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Chapter 6 : Methods for Cyclic Di-GMP Detection

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Abstract:

The first decade of the 21st century has seen an explosion of interest in the bacterial second messenger signal cyclic di-GMP (c-di-GMP). The study of c-di-GMP during this period has been fueled by the development of methods to visualize and quantify this molecule. This chapter reviews the history of the development of methods for c-di-GMP detection, summarizes the currently available technologies, and highlights the new challenges that must be overcome to deepen one's understanding of this molecule and its actions. The methods to measure and quantify c-di-GMP from living cells have sprung forth from the early work of the Benziman laboratory. Two major approaches, two-dimensional thin-layer chromatography (2D-TLC) and HPLC-MS-MS, currently dominate the approaches to quantify the intracellular levels of c-di-GMP, and it is likely that these two techniques will remain important tools in the toolboxes of researchers in this field. However, to gain a further understanding of this signaling molecule, new technologies measuring c-di-GMP at the single-cell level and in localized regions of the cells must be developed. The development of the new technologies will ensure the continuing renaissance in the study of c-di-GMP signaling.

Citation: Waters C. 2010. Methods for Cyclic Di-GMP Detection, p 68-75. In Wolfe A, Visick K (ed), The Second Messenger Cyclic Di-GMP. ASM Press, Washington, DC. doi: 10.1128/9781555816667.ch6

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High-Performance Liquid Chromatography
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Figures

Image of Figure 1.
Figure 1.

Measurement of c-di-GMP by 2D-TLC. Nucleotides from labeled with P were first resolved using 0.2 MNHHCO, pH 7.8. After drying, the plate was turned counterclockwise and exposed to 1.5 M KHPO, pH 3.65. Radioactive molecules were detected by exposing the plate to film. The c-di-GMP spot is indicated by the solid arrow, while the GDP spot is shown by the dotted arrow. In panel A, the extract from wild-type is shown. This condition is known to be low in c-di-GMP due to the phosphodiesterase activity of the EAL type protein, VieA, and thus, no visible c-di-GMP is observed. In panel B, VieA has been rendered inactive through site-directed mutagenesis, and a c-di-GMP spot is now visible. In panel C, a GGDEF protein, VCA0956, is overexpressed, and a large spot of c-di-GMP can be observed. Reprinted from ( ) with permission.

Citation: Waters C. 2010. Methods for Cyclic Di-GMP Detection, p 68-75. In Wolfe A, Visick K (ed), The Second Messenger Cyclic Di-GMP. ASM Press, Washington, DC. doi: 10.1128/9781555816667.ch6
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Image of Figure 2.
Figure 2.

HPLC-MS-MS. (A) This cartoon depicts an HPLC-MS-MS triple quadrupole instrument. Nucleotide extracts are separated based on polarity via HPLC and analyzed in real time. The first quadrupole (Q1) isolates molecules of a specific the molecules are fragmented in the collision cell, and the fragmentation products are analyzed in the third quadrupole (Q3). (B) c-di-GMP is predicted to fragment into three specific products with / values of 540, 344, and 150.

Citation: Waters C. 2010. Methods for Cyclic Di-GMP Detection, p 68-75. In Wolfe A, Visick K (ed), The Second Messenger Cyclic Di-GMP. ASM Press, Washington, DC. doi: 10.1128/9781555816667.ch6
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Image of Figure 3.
Figure 3.

c-di-GMP measured by HPLC-MS-MS. A chemically synthesized c-di-GMP standard (top two boxes) is compared next to nucleotide extracts from (bottom two boxes). The left panels show fragmentation products identified by selected reaction monitoring (SRM) analysis with an / of 344 originating from ions with an / of 689. Likewise, the right panels show fragmentation products with an of 150. The axis indicates the time that each molecule was retained on the HPLC. Notice that the c-di-GMP peak from the extracts elutes from the HPLC at the identical time as the standard. The relative amount of c-di-GMP can be quantified by determining the area under the peak. The molecule in the starred peak also generates both fragmentation products, but it elutes from the HPLC at a different time than the chemical standard. Orbitrap MS analysis showed that this molecule is not c-di-GMP. Reprinted from ( ) with permission.

Citation: Waters C. 2010. Methods for Cyclic Di-GMP Detection, p 68-75. In Wolfe A, Visick K (ed), The Second Messenger Cyclic Di-GMP. ASM Press, Washington, DC. doi: 10.1128/9781555816667.ch6
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Image of Figure 4.
Figure 4.

Putative fluorescence-based c-di-GMP sensor. The typical architecture of fluorescence-based sensors is the attachment of two compatible FRET-based protein partners to each end of a binding domain (CFP, cyan fluorescent protein; YFP, yellow fluorescent protein). In panel A, c-di-GMP is not bound to the receptor component of the sensor protein, resulting in no FRET signal because of the large distance between CFP and YFP. In panel B, binding of c-di-GMP to the receptor component decreases the distance of the FRET protein pairs, resulting in an increased FRET signal.

Citation: Waters C. 2010. Methods for Cyclic Di-GMP Detection, p 68-75. In Wolfe A, Visick K (ed), The Second Messenger Cyclic Di-GMP. ASM Press, Washington, DC. doi: 10.1128/9781555816667.ch6
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References

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1. Amikam, D., and, M. Benziman. 1989. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 171:66496655.
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8. Sudarsan, N.,, E. R. Lee,, Z. Weinberg,, R. H. Moy,, J. N. Kim,, K. H. Link, and, R. R. Breaker. 2008. Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321:411413.
9. Tischler, A. D., and, A. Camilli. 2004. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Mol. Microbiol. 53:857869.
10. Tischler, A. D., and, A. Camilli. 2005. Cyclic diguanylate regulates Vibrio cholerae virulence gene expression. Infect. Immun. 73:58735882.
11. Waters, C. M.,, W. Lu,, J. D. Rabinowitz, and, B. L. Bassler. 2008. Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cyclic di-GMP levels and repression of vpsT. J. Bacteriol. 190:25272536.
12. Weinhouse, H.,, S. Sapir,, D. Amikam,, Y. Shilo,, G. Volman,, P. Ohana, and, M. Benziman. 1997. c-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum. FEBS Lett. 416:207211.

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