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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.


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PQS is not required for MV production in interstitial biofilms.(a) f3D-SIM of PA14pqsA and PAO1pqsA biofilms cultured in the presence of FM1-43FX (white) showing MV patches; scale bar, 1 μm. (b) Quantification of MVs in random fields of view (40 μm × 40 μm) of PA14 (n=54), PA14pqsA (n=60), PAO1 (n=22) and PAO1pqsA (n=24); #P<0.0001, unpaired t-test with Welch's correction.
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f8: PQS is not required for MV production in interstitial biofilms.(a) f3D-SIM of PA14pqsA and PAO1pqsA biofilms cultured in the presence of FM1-43FX (white) showing MV patches; scale bar, 1 μm. (b) Quantification of MVs in random fields of view (40 μm × 40 μm) of PA14 (n=54), PA14pqsA (n=60), PAO1 (n=22) and PAO1pqsA (n=24); #P<0.0001, unpaired t-test with Welch's correction.

Mentions: We have found that explosive cell lysis is associated with the production of MVs in interstitial biofilms and planktonic cultures of P. aeruginosa. The Pseudomonas Quinolone Signal (PQS) has been reported to be crucial for MV production in P. aeruginosa30; whereas a number of other studies have found that PQS is not required for MV production in planktonic cultures under stressed or unstressed conditions313233. Therefore, to further explore the contribution of PQS to MV biogenesis we analysed MV production in interstitial biofilms of pqsA mutants which do not produce PQS34. These assays showed that the pqsA mutants of P. aeruginosa strains PAO1 and PA14 were not defective in the production of MVs in interstitial biofilms relative to their isogenic parent strains (Fig. 8). In fact PA14pqsA produced significantly more MVs than its isogenic parent in this assay. Furthermore, our RNA-seq analyses of MVs obtained from unstressed planktonic cultures revealed that the small RNA PA3305.1 (PhrS), which has been shown to stimulate Pseudomonas quinolone signal (PQS) production35, is less abundant in planktonic MVs than planktonic cells. This is consistent with PQS not having a critical role in the production of MVs with RNA cargo which have presumably been derived through explosive cell lysis. These observations indicate that explosive cell lysis-mediated MV production in biofilms and planktonic cultures is independent of PQS.


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)

PQS is not required for MV production in interstitial biofilms.(a) f3D-SIM of PA14pqsA and PAO1pqsA biofilms cultured in the presence of FM1-43FX (white) showing MV patches; scale bar, 1 μm. (b) Quantification of MVs in random fields of view (40 μm × 40 μm) of PA14 (n=54), PA14pqsA (n=60), PAO1 (n=22) and PAO1pqsA (n=24); #P<0.0001, unpaired t-test with Welch's correction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: PQS is not required for MV production in interstitial biofilms.(a) f3D-SIM of PA14pqsA and PAO1pqsA biofilms cultured in the presence of FM1-43FX (white) showing MV patches; scale bar, 1 μm. (b) Quantification of MVs in random fields of view (40 μm × 40 μm) of PA14 (n=54), PA14pqsA (n=60), PAO1 (n=22) and PAO1pqsA (n=24); #P<0.0001, unpaired t-test with Welch's correction.
Mentions: We have found that explosive cell lysis is associated with the production of MVs in interstitial biofilms and planktonic cultures of P. aeruginosa. The Pseudomonas Quinolone Signal (PQS) has been reported to be crucial for MV production in P. aeruginosa30; whereas a number of other studies have found that PQS is not required for MV production in planktonic cultures under stressed or unstressed conditions313233. Therefore, to further explore the contribution of PQS to MV biogenesis we analysed MV production in interstitial biofilms of pqsA mutants which do not produce PQS34. These assays showed that the pqsA mutants of P. aeruginosa strains PAO1 and PA14 were not defective in the production of MVs in interstitial biofilms relative to their isogenic parent strains (Fig. 8). In fact PA14pqsA produced significantly more MVs than its isogenic parent in this assay. Furthermore, our RNA-seq analyses of MVs obtained from unstressed planktonic cultures revealed that the small RNA PA3305.1 (PhrS), which has been shown to stimulate Pseudomonas quinolone signal (PQS) production35, is less abundant in planktonic MVs than planktonic cells. This is consistent with PQS not having a critical role in the production of MVs with RNA cargo which have presumably been derived through explosive cell lysis. These observations indicate that explosive cell lysis-mediated MV production in biofilms and planktonic cultures is independent of PQS.

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