Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa

Morten T Rybtke, Bradley R Borlee, Keiji Murakami, Yasuhiko Irie, Morten Hentzer, Thomas E Nielsen, Michael Givskov, Matthew R Parsek, Tim Tolker-Nielsen

Research output: Contribution to journalArticle

101 Citations (Scopus)

Abstract

The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in P. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive cdrA promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in P. aeruginosa strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa.
Original languageEnglish
Pages (from-to)5060-5069
Number of pages10
JournalApplied and Environmental Microbiology
Volume78
Issue number15
DOIs
Publication statusPublished - Aug 2012

Keywords

  • Adhesins, Bacterial
  • Biofilms
  • Cyclic GMP
  • Fluorescence
  • Genes, Reporter
  • Green Fluorescent Proteins
  • Plasmids
  • Promoter Regions, Genetic
  • Pseudomonas aeruginosa

Fingerprint Dive into the research topics of 'Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa'. Together they form a unique fingerprint.

  • Cite this

    Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., & Tolker-Nielsen, T. (2012). Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa. Applied and Environmental Microbiology, 78(15), 5060-5069. https://doi.org/10.1128/AEM.00414-12