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

Nair, HAS; Periasamy, S; Yang, L; Kjelleberg, S; Rice, SA

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

Nair, HAS Periasamy, S Yang, L Kjelleberg, S Rice, SA

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Publication Type:: Journal Article
Citation:: Journal of Biological Chemistry, 2017, 292 (2), pp. 477 - 487
Issue Date:: 2017-01-13

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Field	Value	Language
dc.contributor.author	Nair, HAS	en_US
dc.contributor.author	Periasamy, S	en_US
dc.contributor.author	Yang, L	en_US
dc.contributor.author	Kjelleberg, S	en_US
dc.contributor.author	Rice, SA https://orcid.org/0000-0002-9486-2343	en_US
dc.date.available	2020-05-25T19:18:12Z
dc.date.issued	2017-01-13	en_US
dc.identifier.citation	Journal of Biological Chemistry, 2017, 292 (2), pp. 477 - 487	en_US
dc.identifier.issn	0021-9258	en_US
dc.identifier.uri	http://hdl.handle.net/10453/114721
dc.description.abstract	© 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.	en_US
dc.relation.ispartof	Journal of Biological Chemistry	en_US
dc.relation.isbasedon	10.1074/jbc.M116.746743	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Biofilms	en_US
dc.subject.mesh	Pseudomonas aeruginosa	en_US
dc.subject.mesh	Green Fluorescent Proteins	en_US
dc.subject.mesh	Cyclic GMP	en_US
dc.title	Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development	en_US
dc.type	Journal Article
utslib.citation.volume	2	en_US
utslib.citation.volume	292	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	1101 Medical Biochemistry and Metabolomics	en_US
utslib.for	0301 Analytical Chemistry	en_US
utslib.for	03 Chemical Sciences	en_US
utslib.for	06 Biological Sciences	en_US
utslib.for	11 Medical and Health Sciences	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	open_access
pubs.issue	2	en_US
pubs.publication-status	Published	en_US
pubs.volume	292	en_US

Abstract:

© 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.

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http://hdl.handle.net/10453/114721