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      Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

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          Abstract

          Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

          Abstract

          Many bacteria release DNA and membrane vesicles through unclear mechanisms. Here, the authors show that a prophage endolysin is involved in the explosive lysis of a sub-population of cells in Pseudomonas aeruginosa, releasing cytoplasmic content and membrane fragments that rapidly form membrane vesicles.

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          Most cited references36

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          Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion.

          Pseudomonas aeruginosa blebs-off membrane vesicles (MVs) into culture medium during normal growth. Release of these vesicles increased approximately threefold after exposure of the organism to four times the MIC of gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs and g-MVs, respectively) were isolated by filtration and differential centrifugation, and several of their biological activities were characterized. Electron microscopy of both n-MVs and g-MVs revealed that they were spherical bilayer MVs with a diameter of 50 to 150 nm. Immunoelectron microscopy and Western blot (immunoblot) analysis of the vesicles demonstrated the presence of B-band lipopolysaccharide (LPS), with a slightly higher proportion of B-band LPS in g-MVs than in n-MVs. A-band LPS was occasionally detected in g-MVs but not in n-MVs. In addition to LPS, several enzymes, such as phospholipase C, protease, hemolysin, and alkaline phosphatase, which are known to contribute to the pathogenicity of Pseudomonas infections were found to be present in both vesicle types. Both types of vesicles contained DNA, with a significantly higher content in g-MVs. These vesicles could thus play an important role in genetic transformation and disease by serving as a transport vehicle for DNA and virulence factors and are presumably involved in septic shock.
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            The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms.

            Abstract The significance of extracellular DNA (eDNA) in biofilms was overlooked until researchers added DNAse to a Pseudomonas aeruginosa biofilm and watched the biofilm disappear. Now, a decade later, the widespread importance of eDNA in biofilm formation is undisputed, but detailed knowledge about how it promotes biofilm formation and conveys antimicrobial resistance is only just starting to emerge. In this review, we discuss how eDNA is produced, how it aids bacterial adhesion, secures the structural stability of biofilms and contributes to antimicrobial resistance. The appearance of eDNA in biofilms is no accident: It is produced by active secretion or controlled cell lysis - sometimes linked to competence development. eDNA adsorbs to and extends from the cell surface, promoting adhesion to abiotic surfaces through acid-base interactions. In the biofilm, is it less clear how eDNA interacts with cells and matrix components. A few eDNA-binding biomolecules have been identified, revealing new concepts in biofilm formation. Being anionic, eDNA chelates cations and restricts diffusion of cationic antimicrobials. Furthermore, chelation of Mg(2+) triggers a genetic response that further increases resistance. The multifaceted role of eDNA makes it an attractive target to sensitize biofilms to conventional antimicrobial treatment or development of new strategies to combat biofilms.
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              Pervasive domestication of defective prophages by bacteria.

              Integrated phages (prophages) are major contributors to the diversity of bacterial gene repertoires. Domestication of their components is thought to have endowed bacteria with molecular systems involved in secretion, defense, warfare, and gene transfer. However, the rates and mechanisms of domestication remain unknown. We used comparative genomics to study the evolution of prophages within the bacterial genome. We identified over 300 vertically inherited prophages within enterobacterial genomes. Some of these elements are very old and might predate the split between Escherichia coli and Salmonella enterica. The size distribution of prophage elements is bimodal, suggestive of rapid prophage inactivation followed by much slower genetic degradation. Accordingly, we observed a pervasive pattern of systematic counterselection of nonsynonymous mutations in prophage genes. Importantly, such patterns of purifying selection are observed not only on accessory regions but also in core phage genes, such as those encoding structural and lysis components. This suggests that bacterial hosts select for phage-associated functions. Several of these conserved prophages have gene repertoires compatible with described functions of adaptive prophage-derived elements such as bacteriocins, killer particles, gene transfer agents, or satellite prophages. We suggest that bacteria frequently domesticate their prophages. Most such domesticated elements end up deleted from the bacterial genome because they are replaced by analogous functions carried by new prophages. This puts the bacterial genome in a state of continuous flux of acquisition and loss of phage-derived adaptive genes.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                14 April 2016
                2016
                : 7
                : 11220
                Affiliations
                [1 ]The ithree institute, University of Technology Sydney , Ultimo, New South Wales 2007, Australia
                [2 ]Department of Life and Environmental Sciences, University of Tsukuba , Tsukuba, Ibaraki 305-8572, Japan
                [3 ]Department of Plant and Microbial Biology, University of Zurich , Zürich 8008, Switzerland
                [4 ]Department of Biology, Institute of Molecular Systems Biology, ETH Zurich , Zürich 8093, Switzerland
                [5 ]Agroscope, Institute for Plant Production Sciences, Research Group Molecular Diagnostics, Genomics and Bioinformatics, & Swiss Institute of Bioinformatics (SIB) , Wädenswil 8820, Switzerland
                Author notes
                Article
                ncomms11220
                10.1038/ncomms11220
                4834629
                27075392
                cfb207ad-ba1c-44df-995d-bea67a825fe8
                Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 06 September 2015
                : 02 March 2016
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