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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

Stress induces explosive cell lysis.(a) Frequency of explosive cell lysis events in the absence (−FL) or presence (+FL) of fluorescence imaging, mean±s.e.m., #P<0.0001, Unpaired t-test with Welch's correction. (b) Phase-contrast (top) and TOTO-1-stained eDNA (green, bottom) of P. aeruginosa PAO1 interstitial biofilms cultured in the presence of filter discs saturated in water, MMC or CPFLX; scale bar, 5 μm. (c) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of P. aeruginosa PAO1 and PAO1ΔrecA interstitial biofilms cultured in the presence of filter discs saturated in water, or MMC; scale bar, 20 μm.
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f2: Stress induces explosive cell lysis.(a) Frequency of explosive cell lysis events in the absence (−FL) or presence (+FL) of fluorescence imaging, mean±s.e.m., #P<0.0001, Unpaired t-test with Welch's correction. (b) Phase-contrast (top) and TOTO-1-stained eDNA (green, bottom) of P. aeruginosa PAO1 interstitial biofilms cultured in the presence of filter discs saturated in water, MMC or CPFLX; scale bar, 5 μm. (c) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of P. aeruginosa PAO1 and PAO1ΔrecA interstitial biofilms cultured in the presence of filter discs saturated in water, or MMC; scale bar, 20 μm.

Mentions: Interestingly, we noted that fluorescence imaging in the presence of the eDNA stain TOTO-1 to detect eDNA release events yielded more frequent explosive cell lysis events than when fluorescence imaging was not involved (Fig. 2a) although the process appeared identical (Supplementary Movie 4). This suggests that phototoxicity due to exposure to high-intensity excitation light stimulated explosive cell lysis. To determine if other sources of exogenous stress induce explosive cell lysis, we examined the effect of exposing P. aeruginosa interstitial biofilms to antibiotic stress (ciprofloxacin; CPFLX) or genotoxic stress (mitomycin C; MMC). We found that as the biofilm approached CPFLX or MMC gradients, round cells were induced and large quantities of eDNA released through explosive cell lysis (Fig. 1b). To determine if induction of explosive cell lysis by these exogenous stresses is mediated by the RecA-mediated SOS stress response regulon of P. aeruginosa, we examined the response of a PAO1ΔrecA mutant and found no evidence of explosive cell lysis in this strain under either inducing or non-inducing conditions (Fig. 2c).


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)

Stress induces explosive cell lysis.(a) Frequency of explosive cell lysis events in the absence (−FL) or presence (+FL) of fluorescence imaging, mean±s.e.m., #P<0.0001, Unpaired t-test with Welch's correction. (b) Phase-contrast (top) and TOTO-1-stained eDNA (green, bottom) of P. aeruginosa PAO1 interstitial biofilms cultured in the presence of filter discs saturated in water, MMC or CPFLX; scale bar, 5 μm. (c) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of P. aeruginosa PAO1 and PAO1ΔrecA interstitial biofilms cultured in the presence of filter discs saturated in water, or MMC; scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Stress induces explosive cell lysis.(a) Frequency of explosive cell lysis events in the absence (−FL) or presence (+FL) of fluorescence imaging, mean±s.e.m., #P<0.0001, Unpaired t-test with Welch's correction. (b) Phase-contrast (top) and TOTO-1-stained eDNA (green, bottom) of P. aeruginosa PAO1 interstitial biofilms cultured in the presence of filter discs saturated in water, MMC or CPFLX; scale bar, 5 μm. (c) Phase-contrast (left) and TOTO-1-stained eDNA (green, right) of P. aeruginosa PAO1 and PAO1ΔrecA interstitial biofilms cultured in the presence of filter discs saturated in water, or MMC; scale bar, 20 μm.
Mentions: Interestingly, we noted that fluorescence imaging in the presence of the eDNA stain TOTO-1 to detect eDNA release events yielded more frequent explosive cell lysis events than when fluorescence imaging was not involved (Fig. 2a) although the process appeared identical (Supplementary Movie 4). This suggests that phototoxicity due to exposure to high-intensity excitation light stimulated explosive cell lysis. To determine if other sources of exogenous stress induce explosive cell lysis, we examined the effect of exposing P. aeruginosa interstitial biofilms to antibiotic stress (ciprofloxacin; CPFLX) or genotoxic stress (mitomycin C; MMC). We found that as the biofilm approached CPFLX or MMC gradients, round cells were induced and large quantities of eDNA released through explosive cell lysis (Fig. 1b). To determine if induction of explosive cell lysis by these exogenous stresses is mediated by the RecA-mediated SOS stress response regulon of P. aeruginosa, we examined the response of a PAO1ΔrecA mutant and found no evidence of explosive cell lysis in this strain under either inducing or non-inducing conditions (Fig. 2c).

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