1887

Microbial Genomics logo

Volume 7, Issue 11

Massively parallel transposon mutagenesis identifies temporally essential genes for biofilm formation in Open Access

Abstract

Biofilms complete a life cycle where cells aggregate, grow and produce a structured community before dispersing to colonize new environments. Progression through this life cycle requires temporally controlled gene expression to maximize fitness at each stage. Previous studies have largely focused on identifying genes essential for the formation of a mature biofilm; here, we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-, a massively parallel transposon mutagenesis approach using transposon-located promoters to assay the impact of disruption or altered expression of all genes in the genome on biofilm formation. We identified 48 genes that affected the fitness of cells growing in a biofilm, including genes with known roles and those not previously implicated in biofilm formation. Regulation of type 1 fimbriae and motility were important at all time points, adhesion and motility were important for the early biofilm, whereas matrix production and purine biosynthesis were only important as the biofilm matured. We found strong temporal contributions to biofilm fitness for some genes, including some where expression changed between being beneficial or detrimental depending on the stage at which they are expressed, including and . Novel genes implicated in biofilm formation included and involved in cell division, in chromosome organization, and and of unknown function. This work provides new insights into the requirements for successful biofilm formation through the biofilm life cycle and demonstrates the importance of understanding expression and fitness through time.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): adhesion , functional genomics and TraDIS
Funding
This study was supported by the:
  • Biotechnology and Biological Sciences Research Council (Award BB/R012504/1)
    • Principle Award Recipient: MarkA Webber
© 2021 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000673
2021-11-16
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/mgen/7/11/mgen000673.html?itemId=/content/journal/mgen/10.1099/mgen.0.000673&mimeType=html&fmt=ahah

References

  1. Berlanga M, Guerrero R. Living together in biofilms: the microbial cell factory and its biotechnological implications. Microb Cell Fact 2016; 15:165 [View Article]
     [Google Scholar]
  2. Bjarnsholt T, Buhlin K, Dufrêne YF, Gomelsky M, Moroni A et al. Biofilm formation – what we can learn from recent developments. J Intern Med 2018; 284:332–345 [View Article]
     [Google Scholar]
  3. Gbejuade HO, Lovering AM, Webb JC. The role of microbial biofilms in prosthetic joint infections. Acta Orthop 2015; 86:147–158 [View Article]
     [Google Scholar]
  4. Davis SC, Martinez L, Kirsner R. The diabetic foot: the importance of biofilms and wound bed preparation. Curr Diab Rep 2006; 6:439–445 [View Article] [PubMed]
     [Google Scholar]
  5. Vestby LK, Grønseth T, Simm R, Nesse LL. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics 2020; 9:59 [View Article]
     [Google Scholar]
  6. Wang H, Tay M, Palmer J, Flint S. Biofilm formation of Yersinia enterocolitica and its persistence following treatment with different sanitation agents. Food Control 2017; 73:433–437 [View Article]
     [Google Scholar]
  7. Mah TF, Pitts B, Pellock B, Walker GC, Stewart PS et al. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 2003; 426:306–310 [View Article]
     [Google Scholar]
  8. Hoyle BD, Costerton JW. Bacterial resistance to antibiotics: the role of biofilms. Prog Drug Res 1991; 37:91–105 [View Article] [PubMed]
     [Google Scholar]
  9. Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA et al. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 2016; 14:563–575 [View Article]
     [Google Scholar]
  10. Kostakioti M, Hadjifrangiskou M, Hultgren SJ. Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harb Perspect Med 2013; 3:a010306 [View Article]
     [Google Scholar]
  11. Flemming H-C, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010; 8:623–633 [View Article]
     [Google Scholar]
  12. Barnhart MM, Chapman MR. Curli biogenesis and function. Annu Rev Microbiol 2006; 60:131–147 [View Article]
     [Google Scholar]
  13. Serra DO, Hengge R. Cellulose in bacterial biofilms. Cohen E, Merzendorfer H. eds In Extracellular Sugar-Based Biopolymers Matrices Cham: Springer; 2019 pp 355–392
     [Google Scholar]
  14. Jubelin G, Vianney A, Beloin C, Ghigo J-M, Lazzaroni J-C et al. CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli. J Bacteriol 2005; 187:2038–2049 [View Article]
     [Google Scholar]
  15. Vidal O, Longin R, Prigent-Combaret C, Dorel C, Hooreman M et al. Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new OmpR allele that increases curli expression. J Bacteriol 1998; 180:2442–2449 [View Article] [PubMed]
     [Google Scholar]
  16. Dorel C, Vidal O, Prigent-Combaret C, Vallet I, Lejeune P. Involvement of the Cpx signal transduction pathway of E. coli in biofilm formation. FEMS Microbiol Lett 1999; 178:169–175 [View Article]
     [Google Scholar]
  17. Otto K, Silhavy TJ. Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc Natl Acad Sci USA 2002; 99:2287–2292 [View Article]
     [Google Scholar]
  18. Adams JL, McLean RJ. Impact of rpoS deletion on Escherichia coli biofilms. Appl Environ Microbiol 1999; 65:4285–4287 [View Article] [PubMed]
     [Google Scholar]
  19. Corona-Izquierdo FP, Membrillo-Hernández J. A mutation in rpoS enhances biofilm formation in Escherichia coli during exponential phase of growth. FEMS Microbiol Lett 2002; 211:105–110 [View Article] [PubMed]
     [Google Scholar]
  20. Gerstel U, Park C, Römling U. Complex regulation of csgD promoter activity by global regulatory proteins. Mol Microbiol 2003; 49:639–654 [View Article]
     [Google Scholar]
  21. Gerstel U, Römling U. The csgD promoter, a control unit for biofilm formation in Salmonella typhimurium. Res Microbiol 2003; 154:659–667 [View Article]
     [Google Scholar]
  22. Amores GR, de Las Heras A, Sanches-Medeiros A, Elfick A, Silva-Rocha R. Systematic identification of novel regulatory interactions controlling biofilm formation in the bacterium Escherichia coli. Sci Rep 2017; 7:16768 [View Article]
     [Google Scholar]
  23. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science 2002; 295:1487 [View Article]
     [Google Scholar]
  24. Vilain S, Pretorius JM, Theron J, Brözel VS. DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilms. Appl Environ Microbiol 2009; 75:2861–2868 [View Article]
     [Google Scholar]
  25. Tetz GV, Artemenko NK, Tetz VV. Effect of DNase and antibiotics on biofilm characteristics. Antimicrob Agents Chemother 2009; 53:1204–1209 [View Article]
     [Google Scholar]
  26. Niba ETE, Naka Y, Nagase M, Mori H, Kitakawa M. A genome-wide approach to identify the genes involved in biofilm formation in E. coli. DNA Res 2007; 14:237–246 [View Article]
     [Google Scholar]
  27. Aedo SJ, Ma HR, Brynildsen MP. Checks and balances with use of the Keio collection for phenotype testing. Methods Mol Biol 2019; 1927:125–138 [View Article]
     [Google Scholar]
  28. Domka J, Lee J, Bansal T, Wood TK. Temporal gene-expression in Escherichia coli K-12 biofilms. Environ Microbiol 2007; 9:332–346 [View Article]
     [Google Scholar]
  29. Schembri MA, Kjaergaard K, Klemm P. Global gene expression in Escherichia coli biofilms. Mol Microbiol 2003; 48:253–267 [View Article]
     [Google Scholar]
  30. Puttamreddy S, Cornick NA, Minion FC. Genome-wide transposon mutagenesis reveals a role for pO157 genes in biofilm development in Escherichia coli O157:H7 EDL933. Infect Immun 2010; 78:2377–2384 [View Article]
     [Google Scholar]
  31. Goh KGK, Phan M-D, Forde BM, Chong TM, Yin W-F et al. Genome-wide discovery of genes required for capsule production by uropathogenic Escherichia coli. mBio 2017; 8:e01558-17 [View Article]
     [Google Scholar]
  32. Nhu NTK, Phan M-D, Peters KM, Lo AW, Forde BM et al. Discovery of new genes involved in curli production by a uropathogenic Escherichia coli strain from the highly virulent O45:K1:H7 lineage. mBio 2018; 9:e01462-18 [View Article]
     [Google Scholar]
  33. Yasir M, Turner AK, Bastkowski S, Baker D, Page AJ et al. TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance. Genome Res 2020; 30:239–249 [View Article]
     [Google Scholar]
  34. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006; 2:2006.0008 [View Article]
     [Google Scholar]
  35. Marteyn BS, Karimova G, Fenton AK, Gazi AD, West N et al. ZapE is a novel cell division protein interacting with FtsZ and modulating the z-ring dynamics. mBio 2014; 5:e00022–14 [View Article]
     [Google Scholar]
  36. Tsui HC, Arps PJ, Connolly DM, Winkler ME. Absence of hisT-mediated tRNA pseudouridylation results in a uracil requirement that interferes with Escherichia coli K-12 cell division. J Bacteriol 1991; 173:7395–7400 [View Article]
     [Google Scholar]
  37. Valens M, Thiel A, Boccard F. The MaoP/maoS site-specific system organizes the Ori region of the E. coli chromosome into a macrodomain. PLoS Genet 2016; 12:e1006309 [View Article]
     [Google Scholar]
  38. Trampari E, Holden ER, Wickham GJ, Ravi A, de Oliveira Martins L et al. Exposure of Salmonella biofilms to antibiotic concentrations rapidly selects resistance with collateral tradeoffs. NPJ Biofilms Microbiomes 2021; 7:3 [View Article]
     [Google Scholar]
  39. Barquist L, Mayho M, Cummins C, Cain AK, Boinett CJ et al. The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 2016; 32:1109–1111 [View Article]
     [Google Scholar]
  40. Carver T, Harris SR, Berriman M, Parkhill J, McQuillan JA. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics 2012; 28:464–469 [View Article]
     [Google Scholar]
  41. McClain MS, Blomfield IC, Eberhardt KJ, Eisenstein BI. Inversion-independent phase variation of type 1 fimbriae in Escherichia coli. J Bacteriol 1993; 175:4335–4344 [View Article]
     [Google Scholar]
  42. Klemm P. Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J 1986; 5:1389–1393 [View Article]
     [Google Scholar]
  43. Lehnen D, Blumer C, Polen T, Wackwitz B, Wendisch VF et al. LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli. Mol Microbiol 2002; 45:521–532 [View Article]
     [Google Scholar]
  44. Blumer C, Kleefeld A, Lehnen D, Heintz M, Dobrindt U et al. Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli. Microbiology 2005; 151:3287–3298 [View Article]
     [Google Scholar]
  45. Ko M, Park C. H-NS-dependent regulation of flagellar synthesis is mediated by a LysR family protein. J Bacteriol 2000; 182:4670–4672 [View Article]
     [Google Scholar]
  46. Alekshun MN, Levy SB. Alteration of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro. J Bacteriol 1999; 181:4669–4672 [View Article] [PubMed]
     [Google Scholar]
  47. Herzberg M, Kaye IK, Peti W, Wood TK. YdgG (TqsA) controls biofilm formation in Escherichia coli K-12 through autoinducer 2 transport. J Bacteriol 2006; 188:587–598 [View Article]
     [Google Scholar]
  48. Holden ER, Webber MA. MarA, RamA, and SoxS as mediators of the stress response: survival at a cost. Front Microbiol 2020; 11:828 [View Article] [PubMed]
     [Google Scholar]
  49. Kettles RA, Tschowri N, Lyons KJ, Sharma P, Hengge R et al. The Escherichia coli MarA protein regulates the ycgZ-ymgABC operon to inhibit biofilm formation. Mol Microbiol 2019; 112:1609–1625 [View Article]
     [Google Scholar]
  50. Szyf M, Avraham-Haetzni K, Reifman A, Shlomai J, Kaplan F et al. DNA methylation pattern is determined by the intracellular level of the methylase. Proc Natl Acad Sci USA 1984; 81:3278–3282 [View Article]
     [Google Scholar]
  51. Lee Y, Kim Y, Yeom S, Kim S, Park S et al. The role of disulfide bond isomerase A (DsbA) of Escherichia coli O157:H7 in biofilm formation and virulence. FEMS Microbiol Lett 2008; 278:213–222 [View Article]
     [Google Scholar]
  52. Bringer MA, Rolhion N, Glasser AL, Darfeuille-Michaud A. The oxidoreductase DsbA plays a key role in the ability of the Crohn’s disease-associated adherent-invasive Escherichia coli strain LF82 to resist macrophage killing. J Bacteriol 2007; 189:4860–4871 [View Article]
     [Google Scholar]
  53. Zhang Y, Morar M, Ealick SE. Structural biology of the purine biosynthetic pathway. Cell Mol Life Sci 2008; 65:3699–3724 [View Article]
     [Google Scholar]
  54. Hamma T, Ferré-D’Amaré AR. Pseudouridine synthases. Chem Biol 2006; 13:1125–1135 [View Article]
     [Google Scholar]
  55. Pratt LA, Kolter R. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol 1998; 30:285–293 [View Article]
     [Google Scholar]
  56. Wang F, Deng L, Huang F, Wang Z, Lu Q et al. Flagellar motility is critical for Salmonella enterica serovar Typhimurium biofilm development. Front Microbiol 2020; 11:1695 [View Article]
     [Google Scholar]
  57. Shimada T, Bridier A, Briandet R, Ishihama A. Novel roles of LeuO in transcription regulation of E. coli genome: antagonistic interplay with the universal silencer H-NS. Mol Microbiol 2011; 82:378–397 [View Article]
     [Google Scholar]
  58. Kroner GM, Wolfe MB, Freddolino PL. Escherichia coli Lrp regulates one-third of the genome via direct, cooperative, and indirect routes. J Bacteriol 2019; 201:00418 [View Article]
     [Google Scholar]
  59. Tramonti A, De Canio M, De Biase D. GadX/GadW-dependent regulation of the Escherichia coli acid fitness island: transcriptional control at the gadY-gadW divergent promoters and identification of four novel 42 bp GadX/GadW-specific binding sites. Mol Microbiol 2008; 70:965–982 [View Article]
     [Google Scholar]
  60. Eichelberger KR, Sepúlveda VE, Ford J, Selitsky SR, Mieczkowski PA et al. Tn-seq analysis identifies genes important for Yersinia pestis adherence during primary pneumonic plague. mSphere 2020; 5:e00715-20 [View Article]
     [Google Scholar]
  61. Yang X, Wang J, Feng Z, Zhang X, Wang X et al. Relation of the pdxB-usg-truA-dedA operon and the truA gene to the intracellular survival of Salmonella enterica serovar Typhimurium. Int J Mol Sci 2019; 20:380 [View Article]
     [Google Scholar]
  62. Wucher BR, Bartlett TM, Hoyos M, Papenfort K, Persat A et al. Vibrio cholerae filamentation promotes chitin surface attachment at the expense of competition in biofilms. Proc Natl Acad Sci USA 2019; 116:14216–14221 [View Article]
     [Google Scholar]
  63. Anwar N, Rouf SF, Römling U, Rhen M. Modulation of biofilm-formation in Salmonella enterica serovar Typhimurium by the periplasmic DsbA/DsbB oxidoreductase system requires the GGDEF-EAL domain protein STM3615. PLoS One 2014; 9:e106095 [View Article]
     [Google Scholar]
  64. Magnusson LU, Gummesson B, Joksimović P, Farewell A, Nyström TI. Identical, independent, and opposing roles of ppGpp and DksA in Escherichia coli. J Bacteriol 2007; 189:5193–5202 [View Article]
     [Google Scholar]
  65. Smith DR, Price JE, Burby PE, Blanco LP, Chamberlain J et al. The production of curli amyloid fibers is deeply integrated into the biology of Escherichia coli. Biomolecules 2017; 7:E75 [View Article]
     [Google Scholar]
  66. Hengge R. Linking bacterial growth, survival, and multicellularity – small signaling molecules as triggers and drivers. Curr Opin Microbiol 2020; 55:57–66 [View Article]
     [Google Scholar]
  67. Garavaglia M, Rossi E, Landini P. The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli. PLoS One 2012; 7:e31252 [View Article]
     [Google Scholar]
  68. Cepas V, Ballén V, Gabasa Y, Ramírez M, López Y et al. Transposon insertion in the purL gene induces biofilm depletion in Escherichia coli ATCC 25922. Pathogens 2020; 9:E774 [View Article]
     [Google Scholar]
  69. Pfiffer V, Sarenko O, Possling A, Hengge R. Genetic dissection of Escherichia coli’s master diguanylate cyclase DgcE: role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system. PLoS Genet 2019; 15:e1008059 [View Article]
     [Google Scholar]
  70. Pesavento C, Becker G, Sommerfeldt N, Possling A, Tschowri N et al. Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli. Genes Dev 2008; 22:2434–2446 [View Article]
     [Google Scholar]
  71. Fang X, Gomelsky M. A post-translational, c-di-GMP-dependent mechanism regulating flagellar motility. Mol Microbiol 2010; 76:1295–1305 [View Article]
     [Google Scholar]
  72. Wood TK, González Barrios AF, Herzberg M, Lee J. Motility influences biofilm architecture in Escherichia coli. Appl Microbiol Biotechnol 2006; 72:361–367 [View Article]
     [Google Scholar]
  73. Li S, Liang H, Wei Z, Bai H, Li M et al. An osmoregulatory mechanism operating through OmpR and LrhA controls the motile-sessile switch in the plant growth-promoting bacterium Pantoea alhagi. Appl Environ Microbiol 2019; 85:e00077-19 [View Article]
     [Google Scholar]
  74. Barrios AFG, Zuo R, Ren D, Wood TK. Hha, YbaJ, and OmpA regulate Escherichia coli K12 biofilm formation and conjugation plasmids abolish motility. Biotechnol Bioeng 2006; 93:188–200 [View Article]
     [Google Scholar]
  75. García-Contreras R, Zhang X-S, Kim Y, Wood TK. Protein translation and cell death: the role of rare tRNAs in biofilm formation and in activating dormant phage killer genes. PLoS One 2008; 3:e2394 [View Article]
     [Google Scholar]
  76. Sharma VK, Bearson BL. Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD. Appl Environ Microbiol 2013; 79:2384–2396 [View Article]
     [Google Scholar]
  77. Prouty AM, Gunn JS. Comparative analysis of Salmonella enterica serovar Typhimurium biofilm formation on gallstones and on glass. Infect Immun 2003; 71:7154–7158 [View Article] [PubMed]
     [Google Scholar]
  78. Hammar M, Bian Z, Normark S. Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli. Proc Natl Acad Sci USA 1996; 93:6562–6566 [View Article]
     [Google Scholar]
  79. Chauhan A, Sakamoto C, Ghigo J-M, Beloin C. Did I pick the right colony? Pitfalls in the study of regulation of the phase variable antigen 43 adhesin. PLoS One 2013; 8:e73568 [View Article]
     [Google Scholar]
  80. Danese PN, Pratt LA, Dove SL, Kolter R. The outer membrane protein, antigen 43, mediates cell-to-cell interactions within Escherichia coli biofilms. Mol Microbiol 2000; 37:424–432 [View Article]
     [Google Scholar]
  81. Samanta P, Clark ER, Knutson K, Horne SM, Prüß BM. OmpR and RcsB abolish temporal and spatial changes in expression of flhD in Escherichia coli biofilm. BMC Microbiol 2013; 13:182 [View Article]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000673
Loading
Massively parallel transposon mutagenesis identifies temporally essential genes for biofilm formation in Escherichia coli
M Gen 7, 000673 (2021); https://doi.org/10.1099/mgen.0.000673
/content/journal/mgen/10.1099/mgen.0.000673
/content/journal/mgen/10.1099/mgen.0.000673
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error