title: Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development.
abstract: 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-GMP-responsive 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 biofilm-regulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time.
authors.affiliation: From the Singapore Centre for Environmental Life Sciences Engineering.|Interdisciplinary Graduate School, and.
authors.forename: Harikrishnan A S
authors.initials: HA
authors.lastname: Nair
authors.affiliation: From the Singapore Centre for Environmental Life Sciences Engineering.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Periasamy
authors.affiliation: From the Singapore Centre for Environmental Life Sciences Engineering.|School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
authors.forename: Liang
authors.initials: L
authors.lastname: Yang
authors.affiliation: From the Singapore Centre for Environmental Life Sciences Engineering.|School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
authors.forename: Staffan
authors.initials: S
authors.lastname: Kjelleberg
authors.affiliation: From the Singapore Centre for Environmental Life Sciences Engineering, rscott@ntu.edu.sg.|School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
authors.forename: Scott A
authors.identifier: http://orcid.org/0000-0002-9486-2343
authors.initials: SA
authors.lastname: Rice
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP
keywords: Pseudomonas aeruginosa (P. aeruginosa); bacteria; biofilm development; bioreporter; cyclic di-GMP (c-di-GMP); development; image based quantification; nitric oxide