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HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). University of Tartu, Institute of Technology, Nooruse 1, 50411, Tartu, Estonia.ORCID iD: 0000-0001-6900-6986
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). University of Tartu, Institute of Technology, Nooruse 1, 50411, Tartu, Estonia.
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 11022Article in journal (Refereed) Published
Abstract [en]

Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved experiments feasible. The method combines cell harvesting by rapid filtration, followed by acid extraction, freeze-drying with chromatographic separation. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labelling with 32P-labelled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40-and >= 8-fold, respectively) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017. Vol. 7, article id 11022
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Microbiology
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URN: urn:nbn:se:umu:diva-139786DOI: 10.1038/s41598-017-10988-6ISI: 000409841100084PubMedID: 28887466OAI: oai:DiVA.org:umu-139786DiVA, id: diva2:1146703
Available from: 2017-10-03 Created: 2017-10-03 Last updated: 2017-10-03Bibliographically approved

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Varik, ValloHauryliuk, Vasili
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