Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development
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. C...
| Main Authors: | , , , , |
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| Format: | Journal Article |
| Language: | English |
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2018
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| Online Access: | https://hdl.handle.net/10356/87406 http://hdl.handle.net/10220/44416 |
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| author | Nair, Harikrishnan A. S. Periasamy, Saravanan Yang, Liang Kjelleberg, Staffan Rice, Scott A. |
| author2 | Interdisciplinary Graduate School (IGS) |
| author_facet | Interdisciplinary Graduate School (IGS) Nair, Harikrishnan A. S. Periasamy, Saravanan Yang, Liang Kjelleberg, Staffan Rice, Scott A. |
| author_sort | Nair, Harikrishnan A. S. |
| collection | NTU |
| description | 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. |
| first_indexed | 2024-10-01T02:44:08Z |
| format | Journal Article |
| id | ntu-10356/87406 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T02:44:08Z |
| publishDate | 2018 |
| record_format | dspace |
| spelling | ntu-10356/874062020-09-21T11:31:58Z Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development Nair, Harikrishnan A. S. Periasamy, Saravanan Yang, Liang Kjelleberg, Staffan Rice, Scott A. Interdisciplinary Graduate School (IGS) School of Biological Sciences Singapore Centre for Environmental Life Sciences Engineering Bacteria Cyclic di-GMP (c-di-GMP) 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. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version 2018-02-07T03:48:55Z 2019-12-06T16:41:10Z 2018-02-07T03:48:55Z 2019-12-06T16:41:10Z 2017 Journal Article Nair, H. A. S., Periasamy, S., Yang, L., Kjelleberg, S., & Rice, S. A. (2017). Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development. Journal of Biological Chemistry, 292(2), 477-487. 0021-9258 https://hdl.handle.net/10356/87406 http://hdl.handle.net/10220/44416 10.1074/jbc.M116.746743 en Journal of Biological Chemistry This research was originally published in Journal of Biological Chemistry. Nair, H. A. S., Periasamy, S., Yang, L., Kjelleberg, S., & Rice, S. A. Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development. Journal of Biological Chemistry. 2017; 292(2): 477-487. © 2017 The American Society for Biochemistry and Molecular Biology (ASBMB). The published version is available at: [http://dx.doi.org/10.1074/jbc.M116.746743]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law. 11 p. application/pdf |
| spellingShingle | Bacteria Cyclic di-GMP (c-di-GMP) Nair, Harikrishnan A. S. Periasamy, Saravanan Yang, Liang Kjelleberg, Staffan Rice, Scott A. Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development |
| title | Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development |
| title_full | Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development |
| title_fullStr | Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development |
| title_full_unstemmed | Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development |
| title_short | Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development |
| title_sort | real time spatial and temporal mapping of the distribution of c di gmp during biofilm development |
| topic | Bacteria Cyclic di-GMP (c-di-GMP) |
| url | https://hdl.handle.net/10356/87406 http://hdl.handle.net/10220/44416 |
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