A Suppressor of Cell Death Caused by the Loss of σE Downregulates Extracytoplasmic Stress Responses and Outer Membrane Vesicle Production in Escherichia coli

ABSTRACT When envelope biogenesis is compromised or damage to envelope components occurs, bacteria trigger signaling cascades, which lead to the production of proteins that combat such extracytoplasmic stresses. In Escherichia coli, there are three pathways known to deal with envelope stresses: the Bae, Cpx, and σE responses. Although the effectors of the Bae and Cpx responses are not essential in E. coli, the effector of the σE response, the sigma factor RpoE (σE), is essential for viability. However, mutations that suppress the lethality of an rpoE-null allele can be easily obtained, and here we describe how we have isolated at least four classes of these suppressors. We present the first description of one such suppressor class, loss-of-function mutations in ydcQ, a gene encoding a putative DNA-binding protein. In wild-type rpoE + strains, ydcQ mutants have two distinct phenotypes: extracytoplasmic stress responses are significantly downregulated, and the production of outer membrane vesicles is severely reduced. We present a model in which σE is not essential per se but, rather, we propose that rpoE mutant cells die, possibly because they overreact to the absence of this σ factor by triggering a cell death signal.

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Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

Journal of Bacteriology ◽

10.1128/jb.00498-06 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5385-5392 ◽

Cited By ~ 191

Author(s):

Amanda J. McBroom ◽

Alexandra P. Johnson ◽

Sreekanth Vemulapalli ◽

Meta J. Kuehn

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Cell Envelope ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Fold Increase ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Physiological Basis ◽

Gram Negative

ABSTRACT It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the σE cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.

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Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content

Frontiers in Microbiology ◽

10.3389/fmicb.2021.628801 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shelby L. Reimer ◽

Daniel R. Beniac ◽

Shannon L. Hiebert ◽

Timothy F. Booth ◽

Patrick M. Chong ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Size Distributions ◽

Transmission Electron ◽

Isolation Methods ◽

Morphological Differences ◽

K 12

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating virulence factor dissemination and resistance to antimicrobials. Studies of OMV properties often focus on hypervesiculating Escherichia coli mutants that have increased OMV production when compared to their corresponding wild-type (WT) strains. Currently, two conventional techniques, ultracentrifugation (UC) and ultradiafiltration (UF), are used interchangeably to isolate OMVs, however, there is concern that each technique may inadvertently alter the properties of isolated OMVs during study. To address this concern, we compared two OMV isolation methods, UC and UF, with respect to final OMV quantities, size distributions, and morphologies using a hypervesiculating Escherichia coli K-12 ΔtolA mutant. Nanoparticle tracking analysis (NTA) indicated that UC techniques result in lower vesicle yields compared to UF. However, UF permitted isolation of OMVs with smaller average sizes than UC, highlighting a potential OMV isolation size bias by each technique. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between WT and ΔtolA OMVs, where ΔtolA OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes. Proteomic OMV analysis of WT and ΔtolA OMVs confirmed that ΔtolA enhances inner plasma membrane carryover in multi-lamellar OMVs. This study demonstrates that UC and UF are useful techniques for OMV isolation, where UF may be preferable due to faster isolation, higher OMV yields and enrichment of smaller sized vesicles.

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Abiotic stressors impact outer membrane vesicle composition in a beneficial rhizobacterium: Raman spectroscopy characterization

Scientific Reports ◽

10.1038/s41598-020-78357-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Matthew Potter ◽

Cynthia Hanson ◽

Anne J. Anderson ◽

Elizabeth Vargis ◽

David W. Britt

Keyword(s):

Raman Spectroscopy ◽

Nucleic Acids ◽

Outer Membrane ◽

Stress Responses ◽

Membrane Vesicle ◽

Plant Stress ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Linear Discriminant ◽

Abiotic Stressors

AbstractOuter membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacterium Pseudomonas chlororaphis O6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H2O2-released from roots during plant stress responses. Atomic force microscopy revealed 40–300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles of PcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids than PcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H2O2-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents.

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Active Cytotoxic Necrotizing Factor 1 Associated with Outer Membrane Vesicles from Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.74.4.2022-2030.2006 ◽

2006 ◽

Vol 74 (4) ◽

pp. 2022-2030 ◽

Cited By ~ 64

Author(s):

J. Clavin Kouokam ◽

Sun Nyunt Wai ◽

Maria Fällman ◽

Ulrich Dobrindt ◽

Jörg Hacker ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicle ◽

Active Form ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Upec Strain ◽

Cytotoxic Necrotizing Factor ◽

Factor Type ◽

Bacterial Cytoplasm

ABSTRACT Cytotoxic necrotizing factor type 1 (CNF1) is one of the virulence factors produced by uropathogenic Escherichia coli (UPEC). How this toxin is translocated from the bacterial cytoplasm to the surrounding environment is not well understood. Our data suggest that CNF1 may be regarded as a secreted protein, since it could be detected in culture supernatants. Furthermore, we found that CNF1 was tightly associated to outer membrane vesicles, suggesting that such vesicles play a role in the secretion of this protein. Interestingly, vesicle samples containing CNF1 could exert the effects known for this protein on HeLa cell cultures, showing that CNF1 is transported by vesicles in its active form. Taken together, our results strongly suggest that outer membrane vesicles could be a means for the bacteria to deliver CNF1 to the environment and to the infected tissue. In addition, our results indicate that the histone-like nucleoid structuring protein H-NS has a role in the downregulation of CNF1 production and that it affects the outer membrane vesicle release in UPEC strain J96.

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Outer membrane vesicles mediated horizontal transfer of an aerobic denitrification gene between Escherichia coli

Biodegradation ◽

10.1007/s10532-021-09945-y ◽

2021 ◽

Author(s):

Weichuan Qiao ◽

Lianjie Wang ◽

Yang Luo ◽

Jiahui Miao

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Horizontal Transfer ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Aerobic Denitrification ◽

Denitrification Gene

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Proteome‐minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform

Journal of Extracellular Vesicles ◽

10.1002/jev2.12066 ◽

2021 ◽

Vol 10 (4) ◽

Author(s):

Ilaria Zanella ◽

Enrico König ◽

Michele Tomasi ◽

Assunta Gagliardi ◽

Luca Frattini ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Vaccine Platform

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Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽

10.3390/nano11071661 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1661

Author(s):

Mei-Hsiu Chen ◽

Tse-Ying Liu ◽

Yu-Chiao Chen ◽

Ming-Hong Chen

Keyword(s):

Outer Membrane ◽

Membrane Vesicle ◽

Glioma Cells ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

E Coli ◽

Tumor Bearing ◽

Nano Gold ◽

Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

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Outer Membrane Machinery and Alginate Synthesis Regulators Control Membrane Vesicle Production in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00722-09 ◽

2009 ◽

Vol 191 (24) ◽

pp. 7509-7519 ◽

Cited By ~ 50

Author(s):

Yosuke Tashiro ◽

Ryosuke Sakai ◽

Masanori Toyofuku ◽

Isao Sawada ◽

Toshiaki Nakajima-Kambe ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Serine Proteases ◽

Membrane Vesicle ◽

Bacterial Pathogen ◽

Membrane Vesicles ◽

Production Mechanism ◽

Gene Encoding ◽

Surrounding Environment ◽

Regulatory Machinery

ABSTRACT The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.

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