Document Detail

Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa.
MedLine Citation:
PMID:  22582064     Owner:  NLM     Status:  MEDLINE    
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.
Morten T Rybtke; Bradley R Borlee; Keiji Murakami; Yasuhiko Irie; Morten Hentzer; Thomas E Nielsen; Michael Givskov; Matthew R Parsek; Tim Tolker-Nielsen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-05-11
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  78     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-07-19     Completed Date:  2012-12-10     Revised Date:  2013-06-25    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5060-9     Citation Subset:  IM    
Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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MeSH Terms
Adhesins, Bacterial / genetics
Cyclic GMP / analysis*
Genes, Reporter / genetics*
Green Fluorescent Proteins / genetics
Plasmids / genetics
Promoter Regions, Genetic / genetics
Pseudomonas aeruginosa / chemistry*
Grant Support
Reg. No./Substance:
0/Adhesins, Bacterial; 0/CdrA protein, Pseudomonas aeruginosa; 147336-22-9/Green Fluorescent Proteins; 7665-99-8/Cyclic GMP

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