Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development
- Publication Type:
- Journal Article
- Journal of Biological Chemistry, 2017, 292 (2), pp. 477 - 487
- Issue Date:
Files in This Item:
|Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development.pdf||Published Version||4.18 MB|
Copyright Clearance Process
- Recently Added
- In Progress
- Open Access
This item is open access.
The embargo period expires on 1 Feb 2018
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di- GMP) is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. Current methodologies for the quantification of c-di-GMP are typically based on chemical extraction, representing end point measurements. Chemical methodologies also fail to take into consideration the physiological heterogeneity of the biofilm and thus represent an average c-di-GMP concentration across the entire biofilm. To address these problems, a ratiometric, image-based quantification method has been developed based on expression of the green fluorescence protein (GFP) under the control of the c-di-GMPresponsive cdrA promoter (Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., and Tolker-Nielsen, T. (2012) Appl. Environ. Microbiol. 78, 5060-5069). The methodology uses the cyan fluorescent protein (CFP) as a biomass indicator and the GFP as a c-di-GMP reporter. Thus, the CFP/GFP ratio gives the effective c-di-GMP per biomass. A binary mask was applied to alleviate background fluorescence, and fluorescence was calibrated against known c-di-GMP concentrations. Using flow cells for biofilm formation, c-di-GMP showed a non-uniform distribution across the biofilm, with concentrated hot spots of c-di- GMP. Additionally, c-di-GMP was found to be localized at the outer boundary of mature colonies in contrast to a uniform distribution in early stage, small colonies. These data demonstrate the application of a method for the in situ, real time quantification of c-di-GMP and show that the amount of this biofilmregulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time.
Please use this identifier to cite or link to this item: