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

Explosive cell lysis occurs in P. aeruginosa interstitial biofilms.(a) Phase-contrast (left) and TOTO-1-stained eDNA (green, right); scale bar, 50 μm. (b) Time series of a rod-to-round cell transition (dotted white line, upper panels) and subsequent lysis releasing eDNA stained by TOTO-1 (green, lower panels). Time in seconds (top right); scale bar, 1 μm. (c) P. aeruginosa PAK-expressing cytoplasmic CFP (magenta) cultured in the presence of the eDNA stain TOTO-1 (yellow) showing that sites of eDNA release (arrow, left panel) contain extracellular CFP (arrow, right panel); scale bar, 2 μm. (d) Frequency distribution of survival times in seconds (s) of round cells from formation to explosion (n=150, bin size 60s). Another 12 round cells were observed that had either formed within a time-series or were present at the start of a time-series and which did not explode by the end of the time-series. Survival times of these cells were at least 10–45 min including one that we tracked for several hours. (e) Frequency distribution of round cell survival for those cells surviving <60 s (n=129, bin size 5s). (f) A round cell (dotted white line) cultured in the presence of FM-143FX (green), tracked over 20 min. Time in min (top right); scale bar, 1 μm. These round cells with long survival times had malleable cell walls and were able to withstand being pushed out of shape by surrounding cells and rapidly re-formed the default round shape when the neighbouring cells moved away. See Supplementary Movie 2. (g) Cell lengths of round cells that became rods (round cell rod; n=120) and neighbouring rod cells (n=750). Box is 25th–75th percentiles, line in box is median, whisker limits are minimum and maximum values; #P<0.0001, unpaired t-test with Welch's correction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Explosive cell lysis occurs in P. aeruginosa interstitial biofilms.(a) Phase-contrast (left) and TOTO-1-stained eDNA (green, right); scale bar, 50 μm. (b) Time series of a rod-to-round cell transition (dotted white line, upper panels) and subsequent lysis releasing eDNA stained by TOTO-1 (green, lower panels). Time in seconds (top right); scale bar, 1 μm. (c) P. aeruginosa PAK-expressing cytoplasmic CFP (magenta) cultured in the presence of the eDNA stain TOTO-1 (yellow) showing that sites of eDNA release (arrow, left panel) contain extracellular CFP (arrow, right panel); scale bar, 2 μm. (d) Frequency distribution of survival times in seconds (s) of round cells from formation to explosion (n=150, bin size 60s). Another 12 round cells were observed that had either formed within a time-series or were present at the start of a time-series and which did not explode by the end of the time-series. Survival times of these cells were at least 10–45 min including one that we tracked for several hours. (e) Frequency distribution of round cell survival for those cells surviving <60 s (n=129, bin size 5s). (f) A round cell (dotted white line) cultured in the presence of FM-143FX (green), tracked over 20 min. Time in min (top right); scale bar, 1 μm. These round cells with long survival times had malleable cell walls and were able to withstand being pushed out of shape by surrounding cells and rapidly re-formed the default round shape when the neighbouring cells moved away. See Supplementary Movie 2. (g) Cell lengths of round cells that became rods (round cell rod; n=120) and neighbouring rod cells (n=750). Box is 25th–75th percentiles, line in box is median, whisker limits are minimum and maximum values; #P<0.0001, unpaired t-test with Welch's correction.

Mentions: We have observed that in actively expanding interstitial biofilms of P. aeruginosa strain K (PAK), eDNA is distributed as numerous bright foci throughout the active migration zone of the biofilm7 (Fig. 1a). This pattern suggests that eDNA is produced by discrete cells rather than generalized secretion by the whole population. To determine the mechanism by which eDNA is produced in interstitial biofilms we followed 150 eDNA release events and found that all were associated with the rapid transition of rod-shaped cells to round cells that subsequently exploded resulting in the annihilation of the cell and release of eDNA into the environment (Fig. 1b; Supplementary Movie 1).


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 occurs in P. aeruginosa interstitial biofilms.(a) Phase-contrast (left) and TOTO-1-stained eDNA (green, right); scale bar, 50 μm. (b) Time series of a rod-to-round cell transition (dotted white line, upper panels) and subsequent lysis releasing eDNA stained by TOTO-1 (green, lower panels). Time in seconds (top right); scale bar, 1 μm. (c) P. aeruginosa PAK-expressing cytoplasmic CFP (magenta) cultured in the presence of the eDNA stain TOTO-1 (yellow) showing that sites of eDNA release (arrow, left panel) contain extracellular CFP (arrow, right panel); scale bar, 2 μm. (d) Frequency distribution of survival times in seconds (s) of round cells from formation to explosion (n=150, bin size 60s). Another 12 round cells were observed that had either formed within a time-series or were present at the start of a time-series and which did not explode by the end of the time-series. Survival times of these cells were at least 10–45 min including one that we tracked for several hours. (e) Frequency distribution of round cell survival for those cells surviving <60 s (n=129, bin size 5s). (f) A round cell (dotted white line) cultured in the presence of FM-143FX (green), tracked over 20 min. Time in min (top right); scale bar, 1 μm. These round cells with long survival times had malleable cell walls and were able to withstand being pushed out of shape by surrounding cells and rapidly re-formed the default round shape when the neighbouring cells moved away. See Supplementary Movie 2. (g) Cell lengths of round cells that became rods (round cell rod; n=120) and neighbouring rod cells (n=750). Box is 25th–75th percentiles, line in box is median, whisker limits are minimum and maximum values; #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

f1: Explosive cell lysis occurs in P. aeruginosa interstitial biofilms.(a) Phase-contrast (left) and TOTO-1-stained eDNA (green, right); scale bar, 50 μm. (b) Time series of a rod-to-round cell transition (dotted white line, upper panels) and subsequent lysis releasing eDNA stained by TOTO-1 (green, lower panels). Time in seconds (top right); scale bar, 1 μm. (c) P. aeruginosa PAK-expressing cytoplasmic CFP (magenta) cultured in the presence of the eDNA stain TOTO-1 (yellow) showing that sites of eDNA release (arrow, left panel) contain extracellular CFP (arrow, right panel); scale bar, 2 μm. (d) Frequency distribution of survival times in seconds (s) of round cells from formation to explosion (n=150, bin size 60s). Another 12 round cells were observed that had either formed within a time-series or were present at the start of a time-series and which did not explode by the end of the time-series. Survival times of these cells were at least 10–45 min including one that we tracked for several hours. (e) Frequency distribution of round cell survival for those cells surviving <60 s (n=129, bin size 5s). (f) A round cell (dotted white line) cultured in the presence of FM-143FX (green), tracked over 20 min. Time in min (top right); scale bar, 1 μm. These round cells with long survival times had malleable cell walls and were able to withstand being pushed out of shape by surrounding cells and rapidly re-formed the default round shape when the neighbouring cells moved away. See Supplementary Movie 2. (g) Cell lengths of round cells that became rods (round cell rod; n=120) and neighbouring rod cells (n=750). Box is 25th–75th percentiles, line in box is median, whisker limits are minimum and maximum values; #P<0.0001, unpaired t-test with Welch's correction.
Mentions: We have observed that in actively expanding interstitial biofilms of P. aeruginosa strain K (PAK), eDNA is distributed as numerous bright foci throughout the active migration zone of the biofilm7 (Fig. 1a). This pattern suggests that eDNA is produced by discrete cells rather than generalized secretion by the whole population. To determine the mechanism by which eDNA is produced in interstitial biofilms we followed 150 eDNA release events and found that all were associated with the rapid transition of rod-shaped cells to round cells that subsequently exploded resulting in the annihilation of the cell and release of eDNA into the environment (Fig. 1b; Supplementary Movie 1).

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