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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 present within P. aeruginosa interstitial biofilms.(a) f3D-SIM of PAK biofilms cultured in the presence of FM1-43FX (white), scale bar, 1 μm. (b) Frequency distribution of diameters of MVs measured in situ in live PAK biofilms (n=268, bin size=50 nm). (c) f3D-SIM of PAK biofilms cultured in the presence FM1-43FX (blue) and EthHD-2 (red); scale bar, 2 μm. (d) Quantification of MVs in random fields of view (40 μm × 40 μm) of PAO1 (n=35) and PAO1Δlys (n=22) biofilms cultured in the presence of FM1-43FX and imaged with f3D-SIM.
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f5: MVs are present within P. aeruginosa interstitial biofilms.(a) f3D-SIM of PAK biofilms cultured in the presence of FM1-43FX (white), scale bar, 1 μm. (b) Frequency distribution of diameters of MVs measured in situ in live PAK biofilms (n=268, bin size=50 nm). (c) f3D-SIM of PAK biofilms cultured in the presence FM1-43FX (blue) and EthHD-2 (red); scale bar, 2 μm. (d) Quantification of MVs in random fields of view (40 μm × 40 μm) of PAO1 (n=35) and PAO1Δlys (n=22) biofilms cultured in the presence of FM1-43FX and imaged with f3D-SIM.

Mentions: To explore the role of explosive cell lysis in mediating eDNA release during the development of submerged biofilms, we examined biofilm formation by wild-type PAO1 and the endolysin mutant PAO1Δlys after 8 h of culture. PAO1 produced numerous microcolony structures that stained with the eDNA stains EtHD-2 (Fig. 4b,c) or TOTO-1 (Supplementary Fig. 4a). Surface-attached round cells were also visible at this time point (Fig. 4b inset). PAO1Δlys was found to be severely defective in biofilm formation and showed no microcolony structures or round cells (Fig. 4b,c). Wild-type PAO1 biofilms cultured in the presence of DNaseI, produced no microcolonies confirming a requirement for eDNA in submerged biofilm development under these assay conditions (Fig. 4b,c). The defects in microcolony development and round cell formation in PAO1Δlys could be complemented with lys provided in trans (Fig. 5d; Supplementary Fig. 4b). These observations indicate that explosive cell lysis mediated via the endolysin Lys is responsible for the release of eDNA required for the formation of submerged biofilms by P. aeruginosa.


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 present within P. aeruginosa interstitial biofilms.(a) f3D-SIM of PAK biofilms cultured in the presence of FM1-43FX (white), scale bar, 1 μm. (b) Frequency distribution of diameters of MVs measured in situ in live PAK biofilms (n=268, bin size=50 nm). (c) f3D-SIM of PAK biofilms cultured in the presence FM1-43FX (blue) and EthHD-2 (red); scale bar, 2 μm. (d) Quantification of MVs in random fields of view (40 μm × 40 μm) of PAO1 (n=35) and PAO1Δlys (n=22) biofilms cultured in the presence of FM1-43FX and imaged with f3D-SIM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: MVs are present within P. aeruginosa interstitial biofilms.(a) f3D-SIM of PAK biofilms cultured in the presence of FM1-43FX (white), scale bar, 1 μm. (b) Frequency distribution of diameters of MVs measured in situ in live PAK biofilms (n=268, bin size=50 nm). (c) f3D-SIM of PAK biofilms cultured in the presence FM1-43FX (blue) and EthHD-2 (red); scale bar, 2 μm. (d) Quantification of MVs in random fields of view (40 μm × 40 μm) of PAO1 (n=35) and PAO1Δlys (n=22) biofilms cultured in the presence of FM1-43FX and imaged with f3D-SIM.
Mentions: To explore the role of explosive cell lysis in mediating eDNA release during the development of submerged biofilms, we examined biofilm formation by wild-type PAO1 and the endolysin mutant PAO1Δlys after 8 h of culture. PAO1 produced numerous microcolony structures that stained with the eDNA stains EtHD-2 (Fig. 4b,c) or TOTO-1 (Supplementary Fig. 4a). Surface-attached round cells were also visible at this time point (Fig. 4b inset). PAO1Δlys was found to be severely defective in biofilm formation and showed no microcolony structures or round cells (Fig. 4b,c). Wild-type PAO1 biofilms cultured in the presence of DNaseI, produced no microcolonies confirming a requirement for eDNA in submerged biofilm development under these assay conditions (Fig. 4b,c). The defects in microcolony development and round cell formation in PAO1Δlys could be complemented with lys provided in trans (Fig. 5d; Supplementary Fig. 4b). These observations indicate that explosive cell lysis mediated via the endolysin Lys is responsible for the release of eDNA required for the formation of submerged biofilms by P. aeruginosa.

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