercalation of old and new walls.Contrary to the synthesis of cell wall at multiple sites in E. coli, S. faecalis replicated new cell wall at only one site per coccus. The new wall segment was initiated and enlarged at the coccal equator, and was followed by the formation of a cross wall, centripetal growth and constriction to separate the daughter cells.

Sign in / Sign up

Export Citation Format

Share Document