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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

Explosive cell lysis is required for microcolony development in submerged hydrated biofilms.(a) Time series of the initial stages of PAO1 biofilm development 1 h after inoculation showing attachment of a rod cell, its transition to round cell morphotype and subsequent explosion releasing eDNA (TOTO-1, green). Time in min (top right); scale bar, 5 μm. (b,c) Microcolonies in 8-h submerged biofilms of PAO1 (upper), PAO1Δlys (middle), and PAO1 cultured in the presence of DNaseI (lower). (b) Representative phase contrast (left) and eDNA (EthHD-2, right) images; scale bar, 10 μm. Inset shows magnified view of round cell at arrow-head (c) Microcolonies in 8-h submerged biofilms per mm2, n=30. Mean±s.e.m. *P<0.0001, unpaired t-test with Welch's correction. (d) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys carrying either pJN105 or pJN105lys, n=30. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (e) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys cultured in the absence or presence of exogenous DNA (exDNA), n=20. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction.
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f4: Explosive cell lysis is required for microcolony development in submerged hydrated biofilms.(a) Time series of the initial stages of PAO1 biofilm development 1 h after inoculation showing attachment of a rod cell, its transition to round cell morphotype and subsequent explosion releasing eDNA (TOTO-1, green). Time in min (top right); scale bar, 5 μm. (b,c) Microcolonies in 8-h submerged biofilms of PAO1 (upper), PAO1Δlys (middle), and PAO1 cultured in the presence of DNaseI (lower). (b) Representative phase contrast (left) and eDNA (EthHD-2, right) images; scale bar, 10 μm. Inset shows magnified view of round cell at arrow-head (c) Microcolonies in 8-h submerged biofilms per mm2, n=30. Mean±s.e.m. *P<0.0001, unpaired t-test with Welch's correction. (d) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys carrying either pJN105 or pJN105lys, n=30. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (e) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys cultured in the absence or presence of exogenous DNA (exDNA), n=20. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction.

Mentions: We have shown previously that eDNA plays an essential role in the early stages of the development of P. aeruginosa biofilms that are formed on abiotic surfaces submerged in liquid nutrient media9. To determine if explosive cell lysis accounts for eDNA release during the initial stages of the development of submerged biofilms, we performed live-cell imaging of the very early stages of biofilm development and observed the formation and explosion of round cells in both the planktonic phase and at the surface (Fig. 4a; Supplementary Movie 6). In contrast, we were unable to observe any round cells or explosive cell lysis events in PAO1Δlys.


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)

Explosive cell lysis is required for microcolony development in submerged hydrated biofilms.(a) Time series of the initial stages of PAO1 biofilm development 1 h after inoculation showing attachment of a rod cell, its transition to round cell morphotype and subsequent explosion releasing eDNA (TOTO-1, green). Time in min (top right); scale bar, 5 μm. (b,c) Microcolonies in 8-h submerged biofilms of PAO1 (upper), PAO1Δlys (middle), and PAO1 cultured in the presence of DNaseI (lower). (b) Representative phase contrast (left) and eDNA (EthHD-2, right) images; scale bar, 10 μm. Inset shows magnified view of round cell at arrow-head (c) Microcolonies in 8-h submerged biofilms per mm2, n=30. Mean±s.e.m. *P<0.0001, unpaired t-test with Welch's correction. (d) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys carrying either pJN105 or pJN105lys, n=30. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (e) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys cultured in the absence or presence of exogenous DNA (exDNA), n=20. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction.
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Related In: Results  -  Collection

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f4: Explosive cell lysis is required for microcolony development in submerged hydrated biofilms.(a) Time series of the initial stages of PAO1 biofilm development 1 h after inoculation showing attachment of a rod cell, its transition to round cell morphotype and subsequent explosion releasing eDNA (TOTO-1, green). Time in min (top right); scale bar, 5 μm. (b,c) Microcolonies in 8-h submerged biofilms of PAO1 (upper), PAO1Δlys (middle), and PAO1 cultured in the presence of DNaseI (lower). (b) Representative phase contrast (left) and eDNA (EthHD-2, right) images; scale bar, 10 μm. Inset shows magnified view of round cell at arrow-head (c) Microcolonies in 8-h submerged biofilms per mm2, n=30. Mean±s.e.m. *P<0.0001, unpaired t-test with Welch's correction. (d) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys carrying either pJN105 or pJN105lys, n=30. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (e) Microcolonies per mm2 in 8-h submerged hydrated biofilms of PAO1 and PAO1Δlys cultured in the absence or presence of exogenous DNA (exDNA), n=20. Mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction.
Mentions: We have shown previously that eDNA plays an essential role in the early stages of the development of P. aeruginosa biofilms that are formed on abiotic surfaces submerged in liquid nutrient media9. To determine if explosive cell lysis accounts for eDNA release during the initial stages of the development of submerged biofilms, we performed live-cell imaging of the very early stages of biofilm development and observed the formation and explosion of round cells in both the planktonic phase and at the surface (Fig. 4a; Supplementary Movie 6). In contrast, we were unable to observe any round cells or explosive cell lysis events in PAO1Δlys.

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