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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

Lys is involved in stress-induced MV formation of planktonic cells.(a) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under oxic planktonic growth conditions. n=3; mean±s.d. (b) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under anoxic planktonic growth conditions. Values indicate the mean±s.d. of three replicates. n=3; mean±s.d. #P<0.001 versus wild type (WT) (Student's t-test). (c) MV production by planktonic P. aeruginosa PAO1 and isogenic mutants cultured in the presence of MMC (200ngmL−1) relative to no MMC, n=3; mean±s.d. #P<0.0005 (Student's t-test). (d) Catalytic activity of Lys is required for genotoxic stress-induced MV formation, n=3; mean±s.d. #P<0.0005 (Student's t test). (e) MA plot showing the comparison of mRNA levels associated with MVs with the transcript levels of stationary phase cells. More and less abundant transcripts in MVs are indicated by red and green dots, respectively (P value <0.02). Transcripts from the pyocin gene cluster (PA0610 to PA0648) are circled in black. (f) Promoter activities of recA, hol and lacZ (control) under non-inducing conditions were monitored by the aid of plasmids containing transcriptional fusions of the respective promoter regions to eGFP. Cells expressing GFP are green; scale bar, 2.5 μm.
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f7: Lys is involved in stress-induced MV formation of planktonic cells.(a) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under oxic planktonic growth conditions. n=3; mean±s.d. (b) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under anoxic planktonic growth conditions. Values indicate the mean±s.d. of three replicates. n=3; mean±s.d. #P<0.001 versus wild type (WT) (Student's t-test). (c) MV production by planktonic P. aeruginosa PAO1 and isogenic mutants cultured in the presence of MMC (200ngmL−1) relative to no MMC, n=3; mean±s.d. #P<0.0005 (Student's t-test). (d) Catalytic activity of Lys is required for genotoxic stress-induced MV formation, n=3; mean±s.d. #P<0.0005 (Student's t test). (e) MA plot showing the comparison of mRNA levels associated with MVs with the transcript levels of stationary phase cells. More and less abundant transcripts in MVs are indicated by red and green dots, respectively (P value <0.02). Transcripts from the pyocin gene cluster (PA0610 to PA0648) are circled in black. (f) Promoter activities of recA, hol and lacZ (control) under non-inducing conditions were monitored by the aid of plasmids containing transcriptional fusions of the respective promoter regions to eGFP. Cells expressing GFP are green; scale bar, 2.5 μm.

Mentions: Our observations have shown that explosive cell lysis mediated by the endolysin Lys is required for MV biogenesis in P. aeruginosa biofilms. We also examined the role of Lys in producing MVs in planktonic culture. Interestingly, PAO1Δlys showed similar levels of MV production as the wild type under normal oxic growth in liquid medium (Fig. 7a). However, the importance of Lys in MV production was evident under conditions that stimulate MV production through induction of the SOS response such as anoxic growth or exposure to MMC4 (Fig. 7b,c). We found that when either recA or lys was inactivated, stress-induced MV formation was also greatly impaired whereas inactivation of pyocin structural components (pyocin tail, tail fibre or tail sheath)4, had no effect on MV formation (Fig. 7b,c). The defects in MMC-induced MV production by PAO1Δlys could be rescued by complementation with the wild-type lys but not the catalytic lys* mutant allele (Fig. 7d). Promoter-reporter eGFP fusions show that PrecA and Phol were also significantly induced by exposure to MMC (Supplementary Fig. 5).


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)

Lys is involved in stress-induced MV formation of planktonic cells.(a) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under oxic planktonic growth conditions. n=3; mean±s.d. (b) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under anoxic planktonic growth conditions. Values indicate the mean±s.d. of three replicates. n=3; mean±s.d. #P<0.001 versus wild type (WT) (Student's t-test). (c) MV production by planktonic P. aeruginosa PAO1 and isogenic mutants cultured in the presence of MMC (200ngmL−1) relative to no MMC, n=3; mean±s.d. #P<0.0005 (Student's t-test). (d) Catalytic activity of Lys is required for genotoxic stress-induced MV formation, n=3; mean±s.d. #P<0.0005 (Student's t test). (e) MA plot showing the comparison of mRNA levels associated with MVs with the transcript levels of stationary phase cells. More and less abundant transcripts in MVs are indicated by red and green dots, respectively (P value <0.02). Transcripts from the pyocin gene cluster (PA0610 to PA0648) are circled in black. (f) Promoter activities of recA, hol and lacZ (control) under non-inducing conditions were monitored by the aid of plasmids containing transcriptional fusions of the respective promoter regions to eGFP. Cells expressing GFP are green; scale bar, 2.5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f7: Lys is involved in stress-induced MV formation of planktonic cells.(a) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under oxic planktonic growth conditions. n=3; mean±s.d. (b) MV production in P. aeruginosa PAO1 and isogenic mutants were analysed after 16 h of incubation under anoxic planktonic growth conditions. Values indicate the mean±s.d. of three replicates. n=3; mean±s.d. #P<0.001 versus wild type (WT) (Student's t-test). (c) MV production by planktonic P. aeruginosa PAO1 and isogenic mutants cultured in the presence of MMC (200ngmL−1) relative to no MMC, n=3; mean±s.d. #P<0.0005 (Student's t-test). (d) Catalytic activity of Lys is required for genotoxic stress-induced MV formation, n=3; mean±s.d. #P<0.0005 (Student's t test). (e) MA plot showing the comparison of mRNA levels associated with MVs with the transcript levels of stationary phase cells. More and less abundant transcripts in MVs are indicated by red and green dots, respectively (P value <0.02). Transcripts from the pyocin gene cluster (PA0610 to PA0648) are circled in black. (f) Promoter activities of recA, hol and lacZ (control) under non-inducing conditions were monitored by the aid of plasmids containing transcriptional fusions of the respective promoter regions to eGFP. Cells expressing GFP are green; scale bar, 2.5 μm.
Mentions: Our observations have shown that explosive cell lysis mediated by the endolysin Lys is required for MV biogenesis in P. aeruginosa biofilms. We also examined the role of Lys in producing MVs in planktonic culture. Interestingly, PAO1Δlys showed similar levels of MV production as the wild type under normal oxic growth in liquid medium (Fig. 7a). However, the importance of Lys in MV production was evident under conditions that stimulate MV production through induction of the SOS response such as anoxic growth or exposure to MMC4 (Fig. 7b,c). We found that when either recA or lys was inactivated, stress-induced MV formation was also greatly impaired whereas inactivation of pyocin structural components (pyocin tail, tail fibre or tail sheath)4, had no effect on MV formation (Fig. 7b,c). The defects in MMC-induced MV production by PAO1Δlys could be rescued by complementation with the wild-type lys but not the catalytic lys* mutant allele (Fig. 7d). Promoter-reporter eGFP fusions show that PrecA and Phol were also significantly induced by exposure to MMC (Supplementary Fig. 5).

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