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Appl Environ Microbiol. 2012 Aug;78(15):5060-9. doi: 10.1128/AEM.00414-12. Epub 2012 May 11.

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

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1
Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

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.

PMID:
22582064
PMCID:
PMC3416407
DOI:
10.1128/AEM.00414-12
[Indexed for MEDLINE]
Free PMC Article
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