Limits...
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

Pyocin endolysin Lys is required for eDNA release in interstitial biofilms under inducing and non-inducing conditions.(a) and (b) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of interstitial biofilms of (a) PAK and PAKΔlys and (b) PAO1 and PAO1Δlys containing either pJN105 (vector control) or pJN105lys cultured in the presence of filter discs saturated in water or MMC; scale bar, 10 μm. (c) Lys catalytic activity is required for eDNA release in P. aeruginosa PAO1 interstitial biofilms; n=30; mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (d) Time series of cells in an interstitial biofilm of P. aeruginosa PAO1 (phase-contrast, upper panels) containing the Phol-eGFP transcriptional fusion on plasmid pM0614-G (green, lower panels). Time in seconds; scale bar, 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4834629&req=5

f3: Pyocin endolysin Lys is required for eDNA release in interstitial biofilms under inducing and non-inducing conditions.(a) and (b) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of interstitial biofilms of (a) PAK and PAKΔlys and (b) PAO1 and PAO1Δlys containing either pJN105 (vector control) or pJN105lys cultured in the presence of filter discs saturated in water or MMC; scale bar, 10 μm. (c) Lys catalytic activity is required for eDNA release in P. aeruginosa PAO1 interstitial biofilms; n=30; mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (d) Time series of cells in an interstitial biofilm of P. aeruginosa PAO1 (phase-contrast, upper panels) containing the Phol-eGFP transcriptional fusion on plasmid pM0614-G (green, lower panels). Time in seconds; scale bar, 1 μm.

Mentions: We therefore explored the possibility that the putative endolysin Lys may be responsible for eDNA release through explosive cell lysis in P. aeruginosa biofilms. We found that PAO1Δlys and PAKΔlys mutants were significantly abrogated in the explosive cell lysis-mediated release of eDNA in interstitial biofilms under both inducing and non-inducing conditions (Fig. 3a,b). Explosive cell lysis was restored with wild-type lys provided in trans but not by the mutant allele lys* that encodes an E51V substitution in the putative active site (Fig. 3b,c; Supplementary Fig. 2), indicating that the endolytic activity of Lys is critical for explosive cell lysis in these interstitial biofilms. Furthermore, PAKΔlys interstitial biofilms lacked the intricate trail networks that are a characteristic feature of wild-type PAK P. aeruginosa interstitial biofilms (Fig. 3a) and appeared morphologically similar to PAK interstitial biofilms cultured in the presence of DNaseI7. This indicates that Lys-mediated explosive cell lysis is likely to be the major source of eDNA that is required for self-organization of these interstitial biofilms.


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)

Pyocin endolysin Lys is required for eDNA release in interstitial biofilms under inducing and non-inducing conditions.(a) and (b) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of interstitial biofilms of (a) PAK and PAKΔlys and (b) PAO1 and PAO1Δlys containing either pJN105 (vector control) or pJN105lys cultured in the presence of filter discs saturated in water or MMC; scale bar, 10 μm. (c) Lys catalytic activity is required for eDNA release in P. aeruginosa PAO1 interstitial biofilms; n=30; mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (d) Time series of cells in an interstitial biofilm of P. aeruginosa PAO1 (phase-contrast, upper panels) containing the Phol-eGFP transcriptional fusion on plasmid pM0614-G (green, lower panels). Time in seconds; scale bar, 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Pyocin endolysin Lys is required for eDNA release in interstitial biofilms under inducing and non-inducing conditions.(a) and (b) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of interstitial biofilms of (a) PAK and PAKΔlys and (b) PAO1 and PAO1Δlys containing either pJN105 (vector control) or pJN105lys cultured in the presence of filter discs saturated in water or MMC; scale bar, 10 μm. (c) Lys catalytic activity is required for eDNA release in P. aeruginosa PAO1 interstitial biofilms; n=30; mean±s.e.m. #P<0.0001, unpaired t-test with Welch's correction. (d) Time series of cells in an interstitial biofilm of P. aeruginosa PAO1 (phase-contrast, upper panels) containing the Phol-eGFP transcriptional fusion on plasmid pM0614-G (green, lower panels). Time in seconds; scale bar, 1 μm.
Mentions: We therefore explored the possibility that the putative endolysin Lys may be responsible for eDNA release through explosive cell lysis in P. aeruginosa biofilms. We found that PAO1Δlys and PAKΔlys mutants were significantly abrogated in the explosive cell lysis-mediated release of eDNA in interstitial biofilms under both inducing and non-inducing conditions (Fig. 3a,b). Explosive cell lysis was restored with wild-type lys provided in trans but not by the mutant allele lys* that encodes an E51V substitution in the putative active site (Fig. 3b,c; Supplementary Fig. 2), indicating that the endolytic activity of Lys is critical for explosive cell lysis in these interstitial biofilms. Furthermore, PAKΔlys interstitial biofilms lacked the intricate trail networks that are a characteristic feature of wild-type PAK P. aeruginosa interstitial biofilms (Fig. 3a) and appeared morphologically similar to PAK interstitial biofilms cultured in the presence of DNaseI7. This indicates that Lys-mediated explosive cell lysis is likely to be the major source of eDNA that is required for self-organization of these interstitial biofilms.

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