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

Turnbull L, Toyofuku M, Hynen AL, Kurosawa M, Pessi G, Petty NK, Osvath SR, Cárcamo-Oyarce G, Gloag ES, Shimoni R, Omasits U, Ito S, Yap X, Monahan LG, Cavaliere R, Ahrens CH, Charles IG, Nomura N, Eberl L, Whitchurch CB - Nat Commun (2016)

Bottom Line: 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.Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs.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.

View Article: PubMed Central - PubMed

Affiliation: The ithree institute, University of Technology Sydney, Ultimo, New South Wales 2007, Australia.

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.

No MeSH data available.


Related in: MedlinePlus

MVs are produced as a consequence of explosive cell lysis in P. aeruginosa biofilms.(a,b) f3D-SIM time-series of live interstitial biofilms in the presence of FM1-43FX (white). Time in seconds, top right; scale bar, 0.5 μm. (c) f3D-SIM of P. aeruginosa PAK-expressing mChFP (red) in the presence of FM1-43FX (blue). xy (left) and corresponding yz (right) views showing a large MV containing mChFP (arrow); scale bar, 0.5 μm. (d) f3D-SIM of live PAK interstitial biofilms in the presence of FM1-43FX (blue) and EthHD-2 (red). xy (upper) and corresponding xz (lower) views showing a large MV containing eDNA (arrow); scale bar, 0.5 μm
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f6: MVs are produced as a consequence of explosive cell lysis in P. aeruginosa biofilms.(a,b) f3D-SIM time-series of live interstitial biofilms in the presence of FM1-43FX (white). Time in seconds, top right; scale bar, 0.5 μm. (c) f3D-SIM of P. aeruginosa PAK-expressing mChFP (red) in the presence of FM1-43FX (blue). xy (left) and corresponding yz (right) views showing a large MV containing mChFP (arrow); scale bar, 0.5 μm. (d) f3D-SIM of live PAK interstitial biofilms in the presence of FM1-43FX (blue) and EthHD-2 (red). xy (upper) and corresponding xz (lower) views showing a large MV containing eDNA (arrow); scale bar, 0.5 μm

Mentions: In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane1. It is conceivable that the transition from rod-shaped to round cells caused by the putative peptidoglycan hydrolase activity of the Lys endolysin is concomitant with weakening of the connection between the outer membrane and the peptidoglycan of the cell wall and could therefore be associated with the release of MVs. To examine the biogenesis of MVs by P. aeruginosa we used f3D-SIM to follow MV production in live interstitial biofilms to ascertain if blebbing occurs during or after round cell transition. Surprisingly, f3D-SIM revealed that MVs did not appear to be formed through membrane blebbing from either rod-shaped or round cells, but instead were derived from shattered membrane fragments that were produced as a consequence of explosive cell lysis (Fig. 6a,b; Supplementary Movies 9 and 10). The rate of vesicularization of membrane fragments produced through cellular explosions was extremely rapid and often too dynamic to capture by f3D-SIM, which requires objects to be stationary within the period of each image acquisition (∼1 s). However, we were occasionally able to capture the formation of MVs that formed more slowly and found that these were formed via the curling and self-annealing of membrane fragments produced after explosive cell lysis (Fig. 6b; Supplementary Movie 10).


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

Turnbull L, Toyofuku M, Hynen AL, Kurosawa M, Pessi G, Petty NK, Osvath SR, Cárcamo-Oyarce G, Gloag ES, Shimoni R, Omasits U, Ito S, Yap X, Monahan LG, Cavaliere R, Ahrens CH, Charles IG, Nomura N, Eberl L, Whitchurch CB - Nat Commun (2016)

MVs are produced as a consequence of explosive cell lysis in P. aeruginosa biofilms.(a,b) f3D-SIM time-series of live interstitial biofilms in the presence of FM1-43FX (white). Time in seconds, top right; scale bar, 0.5 μm. (c) f3D-SIM of P. aeruginosa PAK-expressing mChFP (red) in the presence of FM1-43FX (blue). xy (left) and corresponding yz (right) views showing a large MV containing mChFP (arrow); scale bar, 0.5 μm. (d) f3D-SIM of live PAK interstitial biofilms in the presence of FM1-43FX (blue) and EthHD-2 (red). xy (upper) and corresponding xz (lower) views showing a large MV containing eDNA (arrow); scale bar, 0.5 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834629&req=5

f6: MVs are produced as a consequence of explosive cell lysis in P. aeruginosa biofilms.(a,b) f3D-SIM time-series of live interstitial biofilms in the presence of FM1-43FX (white). Time in seconds, top right; scale bar, 0.5 μm. (c) f3D-SIM of P. aeruginosa PAK-expressing mChFP (red) in the presence of FM1-43FX (blue). xy (left) and corresponding yz (right) views showing a large MV containing mChFP (arrow); scale bar, 0.5 μm. (d) f3D-SIM of live PAK interstitial biofilms in the presence of FM1-43FX (blue) and EthHD-2 (red). xy (upper) and corresponding xz (lower) views showing a large MV containing eDNA (arrow); scale bar, 0.5 μm
Mentions: In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane1. It is conceivable that the transition from rod-shaped to round cells caused by the putative peptidoglycan hydrolase activity of the Lys endolysin is concomitant with weakening of the connection between the outer membrane and the peptidoglycan of the cell wall and could therefore be associated with the release of MVs. To examine the biogenesis of MVs by P. aeruginosa we used f3D-SIM to follow MV production in live interstitial biofilms to ascertain if blebbing occurs during or after round cell transition. Surprisingly, f3D-SIM revealed that MVs did not appear to be formed through membrane blebbing from either rod-shaped or round cells, but instead were derived from shattered membrane fragments that were produced as a consequence of explosive cell lysis (Fig. 6a,b; Supplementary Movies 9 and 10). The rate of vesicularization of membrane fragments produced through cellular explosions was extremely rapid and often too dynamic to capture by f3D-SIM, which requires objects to be stationary within the period of each image acquisition (∼1 s). However, we were occasionally able to capture the formation of MVs that formed more slowly and found that these were formed via the curling and self-annealing of membrane fragments produced after explosive cell lysis (Fig. 6b; Supplementary Movie 10).

Bottom Line: 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.Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs.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.

View Article: PubMed Central - PubMed

Affiliation: The ithree institute, University of Technology Sydney, Ultimo, New South Wales 2007, Australia.

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.

No MeSH data available.


Related in: MedlinePlus