Cell wall synthesis and initiation of deoxyribonucleic acid replication in Bacillus subtilis.

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.

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Mutations of the YidC Insertase alleviate stress from σM-dependent membrane protein overproduction in Bacillus subtilis

10.1101/678235 ◽

2019 ◽

Author(s):

Heng Zhao ◽

Ankita J. Sachla ◽

John D. Helmann

Keyword(s):

Oxidative Stress ◽

Cell Wall ◽

Bacillus Subtilis ◽

Membrane Proteins ◽

Membrane Protein ◽

Substrate Binding ◽

Single Amino Acid ◽

Cell Wall Synthesis ◽

Intrinsic Resistance ◽

Σ Factor

AbstractIn Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in spoIIIJ, which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 SpoIIIJ contains a single amino acid substitution in the substrate-binding channel (Q140K), and this allele suppresses the lethality of high SigM. Analysis of a library of YidC variants reveals that increased charge (+2 or +3) in the substrate-binding channel can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidCQ140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.Author SummaryBacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis. In Bacillus subtilis, the alternative σ factor σM is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis. However, dysregulation of the σM regulon, resulting from loss of the YhdL anti-σM protein, is lethal. We here identify charge variants of the SpoIIIJ(YidC) membrane protein insertase that suppress the lethal effects of high σM activity. Further analyses reveal that induction of the σM regulon leads to high level expression of membrane proteins that trigger envelope stress, and this stress is countered by specific genes in the σM regulon.

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DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped Actinomycete Corynebacterium glutamicum

Journal of Bacteriology ◽

10.1128/jb.01934-07 ◽

2008 ◽

Vol 190 (9) ◽

pp. 3283-3292 ◽

Cited By ~ 94

Author(s):

Michal Letek ◽

Efrén Ordóñez ◽

José Vaquera ◽

William Margolin ◽

Klas Flärdh ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Streptococcus Pneumoniae ◽

Corynebacterium Glutamicum ◽

Chromosome Segregation ◽

Polar Growth ◽

Cell Wall Synthesis ◽

Peptidoglycan Synthesis ◽

Polarized Cell Growth

ABSTRACT The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVACg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVACg-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

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Induction of autolysis in Bacillus subtilis by ochratoxin A

Canadian Journal of Microbiology ◽

10.1139/m78-091 ◽

1978 ◽

Vol 24 (5) ◽

pp. 563-568 ◽

Cited By ~ 4

Author(s):

U. Singer ◽

R. Röschenthaler

Keyword(s):

Cell Wall ◽

Protein Synthesis ◽

Bacillus Subtilis ◽

Ochratoxin A ◽

Optical Density ◽

Growth Phase ◽

Exponential Growth ◽

Rna Synthesis ◽

Exponential Growth Phase ◽

Cell Wall Synthesis

Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 μg/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 μg/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.

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Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis.

Journal of Bacteriology ◽

10.1128/jb.144.3.957-966.1980 ◽

1980 ◽

Vol 144 (3) ◽

pp. 957-966 ◽

Cited By ~ 15

Author(s):

R J Doyle ◽

U N Streips ◽

S Imada ◽

V S Fan ◽

W C Brown

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Genetic Transformation ◽

Deoxyribonucleic Acid

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Bacteriophage SP01 Gene Product 56 (gp56) Inhibits Bacillus subtilis Cell Division by Interacting with DivIC/FtsL to Prevent Pbp2B/FtsW Recruitment

10.1101/2020.02.06.938217 ◽

2020 ◽

Author(s):

Amit Bhambhani ◽

Isabella Iadicicco ◽

Jules Lee ◽

Syed Ahmed ◽

Max Belfatto ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Gene Product ◽

Lytic Bacteriophage ◽

Cell Wall Synthesis ◽

Ftsz Ring ◽

Ring Assembly ◽

Ring Shaped Structure ◽

Two Hybrid

ABSTRACTPrevious work identified gp56, encoded by the lytic bacteriophage SP01, as 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 gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits 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 analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause 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 mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

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MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis

eLife ◽

10.7554/elife.32471 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 75

Author(s):

Saman Hussain ◽

Carl N Wivagg ◽

Piotr Szwedziak ◽

Felix Wong ◽

Kaitlin Schaefer ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Radial Direction ◽

Membrane Curvature ◽

Biophysical Modeling ◽

Cell Wall Synthesis ◽

Peptidoglycan Synthesis ◽

Stable Maintenance ◽

Spherical Cells

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

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Regeneration of Escherichia coli Giant Protoplasts to Their Original Form

Life ◽

10.3390/life9010024 ◽

2019 ◽

Vol 9 (1) ◽

pp. 24 ◽

Cited By ~ 1

Author(s):

Kazuhito V. Tabata ◽

Takao Sogo ◽

Yoshiki Moriizumi ◽

Hiroyuki Noji

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Bacillus Subtilis ◽

Colony Formation ◽

Cell Walls ◽

Microscopic Investigation ◽

Original Form ◽

Cell Wall Synthesis ◽

E Coli ◽

The Relationship

The spheroplasts and protoplasts of cell wall-deficient (CWD) bacteria are able to revert to their original cellular morphologies through the regeneration of their cell walls. However, whether this is true for giant protoplasts (GPs), which can be as large as 10 μm in diameter, is unknown. GPs can be prepared from various bacteria, including Escherichia coli and Bacillus subtilis, and also from fungi, through culture in the presence of inhibitors for cell wall synthesis or mitosis. In this report, we prepared GPs from E. coli and showed that they can return to rod-shaped bacterium, and that they are capable of colony formation. Microscopic investigation revealed that the regeneration process took place through a variety of morphological pathways. We also report the relationship between GP division and GP volume. Finally, we show that FtsZ is crucial for GP division. These results indicate that E. coli is a highly robust organism that can regenerate its original form from an irregular state, such as GP.

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FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division

Nature Communications ◽

10.1038/s41467-021-22526-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kevin D. Whitley ◽

Calum Jukes ◽

Nicholas Tregidgo ◽

Eleni Karinou ◽

Pedro Almada ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Model Organism ◽

Live Cells ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Bacterial Physiology ◽

Z Ring ◽

Ultrahigh Sensitivity

AbstractDespite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

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Discovery of Novel Cell Wall-Active Compounds Using PywaC, a Sensitive Reporter of Cell Wall Stress, in the Model Gram-Positive Bacterium Bacillus subtilis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02352-14 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3261-3269 ◽

Cited By ~ 21

Author(s):

T. L. Czarny ◽

A. L. Perri ◽

S. French ◽

E. D. Brown

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Wall Stress ◽

Cell Wall Synthesis ◽

Reporter System ◽

Positive Bacterium ◽

Gram Positive ◽

Active Compounds ◽

Cell Wall Stress ◽

Antibiotic Drug

ABSTRACTThe emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacteriumBacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter ofywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds.

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