Explosive cell lysis is required for membrane vesicle biogenesis in Pseudomonas aeruginosa biofilms

• Published in: Journal of extracellular vesicles Vol. 7; p. 178
• Main Authors: Hynen, Amelia L, Lazenby, James J, Turnbull, Lynne, Whitchurch, Cynthia B
• Format: Journal Article
• Language: English
• Published: Abingdon John Wiley & Sons, Inc 01.01.2018
• Subjects: Bacterial proteins; Biofilms; Biosynthesis; Cell walls; Clonal deletion; Complementation; Cytoplasm; Deficient mutant; Deletion mutant; Holin; Lysis; Membrane vesicles; Peptidoglycans; Periplasm; Pseudomonas aeruginosa
• ISSN: 2001-3078

Background: We have recently determined that explosive cell lysis events account for the biogenesis of membrane vesicles (MVs) in biofilms by the Gram-negative bacterium Pseudomonas aeruginosa. Livecell super-resolution microscopy (OMX 3D-SIM) revealed that explosive cell lysis liberates shattered membrane fragments that rapidly vesicularize into MVs. This vesicularization process also captures cellular content that has been released into the extracellular milieu, thereby packaging it as MV cargo. We have determined that explosive cell lysis is mediated by the endolysin Lys that degrades the peptidoglycan of the bacterial cell wall. As Lys-deficient mutants are severely abrogated in the formation of MVs, explosive cell lysis appears to be the major mechanism for MV biogenesis, at least in P. aeruginosa biofilms. The endolysin Lys is encoded within the highly conserved R- and F-pyocin gene cluster. The R- and F-pyocins resemble headless bacteriophage tails and are related to lytic bacteriophage. Endolysins of lytic bacteriophage are transported from the cytoplasm to the periplasm via holins that form pores in the inner membrane. P. aeruginosa possesses three putative holins encoded by hol, alpB and cidA. Hol is likely to be the cognate holin for Lys as it is also encoded in the R- and F-pyocin gene cluster and has been previously shown to mediate Lys translocation. However, both AlpB and CidA have also been previously implicated in lytic processes, but an endolysin associated with these systems has not been described. Methods: Isogenic single, double and triple deletion mutants were generated in hol, alpB and cidA by allelic exchange. Results: We found that all three holin systems contribute to explosive cell lysis in P. aeruginosa biofilms. However, each holin appears to have a unique contribution to explosive cell lysis as complementation of a single holin deletion with another of the holins was not always sufficient to restore explosive cell lysis to wild-type levels. Summary/Conclusion: Our findings have revealed that explosive cell lysis is a novel mechanism for the production of MVs and other cell-derived public goods in P. aeruginosa biofilms. Furthermore, we have found that three holin systems contribute to explosive cell lysis in P. aeruginosa.