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 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.
Simm
Roger
R
Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
Morr
Michael
M
Remminghorst
Uwe
U
Andersson
Mats
M
Römling
Ute
U
D001419:Bacteria / Q000737:chemistry*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000032:analysis*; D008722:Methods; D009711:Nucleotides; D012107:Research Design; D015290:Second Messenger Systems; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009711:Nucleotides; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP