1887

Abstract

Bacteria form biofilm as a response to a number of environmental signals that are mediated by global transcription regulators and alarmones. Here we report the involvement of the global transcription regulator Fis in biofilm formation through regulation of and genes. The major component of biofilm is proteinaceous and two large adhesive proteins, LapA and LapF, are known to play a key role in its formation. We have previously shown that Fis overexpression enhances biofilm formation. In this study, we used mini-Tn transposon mutagenesis to select potential Fis-regulated genes involved in biofilm formation. A total of 90 % of the studied transposon mutants carried insertions in the genes. Since our experiments showed that Fis-enhanced biofilm is mostly proteinaceous, the amounts of LapA and LapF from cells lysates were quantified using SDS-PAGE. Fis overexpression increases the quantity of LapA 1.6 times and decreases the amount of LapF at least 4 times compared to the wild-type cells. The increased LapA expression caused by Fis overexpression was confirmed by FACS analysis measuring the amount of LapA-GFP fusion protein. Our results suggest that the profusion of LapA in the Fis-overexpressed cells causes enhanced biofilm formation in mature stages of biofilm and LapF has a minor role in biofilm formation.

Funding
This study was supported by the:
  • Estonian Ministry of Education and Research (Award IUT20-19 and SF0180031s08)
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2014-12-01
2024-04-19
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References

  1. Adams M. H. ( 1959). Bacteriophages New York: Interscience Publishers;
    [Google Scholar]
  2. Ali Azam T., Iwata A., Nishimura A., Ueda S., Ishihama A. ( 1999). Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid. J Bacteriol 181:6361–6370[PubMed]
    [Google Scholar]
  3. Allesen-Holm M., Barken K. B., Yang L., Klausen M., Webb J. S., Kjelleberg S., Molin S., Givskov M., Tolker-Nielsen T. ( 2006). A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 59:1114–1128 [View Article][PubMed]
    [Google Scholar]
  4. Azam T. A., Ishihama A. ( 1999). Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J Biol Chem 274:33105–33113 [View Article][PubMed]
    [Google Scholar]
  5. Bayley S. A., Duggleby C. J., Worsey M. J., Williams P. A., Hardy K. G., Broda P. ( 1977). Two modes of loss of the Tol function from Pseudomonas putida mt-2. Mol Gen Genet 154:203–204 [View Article][PubMed]
    [Google Scholar]
  6. Beach M. B., Osuna R. ( 1998). Identification and characterization of the fis operon in enteric bacteria. J Bacteriol 180:5932–5946[PubMed]
    [Google Scholar]
  7. Boswell S., Mathew J., Beach M., Osuna R., Colón W. ( 2004). Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation. Biochemistry 43:2964–2977 [View Article][PubMed]
    [Google Scholar]
  8. Boyd C. D., Smith T. J., El-Kirat-Chatel S., Newell P. D., Dufrêne Y. F., O'Toole G. A. ( 2014). Structural features of the Pseudomonas fluorescens biofilm adhesin LapA required for LapG-dependent cleavage, biofilm formation, and cell surface localization. J Bacteriol 196:2775–2788 [View Article][PubMed]
    [Google Scholar]
  9. Boyer H. W., Roulland-Dussoix D. ( 1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472 [View Article][PubMed]
    [Google Scholar]
  10. Bradley M. D., Beach M. B., de Koning A. P., Pratt T. S., Osuna R. ( 2007). Effects of Fis on Escherichia coli gene expression during different growth stages. Microbiology 153:2922–2940 [View Article][PubMed]
    [Google Scholar]
  11. Carter P., Bedouelle H., Winter G. ( 1985). Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res 13:4431–4443 [View Article][PubMed]
    [Google Scholar]
  12. Chang W. S., van de Mortel M., Nielsen L., Nino de Guzman G., Li X., Halverson L. J. ( 2007). Alginate production by Pseudomonas putida creates a hydrated microenvironment and contributes to biofilm architecture and stress tolerance under water-limiting conditions. J Bacteriol 189:8290–8299 [View Article][PubMed]
    [Google Scholar]
  13. Chaudhuri R. R., Sebaihia M., Hobman J. L., Webber M. A., Leyton D. L., Goldberg M. D., Cunningham A. F., Scott-Tucker A., Ferguson P. R. & other authors ( 2010). Complete genome sequence and comparative metabolic profiling of the prototypical enteroaggregative Escherichia coli strain 042. PLoS ONE 5:e8801 [View Article][PubMed]
    [Google Scholar]
  14. Chevallet M., Luche S., Rabilloud T. ( 2006). Silver staining of proteins in polyacrylamide gels. Nat Protoc 1:1852–1858 [View Article][PubMed]
    [Google Scholar]
  15. Czeczulin J. R., Balepur S., Hicks S., Phillips A., Hall R., Kothary M. H., Navarro-Garcia F., Nataro J. P. ( 1997). Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli. Infect Immun 65:4135–4145[PubMed]
    [Google Scholar]
  16. Danhorn T., Fuqua C. ( 2007). Biofilm formation by plant-associated bacteria. Annu Rev Microbiol 61:401–422 [View Article][PubMed]
    [Google Scholar]
  17. de Lorenzo V., Cases I., Herrero M., Timmis K. N. ( 1993). Early and late responses of TOL promoters to pathway inducers: identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters. J Bacteriol 175:6902–6907[PubMed]
    [Google Scholar]
  18. Dorgai L., Oberto J., Weisberg R. A. ( 1993). Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022. J Bacteriol 175:693–700[PubMed]
    [Google Scholar]
  19. Duque E., de la Torre J., Bernal P., Molina-Henares M. A., Alaminos M., Espinosa-Urgel M., Roca A., Fernández M., de Bentzmann S., Ramos J. L. ( 2013). Identification of reciprocal adhesion genes in pathogenic and non-pathogenic Pseudomonas. Environ Microbiol 15:36–48 [View Article][PubMed]
    [Google Scholar]
  20. Espinosa-Urgel M., Salido A., Ramos J. L. ( 2000). Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J Bacteriol 182:2363–2369 [View Article][PubMed]
    [Google Scholar]
  21. Figurski D. H., Helinski D. R. ( 1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [View Article][PubMed]
    [Google Scholar]
  22. Finkel S. E., Johnson R. C. ( 1992). The Fis protein: it’s not just for DNA inversion anymore. Mol Microbiol 6:3257–3265 [View Article][PubMed]
    [Google Scholar]
  23. Fletcher A. ( 1977). The effects of culture concentration and age, time and temperature on bacterial attachments to polystyrene. Can J Microbiol 23:1–6 [View Article]
    [Google Scholar]
  24. Fuqua C. ( 2010). Passing the baton between laps: adhesion and cohesion in Pseudomonas putida biofilms. Mol Microbiol 77:533–536 [View Article][PubMed]
    [Google Scholar]
  25. Gjermansen M., Nilsson M., Yang L., Tolker-Nielsen T. ( 2010). Characterization of starvation-induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms. Mol Microbiol 75:815–826 [View Article][PubMed]
    [Google Scholar]
  26. González-Gil G., Bringmann P., Kahmann R. ( 1996). FIS is a regulator of metabolism in Escherichia coli. Mol Microbiol 22:21–29 [View Article][PubMed]
    [Google Scholar]
  27. Herrero M., de Lorenzo V., Timmis K. N. ( 1990). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172:6557–6567[PubMed]
    [Google Scholar]
  28. Hinsa S. M., O'Toole G. A. ( 2006). Biofilm formation by Pseudomonas fluorescens WCS365: a role for LapD. Microbiology 152:1375–1383 [View Article][PubMed]
    [Google Scholar]
  29. Hinsa S. M., Espinosa-Urgel M., Ramos J. L., O'Toole G. A. ( 2003). Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein. Mol Microbiol 49:905–918 [View Article][PubMed]
    [Google Scholar]
  30. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. ( 1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68 [View Article][PubMed]
    [Google Scholar]
  31. Jakovleva J., Teppo A., Velts A., Saumaa S., Moor H., Kivisaar M., Teras R. ( 2012). Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation. Microbiology 158:708–720 [View Article][PubMed]
    [Google Scholar]
  32. Jeong J. Y., Yim H. S., Ryu J. Y., Lee H. S., Lee J. H., Seen D. S., Kang S. G. ( 2012). One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol 78:5440–5443 [View Article][PubMed]
    [Google Scholar]
  33. Johnson R. C., Bruist M. F., Simon M. I. ( 1986). Host protein requirements for in vitro site-specific DNA inversion. Cell 46:531–539 [View Article][PubMed]
    [Google Scholar]
  34. King E. O., Ward M. K., Raney D. E. ( 1954). Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44:301–307[PubMed]
    [Google Scholar]
  35. Kugelberg E., Löfmark S., Wretlind B., Andersson D. I. ( 2005). Reduction of the fitness burden of quinolone resistance in Pseudomonas aeruginosa. J Antimicrob Chemother 55:22–30 [View Article][PubMed]
    [Google Scholar]
  36. Lee D. J., Bingle L. E., Heurlier K., Pallen M. J., Penn C. W., Busby S. J., Hobman J. L. ( 2009). Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiol 9:252 [View Article][PubMed]
    [Google Scholar]
  37. Liberati N. T., Urbach J. M., Miyata S., Lee D. G., Drenkard E., Wu G., Villanueva J., Wei T., Ausubel F. M. ( 2006). An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103:2833–2838 [View Article][PubMed]
    [Google Scholar]
  38. Lugtenberg B. J., Kravchenko L. V., Simons M. ( 1999). Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization. Environ Microbiol 1:439–446 [View Article][PubMed]
    [Google Scholar]
  39. Martínez-García E., de Lorenzo V. ( 2011). Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol 13:2702–2716 [View Article][PubMed]
    [Google Scholar]
  40. Martínez-García E., Calles B., Arévalo-Rodríguez M., de Lorenzo V. ( 2011). pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol 11:38 [View Article][PubMed]
    [Google Scholar]
  41. Martínez-Gil M., Yousef-Coronado F., Espinosa-Urgel M. ( 2010). LapF, the second largest Pseudomonas putida protein, contributes to plant root colonization and determines biofilm architecture. Mol Microbiol 77:549–561 [View Article][PubMed]
    [Google Scholar]
  42. Martínez-Gil M., Quesada J. M., Ramos-González M. I., Soriano M. I., de Cristóbal R. E., Espinosa-Urgel M. ( 2013). Interplay between extracellular matrix components of Pseudomonas putida biofilms. Res Microbiol 164:382–389 [View Article][PubMed]
    [Google Scholar]
  43. Martínez-Gil M., Ramos-González M. I., Espinosa-Urgel M. ( 2014). Roles of cyclic di-GMP and the Gac system in transcriptional control of the genes coding for the Pseudomonas putida adhesins LapA and LapF. J Bacteriol 196:1484–1495 [View Article][PubMed]
    [Google Scholar]
  44. Mathesius U. ( 2009). Comparative proteomic studies of root–microbe interactions. J Proteomics 72:353–366 [View Article][PubMed]
    [Google Scholar]
  45. Matilla M. A., Espinosa-Urgel M., Rodríguez-Herva J. J., Ramos J. L., Ramos-González M. I. ( 2007). Genomic analysis reveals the major driving forces of bacterial life in the rhizosphere. Genome Biol 8:R179 [View Article][PubMed]
    [Google Scholar]
  46. Matilla M. A., Travieso M. L., Ramos J. L., Ramos-González M. I. ( 2011). Cyclic diguanylate turnover mediated by the sole GGDEF/EAL response regulator in Pseudomonas putida: its role in the rhizosphere and an analysis of its target processes. Environ Microbiol 13:1745–1766 [View Article][PubMed]
    [Google Scholar]
  47. Meyer J. M. ( 2000). Pyoverdines: pigments, siderophores and potential taxonomic markers of fluorescent Pseudomonas species. Arch Microbiol 174:135–142 [View Article][PubMed]
    [Google Scholar]
  48. Mikelsaar A. V., Sünter A., Mikelsaar R., Toomik P., Kõiveer A., Mikelsaar I., Juronen E. ( 2012a). Epitope of titin A-band-specific monoclonal antibody Tit1 5 H1.1 is highly conserved in several Fn3 domains of the titin molecule. Centriole staining in human, mouse and zebrafish cells. Cell Div 7:21 [View Article][PubMed]
    [Google Scholar]
  49. Mikelsaar A. V., Sünter A., Toomik P., Karpson K., Juronen E. ( 2012b). Development of new monoclonal antibodies for immunocytochemical characterization of neural stem and differentiated cells. Neural Stem Cells and Therapy93–118 Sun D. T. Rijecka: Intech; [View Article]
    [Google Scholar]
  50. Miller J. H. ( 1992). A Short Course in Bacterial Genetics: a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  51. Navarro M. V., Newell P. D., Krasteva P. V., Chatterjee D., Madden D. R., O'Toole G. A., Sondermann H. ( 2011). Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis. PLoS Biol 9:e1000588 [View Article][PubMed]
    [Google Scholar]
  52. Newell P. D., Monds R. D., O'Toole G. A. ( 2009). LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1. Proc Natl Acad Sci U S A 106:3461–3466 [View Article][PubMed]
    [Google Scholar]
  53. Newell P. D., Yoshioka S., Hvorecny K. L., Monds R. D., O'Toole G. A. ( 2011). Systematic analysis of diguanylate cyclases that promote biofilm formation by Pseudomonas fluorescens Pf0-1. J Bacteriol 193:4685–4698 [View Article][PubMed]
    [Google Scholar]
  54. Nilsson M., Chiang W. C., Fazli M., Gjermansen M., Givskov M., Tolker-Nielsen T. ( 2011). Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability. Environ Microbiol 13:1357–1369 [View Article][PubMed]
    [Google Scholar]
  55. O'Toole G. A., Kolter R. ( 1998). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28:449–461 [View Article][PubMed]
    [Google Scholar]
  56. Osuna R., Lienau D., Hughes K. T., Johnson R. C. ( 1995). Sequence, regulation, and functions of fis in Salmonella typhimurium. J Bacteriol 177:2021–2032[PubMed]
    [Google Scholar]
  57. Perron K., Comte R., van Delden C. ( 2005). DksA represses ribosomal gene transcription in Pseudomonas aeruginosa by interacting with RNA polymerase on ribosomal promoters. Mol Microbiol 56:1087–1102 [View Article][PubMed]
    [Google Scholar]
  58. Prigent-Combaret C., Zghidi-Abouzid O., Effantin G., Lejeune P., Reverchon S., Nasser W. ( 2012). The nucleoid-associated protein Fis directly modulates the synthesis of cellulose, an essential component of pellicle-biofilms in the phytopathogenic bacterium Dickeya dadantii. Mol Microbiol 86:172–186 [View Article][PubMed]
    [Google Scholar]
  59. Ramos-González M. I., Campos M. J., Ramos J. L. ( 2005). Analysis of Pseudomonas putida KT2440 gene expression in the maize rhizosphere: in vivo expression technology capture and identification of root-activated promoters. J Bacteriol 187:4033–4041 [View Article][PubMed]
    [Google Scholar]
  60. Rudrappa T., Quinn W. J., Stanley-Wall N. R., Bais H. P. ( 2007). A degradation product of the salicylic acid pathway triggers oxidative stress resulting in down-regulation of Bacillus subtilis biofilm formation on Arabidopsis thaliana roots. Planta 226:283–297 [View Article][PubMed]
    [Google Scholar]
  61. Saldaña Z., Xicohtencatl-Cortes J., Avelino F., Phillips A. D., Kaper J. B., Puente J. L., Girón J. A. ( 2009). Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli. Environ Microbiol 11:992–1006 [View Article][PubMed]
    [Google Scholar]
  62. Sambrook J., Russel D. W. ( 2001). Molecular Cloning: a Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  63. Sauer K., Camper A. K. ( 2001). Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth. J Bacteriol 183:6579–6589 [View Article][PubMed]
    [Google Scholar]
  64. Schägger H. ( 2006). Tricine-SDS-PAGE. Nat Protoc 1:16–22 [View Article][PubMed]
    [Google Scholar]
  65. Schneider R., Travers A., Kutateladze T., Muskhelishvili G. ( 1999). A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli. Mol Microbiol 34:953–964 [View Article][PubMed]
    [Google Scholar]
  66. Schneider R., Lurz R., Lüder G., Tolksdorf C., Travers A., Muskhelishvili G. ( 2001). An architectural role of the Escherichia coli chromatin protein FIS in organising DNA. Nucleic Acids Res 29:5107–5114 [View Article][PubMed]
    [Google Scholar]
  67. Sharma R. C., Schimke R. T. ( 1996). Preparation of electrocompetent E. coli using salt-free growth medium. Biotechniques 20:42–44[PubMed]
    [Google Scholar]
  68. Sheikh J., Hicks S., Dall’Agnol M., Phillips A. D., Nataro J. P. ( 2001). Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 41:983–997 [View Article][PubMed]
    [Google Scholar]
  69. Studier F. W., Moffatt B. A. ( 1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130 [View Article][PubMed]
    [Google Scholar]
  70. Teras R., Jakovleva J., Kivisaar M. ( 2009). Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652. Microbiology 155:1203–1214 [View Article][PubMed]
    [Google Scholar]
  71. Travers A., Schneider R., Muskhelishvili G. ( 2001). DNA supercoiling and transcription in Escherichia coli: the FIS connection. Biochimie 83:213–217 [View Article][PubMed]
    [Google Scholar]
  72. Winsor G. L., Lam D. K. W., Fleming L., Lo R., Whiteside M. D., Yu N. Y., Hancock R. E. W., Brinkman F. S. L. ( 2011). Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39:Database issueD596–D600 [View Article][PubMed]
    [Google Scholar]
  73. Wiśniewski J. R., Zougman A., Nagaraj N., Mann M. ( 2009). Universal sample preparation method for proteome analysis. Nat Methods 6:359–362 [View Article][PubMed]
    [Google Scholar]
  74. Wong S. M., Mekalanos J. J. ( 2000). Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97:10191–10196 [View Article][PubMed]
    [Google Scholar]
  75. Xu J., Johnson R. C. ( 1995). Identification of genes negatively regulated by Fis: Fis and RpoS comodulate growth-phase-dependent gene expression in Escherichia coli. J Bacteriol 177:938–947[PubMed]
    [Google Scholar]
  76. Yeung A. T., Torfs E. C., Jamshidi F., Bains M., Wiegand I., Hancock R. E., Overhage J. ( 2009). Swarming of Pseudomonas aeruginosa is controlled by a broad spectrum of transcriptional regulators, including MetR. J Bacteriol 191:5592–5602 [View Article][PubMed]
    [Google Scholar]
  77. Yousef-Coronado F., Travieso M. L., Espinosa-Urgel M. ( 2008). Different, overlapping mechanisms for colonization of abiotic and plant surfaces by Pseudomonas putida. FEMS Microbiol Lett 288:118–124 [View Article][PubMed]
    [Google Scholar]
  78. Yousef-Coronado F., Soriano M. I., Yang L., Molin S., Espinosa-Urgel M. ( 2011). Selection of hyperadherent mutants in Pseudomonas putida biofilms. Microbiology 157:2257–2265 [View Article][PubMed]
    [Google Scholar]
  79. Yuste L., Hervás A. B., Canosa I., Tobes R., Jiménez J. I., Nogales J., Pérez-Pérez M. M., Santero E., Díaz E. & other authors ( 2006). Growth phase-dependent expression of the Pseudomonas putida KT2440 transcriptional machinery analysed with a genome-wide DNA microarray. Environ Microbiol 8:165–177 [View Article][PubMed]
    [Google Scholar]
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