In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate).
The intracellular alarmone guanosine 3',5'-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone's complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. (13)C NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp.
Hardiman
Timo
T
Institute of Biochemical Engineering, University of Stuttgart, D-70569 Stuttgart, Germany.
Windeisen
Volker
V
Ewald
Jennifer C
JC
Zibek
Susanne
S
Schlack
Petra
P
Rebell
Jochen
J
Reuss
Matthias
M
Siemann-Herzberg
Martin
M
D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D006159:Guanosine Tetraphosphate / Q000096:biosynthesis* / Q000378:metabolism*; D010766:Phosphorylation; D012266:Ribose / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D006159:Guanosine Tetraphosphate; D012266:Ribose