HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp
Varik, Vallo ; Oliveira, Sofia Raquel Alves ; Hauryliuk, Vasili LU
and Tenson, Tanel
(2017)
In Scientific Reports
7(1).
- Abstract
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... (More)
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.
(Less)
- author
- Varik, Vallo
; Oliveira, Sofia Raquel Alves
; Hauryliuk, Vasili
LU
and Tenson, Tanel
- publishing date
- 2017-12-01
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Scientific Reports
- volume
- 7
- issue
- 1
- article number
- 11022
- publisher
- Nature Publishing Group
- external identifiers
-
- scopus:85029008450
- pmid:28887466
- ISSN
- 2045-2322
- DOI
- 10.1038/s41598-017-10988-6
- language
- English
- LU publication?
- no
- additional info
- Funding Information: We are grateful to Michael Cashel, Andrei Chabes and Lisette Marjavaara for helpful discussions, Stoyan Tankov and Pavel Kudrin for preparing the ppGpp, Martin Lepiku for participating in the earlier stages of this project and Niilo Kaldalu for his expert help with microscopy. This work was supported by the Estonian Research Council (grant IUT2-22 to TT); the European Regional Development Fund through the Centre of Excellence for Molecular Cell Technology (VH and TT); the Swedish Research council (Vetenskapsrådet) (grant 2013-4680 to VH); Kempe and Ragnar Söderberg foundations (VH). Publisher Copyright: © 2017 The Author(s). Copyright: Copyright 2017 Elsevier B.V., All rights reserved.
- id
- 579b6057-901e-4f55-a4ab-f17c49faf9d1
- date added to LUP
- 2021-09-24 20:41:12
- date last changed
- 2024-07-28 21:28:41
@article{579b6057-901e-4f55-a4ab-f17c49faf9d1,
abstract = {{<p>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.</p>}},
author = {{Varik, Vallo and Oliveira, Sofia Raquel Alves and Hauryliuk, Vasili and Tenson, Tanel}},
issn = {{2045-2322}},
language = {{eng}},
month = {{12}},
number = {{1}},
publisher = {{Nature Publishing Group}},
series = {{Scientific Reports}},
title = {{HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp}},
url = {{http://dx.doi.org/10.1038/s41598-017-10988-6}},
doi = {{10.1038/s41598-017-10988-6}},
volume = {{7}},
year = {{2017}},
}