Journal of Microbiology

, Volume 55, Issue 10, pp 800–808 | Cite as

Mutation of the cyclic di-GMP phosphodiesterase gene in Burkholderia lata SK875 attenuates virulence and enhances biofilm formation

  • Hae-In Jung
  • Yun-Jung Kim
  • Yun-Jung Lee
  • Hee-Soo Lee
  • Jung-Kee Lee
  • Soo-Ki KimEmail author
Microbial Genetics, Genomics and Molecular Biology


Burkholderia sp. is a gram-negative bacterium that commonly exists in the environment, and can cause diseases in plants, animals, and humans. Here, a transposon mutant library of a Burkholderia lata isolate from a pig with swine respiratory disease in Korea was screened for strains showing attenuated virulence in Caenorhabditis elegans. One such mutant was obtained, and the Tn5 insertion junction was mapped to rpfR, a gene encoding a cyclic di-GMP phosphodiesterase that functions as a receptor. Mutation of rpfR caused a reduction in growth on CPG agar and swimming motility as well as a rough colony morphology on Congo red agar. TLC analysis showed reduced AHL secretion, which was in agreement with the results from plate-based and bioluminescence assays. The mutant strain produced significantly more biofilm detected by crystal violet staining than the parent strain. SEM of the mutant strain clearly showed that the overproduced biofilm contained a filamentous structure. These results suggest that the cyclic di-GMP phosphodiesterase RpfR plays an important role in quorum sensing modulation of the bacterial virulence and biofilm formation.


quorum sensing Burkholderia lata transposon Caenorhabditis elegans c-di-GMP phosphodiesterase BDSF 


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  1. Asad, S. and Opal, S.M. 2008. Bench-to-bedside review: Quorum sensing and the role of cell-to-cell communication during invasive bacterial infection. Crit. Care 12, 236.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bassis, C.M. and Visick, K.L. 2010. The cyclic-di-GMP phosphodiesterase BinA negatively regulates cellulose-containing biofilms in Vibrio fischeri. J. Bacteriol. 192, 1269–1278.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bassler, B.L., Wright, M., and Silverman, M.R. 1994. Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 13, 273–286.CrossRefPubMedGoogle Scholar
  4. Bi, H., Christensen, Q.H., Feng, Y., Wang, H., and Cronan, J.E. 2012. The Burkholderia cenocepacia BDSF quorum sensing fatty acid is synthesized by a bifunctional crotonase homologue having both dehydratase and thioesterase activities. Mol. Microbiol. 83, 840–855.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Boon, C., Deng, Y., Wang, L.H., He, Y., Xu, J.L., Fan, Y., Pan, S.Q., and Zhang, L.H. 2008. A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition. ISME J 2, 27–36.CrossRefPubMedGoogle Scholar
  6. Chankhamhaengdecha, S., Hongvijit, S., Srichaisupakit, A., Charnchai, P., and Panbangred, W. 2013. Endophytic actinomycetes: a novel source of potential acyl homoserine lactone degrading enzymes. Biomed. Res. Int. 2013, 782847.PubMedPubMedCentralGoogle Scholar
  7. Coenye, T. 2010. Social interactions in the Burkholderia cepacia complex: biofilms and quorum sensing. Future Microbiol. 5, 1087–1099.CrossRefPubMedGoogle Scholar
  8. Coenye, T. and Vandamme, P. 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ. Microbiol. 5, 719–729.CrossRefPubMedGoogle Scholar
  9. Deng, Y., Boon, C., Eberl, L., and Zhang, L.H. 2009. Differential modulation of Burkholderia cenocepacia virulence and energy metabolism by the quorum-sensing signal BDSF and its synthase. J. Bacteriol. 191, 7270–7278.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Deng, Y., Lim, A., Wang, J., Zhou, T., Chen, S., Lee, J., Dong, Y.H., and Zhang, L.H. 2013. Cis-2-dodecenoic acid quorum sensing system modulates N-acyl homoserine lactone production through RpfR and cyclic di-GMP turnover in Burkholderia cenocepacia. BMC Microbiol. 13, 148.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Deng, Y., Schmid, N., Wang, C., Wang, J., Pessi, G., Wu, D., Lee, J., Aguilar, C., Ahrens, C.H., Chang, C., et al. 2012. Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover. Proc. Natl. Acad. Sci. USA 109, 15479–15484.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Deng, Y., Wu, J., Eberl, L., and Zhang, L.H. 2010. Structural and functional characterization of diffusible signal factor family quorum-sensing signals produced by members of the Burkholderia cepacia complex. Appl. Environ. Microbiol. 76, 4675–4683.CrossRefPubMedPubMedCentralGoogle Scholar
  13. Deng, Y., Wu, J., Tao, F., and Zhang, L.H. 2011. Listening to a new language: DSF-based quorum sensing in Gram-negative bacteria. Chem. Rev. 111, 160–173.CrossRefPubMedGoogle Scholar
  14. Eberl, L. 2006. Quorum sensing in the genus Burkholderia. Int. J. Med. Microbiol. 296, 103–110.CrossRefPubMedGoogle Scholar
  15. Friedman, L. and Kolter, R. 2004. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J. Bacteriol. 186, 4457–4465.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Garcia, B., Latasa, C., Solano, C., Garcia-del Portillo, F., Gamazo, C., and Lasa, I. 2004. Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation. Mol. Microbiol. 54, 264–277.CrossRefPubMedGoogle Scholar
  17. Hickman, J.W., Tifrea, D.F., and Harwood, C.S. 2005. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc. Natl. Acad. Sci. USA 102, 14422–14427.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Karki, H.S., Barphagha, I.K., and Ham, J.H. 2012. A conserved twocomponent regulatory system, PidS/PidR, globally regulates pigmentation and virulence-related phenotypes of Burkholderia glumae. Mol. Plant Pathol. 13, 785–794.CrossRefPubMedGoogle Scholar
  19. Khanna, N., Cressman III, C.P., Tatara, C.P., and Williams, P.L. 1997. Tolerance of the nematode Caenorhabditis elegans to pH, salinity, and hardness in aquatic media. Arch. Environ. Contam. Toxicol. 32, 110–114.CrossRefPubMedGoogle Scholar
  20. Kim, S.K. and Lee, J.H. 2016. Biofilm dispersion in Pseudomonas aeruginosa. J. Microbiol. 54, 71–85.CrossRefPubMedGoogle Scholar
  21. Kirillina, O., Fetherston, J.D., Bobrov, A.G., Abney, J., and Perry, R.D. 2004. HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis. Mol. Microbiol. 54, 75–88.CrossRefPubMedGoogle Scholar
  22. Kuchma, S.L., Brothers, K.M., Merritt, J.H., Liberati, N.T., Ausubel, F.M., and O’Toole, G.A. 2007. BifA, a cyclic-di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J. Bacteriol. 189, 8165–8178.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Larsen, R.A., Wilson, M.M., Guss, A.M., and Metcalf, W.W. 2002. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch. Microbiol. 178, 193–201.CrossRefPubMedGoogle Scholar
  24. Lee, H.S., Gu, F., Ching, S.M., Lam, Y., and Chua, K.L. 2010. CdpA is a Burkholderia pseudomallei cyclic di-GMP phosphodiesterase involved in autoaggregation, flagellum synthesis, motility, biofilm formation, cell invasion, and cytotoxicity. Infect. Immun. 78, 1832–1840.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lee, Y., Lee, Y., Lim, S., Park, G., Choi, S., Hong, H., and Ko, J. 2013. Volatile compounds and ultrastructure of petal epidermal cells according to scent intensity in Rosa hybrida. Korean J. Hortic. Sci. Technol. 31, 590–597.CrossRefGoogle Scholar
  26. Mahenthiralingam, E., Urban, T.A., and Goldberg, J.B. 2005. The multifarious, multireplicon Burkholderia cepacia complex. Nat. Rev. Microbiol. 3, 144–156.CrossRefPubMedGoogle Scholar
  27. Marchler-Bauer, A., Derbyshire, M.K., Gonzales, N.R., Lu, S., Chitsaz, F., Geer, L.Y., Geer, R.C., He, J., Gwadz, M., Hurwitz, D.I., et al. 2015. CDD: NCBI’s conserved domain database. Nucleic. Acids. Res. 43, D222–D226.CrossRefPubMedGoogle Scholar
  28. McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft, B.W., et al. 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143, 3703–3711.CrossRefPubMedGoogle Scholar
  29. Mil-Homens, D., Rocha, E.P., and Fialho, A.M. 2010. Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage. Microbiology 156, 1084–1096.CrossRefPubMedGoogle Scholar
  30. Molina, L., Constantinescu, F., Michel, L., Reimmann, C., Duffy, B., and Defago, G. 2003. Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. FEMS Microbiol. Ecol. 45, 71–81.CrossRefPubMedGoogle Scholar
  31. Ryan, R.P., McCarthy, Y., Watt, S.A., Niehaus, K., and Dow, J.M. 2009. Intraspecies signaling involving the diffusible signal factor BDSF (cis-2-dodecenoic acid) influences virulence in Burkholderia cenocepacia. J. Bacteriol. 191, 5013–5019.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Schmid, N., Pessi, G., Deng, Y., Aguilar, C., Carlier, A.L., Grunau, A., Omasits, U., Zhang, L.H., Ahrens, C.H., and Eberl, L. 2012. The AHL-and BDSF-dependent quorum sensing systems control specific and overlapping sets of genes in Burkholderia cenocepacia H111. PLoS One 7, e49966.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Schmid, N., Suppiger, A., Steiner, E., Pessi, G., Kaever, V., Fazli, M., Tolker-Nielsen, T., Jenal, U., and Eberl, L. 2017. High intracellular c-di-GMP levels antagonize quorum sensing and virulence gene expression in Burkholderia cenocepacia H111. Microbiology 163, 754–764.CrossRefPubMedGoogle Scholar
  34. Shaw, P.D., Ping, G., Daly, S.L., Cha, C., Cronan, J.E., Jr., Rinehart, K.L., and Farrand, S.K. 1997. Detecting and characterizing Nacyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94, 6036–6041.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Simm, R., Morr, M., Kader, A., Nimtz, M., and Romling, U. 2004. GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol. Microbiol. 53, 1123–1134.CrossRefPubMedGoogle Scholar
  36. Stanier, R.Y., Palleroni, N.J., and Doudoroff, M. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43, 159–271.CrossRefPubMedGoogle Scholar
  37. Steindler, L. and Venturi, V. 2007. Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors. FEMS Microbiol. Lett. 266, 1–9.CrossRefPubMedGoogle Scholar
  38. Suppiger, A., Aguilar, C., and Eberl, L. 2016a. Evidence for the widespread production of DSF family signal molecules by members of the genus Burkholderia by the aid of novel biosensors. Environ. Microbiol. Rep. 8, 38–44.CrossRefPubMedGoogle Scholar
  39. Suppiger, A., Eshwar, A.K., Stephan, R., Kaever, V., Eberl, L., and Lehner, A. 2016b. The DSF type quorum sensing signalling system RpfF/R regulates diverse phenotypes in the opportunistic pathogen Cronobacter. Sci. Rep. 6, 18753.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Suppiger, A., Schmid, N., Aguilar, C., Pessi, G., and Eberl, L. 2013. Two quorum sensing systems control biofilm formation and virulence in members of the Burkholderia cepacia complex. Virulence 4, 400–409.CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sutphin, G.L. and Kaeberlein, M. 2009. Measuring Caenorhabditis elegans life span on solid media. J. Vis. Exp. 27, 1152.Google Scholar
  42. Tamayo, R., Pratt, J.T., and Camilli, A. 2007. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu. Rev. Microbiol. 61, 131–148.CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tedesco, P., Di Schiavi, E., Esposito, F.P., and de Pascale, D. 2016. Evaluation of Burkholderia cepacia complex bacteria pathogenicity using Caenorhabditis elegans. Bio. Protoc. 6, e1964.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Tischler, A.D. and Camilli, A. 2004. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Mol. Microbiol. 53, 857–869.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Vanlaere, E., Baldwin, A., Gevers, D., Henry, D., De Brandt, E., Li-Puma, J.J., Mahenthiralingam, E., Speert, D.P., Dowson, C., and Vandamme, P. 2009. Taxon K, a complex within the Burkholderia cepacia complex, comprises at least two novel species, Burkholderia contaminans sp. nov. and Burkholderia lata sp. nov. Int. J. Syst. Evol. Microbiol. 59, 102–111.CrossRefPubMedGoogle Scholar
  46. Venturi, V., Friscina, A., Bertani, I., Devescovi, G., and Aguilar, C. 2004. Quorum sensing in the Burkholderia cepacia complex. Res. Microbiol. 155, 238–244.CrossRefPubMedGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Hae-In Jung
    • 1
  • Yun-Jung Kim
    • 1
  • Yun-Jung Lee
    • 1
  • Hee-Soo Lee
    • 2
  • Jung-Kee Lee
    • 3
  • Soo-Ki Kim
    • 1
    Email author
  1. 1.Department of Animal Science and TechnologyKonkuk UniversitySeoulRepublic of Korea
  2. 2.National Veterinary Research and Quarantine ServiceAnyangRepublic of Korea
  3. 3.Department of Life Science and Genetic EngineeringPaichai UniversityDaejeonRepublic of Korea

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