Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development

• Published in: The Journal of biological chemistry Vol. 292; no. 2; pp. 477 - 487
• Main Authors: Nair, Harikrishnan A.S., Periasamy, Saravanan, Yang, Liang, Kjelleberg, Staffan, Rice, Scott A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.01.2017; American Society for Biochemistry and Molecular Biology
• Subjects: bacteria; biofilm development; Biofilms - growth & development; bioreporter; cyclic di-GMP (c-di-GMP); Cyclic GMP - analogs & derivatives; Cyclic GMP - genetics; Cyclic GMP - metabolism; development; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; image based quantification; Microbiology; nitric oxide; Pseudomonas aeruginosa (P. aeruginosa); Pseudomonas aeruginosa - physiology; cyclic di-GMP (c-di-GMP); development; nitric oxide; bacteria; Pseudomonas aeruginosa (P. aeruginosa); bioreporter; biofilm development; image based quantification
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.746743

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.