Nair Harikrishnan A S, Periasamy Saravanan, Yang Liang, Kjelleberg Staffan, Rice Scott A
From the Singapore Centre for Environmental Life Sciences Engineering.
Interdisciplinary Graduate School, and.
J Biol Chem. 2017 Jan 13;292(2):477-487. doi: 10.1074/jbc.M116.746743. Epub 2016 Nov 29.
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.
双（3'-5'）-环二鸟苷单磷酸（c-di-GMP）是一种动态的细胞内信号分子，在生物膜生命周期中起着核心作用。目前用于定量c-di-GMP的方法通常基于化学提取，这是一种终点测量方法。化学方法也没有考虑生物膜的生理异质性，因此代表的是整个生物膜的平均c-di-GMP浓度。为了解决这些问题，基于在c-di-GMP响应性cdrA启动子控制下绿色荧光蛋白（GFP）的表达，开发了一种基于比率、基于图像的定量方法（Rybtke，M. T.，Borlee，B. R.，Murakami，K.，Irie，Y.，Hentzer，M.，Nielsen，T. E.，Givskov，M.，Parsek，M. R.，和Tolker-Nielsen，T.（2012）应用环境微生物学78，5060 - 5069）。该方法使用青色荧光蛋白（CFP）作为生物量指标，GFP作为c-di-GMP报告基因。因此，CFP/GFP比率给出了每生物量的有效c-di-GMP。应用二元掩膜来减轻背景荧光，并针对已知的c-di-GMP浓度对荧光进行校准。使用流动池形成生物膜，c-di-GMP在生物膜中呈现非均匀分布，存在c-di-GMP的集中热点。此外，与早期小菌落中的均匀分布相比，发现c-di-GMP定位于成熟菌落的外边界。这些数据证明了一种用于原位实时定量c-di-GMP方法的应用，并表明这种生物膜调节第二信使的量随时间和菌落大小动态变化，实时反映了生物膜异质性的程度。
