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Abstract

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell–cell and cell–substratum interactions. We have previously demonstrated that eDNA is produced in biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within biofilms.

Funding
This study was supported by the:
  • Wellcome Trust (Award 106064/Z/14/2)
    • Principle Award Recipient: LauraC McCaughey
  • Cystic Fibrosis Trust (Award VIA 070)
    • Principle Award Recipient: LauraM Nolan
  • Biotechnology and Biological Sciences Research Council (Award BB/CCG1860/1)
    • Principle Award Recipient: GeorgeM Savva
  • Biotechnology and Biological Sciences Research Council (Award BB/R012504/1)
    • Principle Award Recipient: CynthiaB Whitchurch
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/content/journal/micro/10.1099/mic.0.000990
2021-01-05
2021-08-02
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References

  1. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002; 15:167–193 [View Article][PubMed]
    [Google Scholar]
  2. Donlan RM. Biofilms and device-associated infections. Emerg Infect Dis 2001; 7:277–281 [View Article][PubMed]
    [Google Scholar]
  3. Hughes G, Webber MA. Novel approaches to the treatment of bacterial biofilm infections. Br J Pharmacol 2017; 174:2237–2246 [View Article][PubMed]
    [Google Scholar]
  4. Xu D, Jia R, Li Y, Gu T. Advances in the treatment of problematic industrial biofilms. World J Microbiol Biotechnol 2017; 33:97 [View Article][PubMed]
    [Google Scholar]
  5. Flemming H-C, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010; 8:623–633 [View Article][PubMed]
    [Google Scholar]
  6. Payne DE, Boles BR. Emerging interactions between matrix components during biofilm development. Curr Genet 2016; 62:137–141 [View Article][PubMed]
    [Google Scholar]
  7. Vu B, Chen M, Crawford RJ, Ivanova EP. Bacterial extracellular polysaccharides involved in biofilm formation. Molecules 2009; 14:2535–2554 [View Article][PubMed]
    [Google Scholar]
  8. Okshevsky M, Meyer RL. The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Crit Rev Microbiol 2015; 41:341–352 [View Article][PubMed]
    [Google Scholar]
  9. Southey-Pillig CJ, Davies DG, Sauer K. Characterization of temporal protein production in Pseudomonas aeruginosa biofilms. J Bacteriol 2005; 187:8114–8126 [View Article][PubMed]
    [Google Scholar]
  10. Palmer J, Flint S, Brooks J. Bacterial cell attachment, the beginning of a biofilm. J Ind Microbiol Biotechnol 2007; 34:577–588 [View Article][PubMed]
    [Google Scholar]
  11. Ma L, Conover M, Lu H, Parsek MR, Bayles K et al. Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoS Pathog 2009; 5:e1000354 [View Article][PubMed]
    [Google Scholar]
  12. McDougald D, Klebensberger J, Tolker-Nielsen T, Webb JS, Conibear T. Pseudomonas aeruginosa: A Model for Biofilm Formation. In Rehm BHA. editor Pseudomonas Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA; 2008 pp 215–253
    [Google Scholar]
  13. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science 2002; 295:1487 [View Article][PubMed]
    [Google Scholar]
  14. Gloag ES, Turnbull L, Huang A, Vallotton P, Wang H et al. Self-Organization of bacterial biofilms is facilitated by extracellular DNA. Proc Natl Acad Sci U S A 2013; 110:11541–11546 [View Article][PubMed]
    [Google Scholar]
  15. Fuxman Bass JI, Russo DM, Gabelloni ML, Geffner JR, Giordano M et al. Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms. J Immunol 2010; 184:6386–6395 [View Article][PubMed]
    [Google Scholar]
  16. Mulcahy H, Charron-Mazenod L, Lewenza S. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog 2008; 4:e1000213 [View Article][PubMed]
    [Google Scholar]
  17. Madsen JS, Burmølle M, Hansen LH, Sørensen SJ. The interconnection between biofilm formation and horizontal gene transfer. FEMS Immunol Med Microbiol 2012; 65:183–195 [View Article][PubMed]
    [Google Scholar]
  18. Ibáñez de Aldecoa AL, Zafra O, González-Pastor JE. Mechanisms and regulation of extracellular DNA release and its biological roles in microbial communities. Front Microbiol 2017; 8:1390 [View Article][PubMed]
    [Google Scholar]
  19. Turnbull L, Toyofuku M, Hynen AL, Kurosawa M, Pessi G et al. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms. Nat Commun 2016; 7:11220 [View Article][PubMed]
    [Google Scholar]
  20. Nakayama K, Takashima K, Ishihara H, Shinomiya T, Kageyama M et al. The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Mol Microbiol 2000; 38:213–231 [View Article][PubMed]
    [Google Scholar]
  21. Amini S, Hottes AK, Smith LE, Tavazoie S. Fitness landscape of antibiotic tolerance in Pseudomonas aeruginosa biofilms. PLoS Pathog 2011; 7:e1002298 [View Article][PubMed]
    [Google Scholar]
  22. Saier MH, Reddy BL. Holins in bacteria, eukaryotes, and archaea: multifunctional xenologues with potential biotechnological and biomedical applications. J Bacteriol 2015; 197:7–17 [View Article][PubMed]
    [Google Scholar]
  23. Young R. Phage lysis: three steps, three choices, one outcome. J Microbiol 2014; 52:243–258 [View Article][PubMed]
    [Google Scholar]
  24. McFarland KA, Dolben EL, LeRoux M, Kambara TK, Ramsey KM et al. A self-lysis pathway that enhances the virulence of a pathogenic bacterium. Proc Natl Acad Sci U S A 2015; 112:8433–8438 [View Article][PubMed]
    [Google Scholar]
  25. Bayles KW. Are the molecular strategies that control apoptosis conserved in bacteria?. Trends Microbiol 2003; 11:306–311 [View Article][PubMed]
    [Google Scholar]
  26. Rice KC, Mann EE, Endres JL, Weiss EC, Cassat JE et al. The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus . Proc Natl Acad Sci U S A 2007; 104:8113–8118 [View Article][PubMed]
    [Google Scholar]
  27. Ranjit DK, Endres JL, Bayles KW. Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties. J Bacteriol 2011; 193:2468–2476 [View Article][PubMed]
    [Google Scholar]
  28. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 1998; 212:77–86 [View Article][PubMed]
    [Google Scholar]
  29. Toyofuku M, Zhou S, Sawada I, Takaya N, Uchiyama H et al. Membrane vesicle formation is associated with pyocin production under denitrifying conditions in Pseudomonas aeruginosa PAO1. Environ Microbiol 2014; 16:2927–2938 [View Article][PubMed]
    [Google Scholar]
  30. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article][PubMed]
    [Google Scholar]
  31. Bates D, Mächler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J Stat Softw 2015; 67: [View Article]
    [Google Scholar]
  32. Kuznetsova A, Brockhoff PB, Christensen RHB. lmertest package: tests in linear mixed effects models. J Stat Softw 2017; 82: [View Article]
    [Google Scholar]
  33. RCT R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2019. https://www.R-project.org/ ; 2019
  34. Wang IN, Smith DL, Young R. Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 2000; 54:799–825 [View Article][PubMed]
    [Google Scholar]
  35. Zhao K, Tseng BS, Beckerman B, Jin F, Gibiansky ML et al. Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms. Nature 2013; 497:388–391 [View Article][PubMed]
    [Google Scholar]
  36. Wang S, Liu X, Liu H, Zhang L, Guo Y et al. The exopolysaccharide Psl-eDNA interaction enables the formation of a biofilm skeleton in Pseudomonas aeruginosa. Environ Microbiol Rep 2015; 7:330–340 [View Article][PubMed]
    [Google Scholar]
  37. Mulcahy H, Lewenza S. Magnesium limitation is an environmental trigger of the Pseudomonas aeruginosa biofilm lifestyle. PLoS One 2011; 6:e23307 [View Article][PubMed]
    [Google Scholar]
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