PMID- 27899451
OWN - NLM
STAT- MEDLINE
DCOM- 20170601
LR  - 20210205
IS  - 1083-351X (Electronic)
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 292
IP  - 2
DP  - 2017 Jan 13
TI  - Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during 
      Biofilm Development.
PG  - 477-487
LID - 10.1074/jbc.M116.746743 [doi]
AB  - 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.
CI  - (c) 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
FAU - Nair, Harikrishnan A S
AU  - Nair HA
AD  - From the Singapore Centre for Environmental Life Sciences Engineering.
AD  - Interdisciplinary Graduate School, and.
FAU - Periasamy, Saravanan
AU  - Periasamy S
AD  - From the Singapore Centre for Environmental Life Sciences Engineering.
FAU - Yang, Liang
AU  - Yang L
AD  - From the Singapore Centre for Environmental Life Sciences Engineering.
AD  - School of Biological Sciences, Nanyang Technological University, Singapore 
      637551, Singapore.
FAU - Kjelleberg, Staffan
AU  - Kjelleberg S
AD  - From the Singapore Centre for Environmental Life Sciences Engineering.
AD  - School of Biological Sciences, Nanyang Technological University, Singapore 
      637551, Singapore.
FAU - Rice, Scott A
AU  - Rice SA
AUID- ORCID: 0000-0002-9486-2343
AD  - From the Singapore Centre for Environmental Life Sciences Engineering, 
      rscott@ntu.edu.sg.
AD  - School of Biological Sciences, Nanyang Technological University, Singapore 
      637551, Singapore.
LA  - eng
PT  - Journal Article
DEP - 20161129
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - 61093-23-0 (bis(3',5')-cyclic diguanylic acid)
RN  - H2D2X058MU (Cyclic GMP)
SB  - IM
MH  - Biofilms/*growth & development
MH  - Cyclic GMP/*analogs & derivatives/genetics/metabolism
MH  - Green Fluorescent Proteins/genetics/metabolism
MH  - Pseudomonas aeruginosa/*physiology
PMC - PMC5241725
OTO - NOTNLM
OT  - Pseudomonas aeruginosa (P. aeruginosa)
OT  - bacteria
OT  - biofilm development
OT  - bioreporter
OT  - cyclic di-GMP (c-di-GMP)
OT  - development
OT  - image based quantification
OT  - nitric oxide
EDAT- 2016/12/03 06:00
MHDA- 2017/06/02 06:00
PMCR- 2018/01/13
CRDT- 2016/12/01 06:00
PHST- 2016/07/05 00:00 [received]
PHST- 2016/11/28 00:00 [revised]
PHST- 2016/12/03 06:00 [pubmed]
PHST- 2017/06/02 06:00 [medline]
PHST- 2016/12/01 06:00 [entrez]
PHST- 2018/01/13 00:00 [pmc-release]
AID - S0021-9258(20)40181-4 [pii]
AID - M116.746743 [pii]
AID - 10.1074/jbc.M116.746743 [doi]
PST - ppublish
SO  - J Biol Chem. 2017 Jan 13;292(2):477-487. doi: 10.1074/jbc.M116.746743. Epub 2016 
      Nov 29.