Quantitative determination of cyclic diguanosine monophosphate concentrations in nucleotide extracts of bacteria by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry
The physiological response to small molecules (secondary messengers) is the outcome of a delicate equilibrium between biosynthesis and degradation of the signal. Cyclic diguanosine monophosphate (c-di-GMP) is a novel secondary messenger present in many bacteria. It has a complex cellular metabolism...
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Veröffentlicht in: | Analytical biochemistry 2009-03, Vol.386 (1), p.53-58 |
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Sprache: | eng |
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Zusammenfassung: | The physiological response to small molecules (secondary messengers) is the outcome of a delicate equilibrium between biosynthesis and degradation of the signal. Cyclic diguanosine monophosphate (c-di-GMP) is a novel secondary messenger present in many bacteria. It has a complex cellular metabolism whereby usually more than one enzyme synthesizing and degrading c-di-GMP is encoded by a bacterial genome. To assess the in vivo conditions of c-di-GMP signaling, we developed a high-performance liquid chromatography (HPLC)–mass spectrometry-based method to detect c-di-GMP with high sensitivity and to quantify the c-di-GMP concentration in the bacterial cell as described here in detail. We successfully used the methodology to determine and compare the c-di-GMP concentrations in bacterial species such as
Salmonella typhimurium,
Escherichia coli,
Pseudomonas aeruginosa, and
Vibrio cholerae. We describe the use of the methodology to assess the change in c-di-GMP concentration during the growth phase and the contribution of a point mutation in
S. typhimurium to the overall cellular c-di-GMP concentration. |
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ISSN: | 0003-2697 1096-0309 1096-0309 |
DOI: | 10.1016/j.ab.2008.12.013 |