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HPLC-based quantifi...
HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp
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- Varik, Vallo (författare)
- Umeå universitet,Institutionen för molekylärbiologi (Medicinska fakulteten),Molekylär Infektionsmedicin, Sverige (MIMS),University of Tartu, Institute of Technology, Nooruse 1, 50411, Tartu, Estonia
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Oliveira, Sofia Raquel Alves (författare)
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- Hauryliuk, Vasili (författare)
- Umeå universitet,Institutionen för molekylärbiologi (Medicinska fakulteten),Molekylär Infektionsmedicin, Sverige (MIMS),University of Tartu, Institute of Technology, Nooruse 1, 50411, Tartu, Estonia
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Tenson, Tanel (författare)
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(creator_code:org_t)
- 2017-09-08
- 2017
- Engelska.
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
- Relaterad länk:
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https://doi.org/10.1...
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https://umu.diva-por... (primary) (Raw object)
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https://doi.org/10.1...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi -- Mikrobiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Microbiology (hsv//eng)
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