Name: using microtiter dish radiolabeling for multiple in vivo measurements of escherichia coli (p)ppgpp followed by thin layer chromatography
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This research was supported the Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH.

1. Media Preparation
<ol>
	<li>For MOPS (3-(N-morpholino)propanesulfonic acid) media25, use 1/10 volume of 10x MOPS salts, 1/100 volume of 100x micronutrients solution, 3 mM sodium phosphate for overnight cultures or 0.2 mM sodium phosphate for uniform labeling with 32P, 0.2% glucose and 1 &#181;g/mL thiamin (vitamin B1). Add amino acids at 40 &#181;g/mL if required.
	<ol>
		<li>For MOPS salts, use 400 mM MOPS, 40 mM Tricine, 0.1 mM FeSO4, 95 mM NH4</s...

<ol>
	<li>Cashel, M., Gentry, D., Hernandez, V., Vinella, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stringent+Response.">The stringent Response.</a> Escherichia coli and Salmonella: Cellular and Molecular Biology. , 1458-1489 (1996).</li><li>Magnusson, L. U., Farewell, A., Nystr&#246;m, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp:+a+global+regulator+in+Escherichia+coli.">ppGpp: a global regulator in Escherichia coli.</a> Trends in Microbiology. 13 (5), 236-242 (2005).</li><li>Cashel, M., Gallant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+Compounds+implicated+in+the+Function+of+the+RC+Gene+of+Escherichia+coli.">Two Compounds implicated in the Function of the RC Gene of Escherichia coli.</a> Nature. 221 (5183), 838-841 (1969).</li><li>Braeken, K., Moris, M., Daniels, R., Vanderleyden, J., Michiels, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+horizons+for+(p)ppGpp+in+bacterial+and+plant+physiology.">New horizons for (p)ppGpp in bacterial and plant physiology.</a> Trends in Microbiology. 14 (1), 45-54 (2006).</li><li>Atkinson, G. C., Tenson, T., Hauryliuk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RelA/SpoT+homolog+(RSH)+superfamily:+distribution+and+functional+evolution+of+ppGpp+synthetases+and+hydrolases+across+the+tree+of+life.">The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life.</a> PloS One. 6 (8), e23479 (2011).</li><li>Mechold, U., Potrykus, K., Murphy, H., Murakami, K. S., Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+by+ppGpp+versus+pppGpp+in+Escherichia+coli.">Differential regulation by ppGpp versus pppGpp in Escherichia coli.</a> Nucleic Acids Research. 41 (12), 6175-6189 (2013).</li><li>Molodtsov, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+Effector+ppGpp+Potentiates+the+Inhibition+of+Transcript+Initiation+by+DksA.">Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA.</a> Molecular Cell. 69 (5), 828-839 (2018).</li><li>Ross, W., Sanchez-Vazquez, P., Chen, A. Y. Y., Lee, J. -. H. H., Burgos, H. L. L., Gourse, R. L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp+Binding+to+a+Site+at+the+RNAP-DksA+Interface+Accounts+for+Its+Dramatic+Effects+on+Transcription+Initiation+during+the+Stringent+Response.">ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response.</a> Molecular Cell. 62 (6), 811-823 (2016).</li><li>Kriel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Regulation+of+GTP+Homeostasis+by+(p)ppGpp:+A+Critical+Component+of+Viability+and+Stress+Resistance.">Direct Regulation of GTP Homeostasis by (p)ppGpp: A Critical Component of Viability and Stress Resistance.</a> Molecular Cell. 48 (2), 231-241 (2012).</li><li>Kriel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP+dysregulation+in+Bacillus+subtilis+cells+lacking+(p)ppGpp+results+in+phenotypic+amino+acid+auxotrophy+and+failure+to+adapt+to+nutrient+downshift+and+regulate+biosynthesis+genes.">GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.</a> Journal of Bacteriology. 196 (1), 189-201 (2014).</li><li>Potrykus, K., Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p)ppGpp:+still+magical.">p)ppGpp: still magical.</a> Annual Review of Microbiology. 62, 35-51 (2008).</li><li>Hauryliuk, V., Atkinson, G. C., Murakami, K. S., Tenson, T., Gerdes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+functional+insights+into+the+role+of+(p)ppGpp+in+bacterial+physiology.">Recent functional insights into the role of (p)ppGpp in bacterial physiology.</a> Nature Reviews Microbiology. 13 (5), 298-309 (2015).</li><li>Arenz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stringent+factor+RelA+adopts+an+open+conformation+on+the+ribosome+to+stimulate+ppGpp+synthesis.">The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis.</a> Nucleic Acids Research. 44 (13), 6471-6481 (2016).</li><li>Loveland, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome&#8226;RelA+structures+reveal+the+mechanism+of+stringent+response+activation.">Ribosome&#8226;RelA structures reveal the mechanism of stringent response activation.</a> eLife. 5, e17029 (2016).</li><li>Brown, A., Fern&#225;ndez, I. S., Gordiyenko, Y., Ramakrishnan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome-dependent+activation+of+stringent+control.">Ribosome-dependent activation of stringent control.</a> Nature. 534 (7606), 277-280 (2016).</li><li>Battesti, A., Bouveret, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyl+carrier+protein/SpoT+interaction,+the+switch+linking+SpoT-dependent+stress+response+to+fatty+acid+metabolism.">Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism.</a> Molecular Microbiology. 62 (4), 1048-1063 (2006).</li><li>Lee, J. -. W., Park, Y. -. H., Seok, Y. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rsd+balances+(p)ppGpp+level+by+stimulating+the+hydrolase+activity+of+SpoT+during+carbon+source+downshift+in+Escherichia+coli.">Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (29), E6845-E6854 (2018).</li><li>Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+(p)ppGpp+Accumulation+Patterns+in+Escherichia+coli+mutants.">Detection of (p)ppGpp Accumulation Patterns in Escherichia coli mutants.</a> Molecular Microbiology Techniques. 3, 341-356 (1994).</li><li>Fern&#225;ndez-Coll, L., Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+SpoT+Hydrolase,+SpoT+Synthetase,+and+RelA+Synthetase+to+Carbon+Source+Diauxic+Growth+Transitions+in+Escherichia+coli.">Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.</a> Frontiers in Microbiology. , (2018).</li><li>Varik, V., Oliveira, S. R. A., Hauryliuk, V., Tenson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC-based+quantification+of+bacterial+housekeeping+nucleotides+and+alarmone+messengers+ppGpp+and+pppGpp.">HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp.</a> Scientific Reports. 7 (1), 11022 (2017).</li><li>Rhee, H. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Fluorescent+Chemosensor+for+the+Bacterial+Alarmone+(p)ppGpp.">Selective Fluorescent Chemosensor for the Bacterial Alarmone (p)ppGpp.</a> J. Am. Chem. Soc. 130 (3), 784-785 (2008).</li><li>Wang, J., Chen, W., Liu, X., Wesdemiotis, C., Pang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mononuclear+Zinc+Complex+for+Selective+Detection+of+Diphosphate+via+Fluorescence+ESIPT+Turn-On.">A Mononuclear Zinc Complex for Selective Detection of Diphosphate via Fluorescence ESIPT Turn-On.</a> Journal of Materials Chemistry B. 2 (21), 3349-3354 (2014).</li><li>Pokhilko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+nutrient+limitation+in+growing+E.+coli:+a+mathematical+model+of+a+ppGpp-based+biosensor.">Monitoring of nutrient limitation in growing E. coli: a mathematical model of a ppGpp-based biosensor.</a> BMC Systems Biology. 11 (1), 106 (2017).</li><li>Funabashi, H., Mie, M., Yanagida, Y., Kobatake, E., Aizawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+monitoring+of+cellular+physiological+status+depending+on+the+accumulation+of+ppGpp.">Fluorescent monitoring of cellular physiological status depending on the accumulation of ppGpp.</a> Biotechnology Letters. 24 (4), 269-273 (2002).</li><li>Neidhardt, F. C., Bloch, P. L., Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+medium+for+enterobacteria.">Culture medium for enterobacteria.</a> Journal of Bacteriology. 119 (3), 736-747 (1974).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Peselis, A., Serganov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ykkC+riboswitches+employ+an+add-on+helix+to+adjust+specificity+for+polyanionic+ligands.">ykkC riboswitches employ an add-on helix to adjust specificity for polyanionic ligands.</a> Nature Chemical Biology. 14 (9), 887-894 (2018).</li><li>Oki, T., Yoshimoto, A., Sato, S., Takamatsu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purine+nucleotide+pyrophosphotransferase+from+Streptomyces+morookaensis,+capable+of+synthesizing+pppApp+and+pppGpp.">Purine nucleotide pyrophosphotransferase from Streptomyces morookaensis, capable of synthesizing pppApp and pppGpp.</a> Biochimica et Biophysica Acta (BBA) - Enzymology. 410 (2), 262-272 (1975).</li><li>Travers, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppApp+alters+transcriptional+selectivity+of+Escherichia+coli+RNA+polymerase.">ppApp alters transcriptional selectivity of Escherichia coli RNA polymerase.</a> FEBS Letters. 94 (2), 345-348 (1978).</li><li>Bruhn-Olszewska, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+comparisons+of+(p)ppApp+vs+(p)ppGpp+for+Escherichia+coli+RNA+polymerase+binding+sites+and+for+rrnB+P1+promoter+regulatory+responses+in+vitro.">Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro.</a> Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1861 (8), 731-742 (2018).</li></ol>

Achieving near uniform labeling of the cells is a critical step for this protocol. Therefore, the use of defined media, such as MOPS or Tris media, is crucial to allow variation of carrier phosphate concentrations and specific activity. Phosphate buffered media, such as M9 or media A, cannot be used. Most undefined media contain variable amounts of phosphate, such as LB, tryptone and casamino acids. The phosphate isotope 33P is a weaker emitter that can be substituted for 32P. Advantages of the subs...

The (p)ppGpp second messenger is a global regulator that modulates the expression of a large number of genes, including genes for synthesizing ribosomes and amino acids1,2. Although initially discovered in Escherichia coli3, (p)ppGpp can be found in both Gram positive and Gram negative bacteria as well as in plant chloroplasts4,5. For E. coli and other Gram-negative bacteria, (p)ppGpp interacts directly with RNA polymerase at two different sites6,7,8. In Gram positives, (p)ppGpp inhibits GTP abundance, which is sensed by CodY, a GTP-binding protein with gene-specific DNA recognition motifs that lead to regulation9,10. (p)ppGpp accumulates in response to starvation for different nutrients and stress conditions, resulting in slow growth and adjustments of gene expression to allow adaptation to stress11,12.
The net amount of ppGpp accumulated reflects a balance between synthetase and hydrolase activities. In E. coli RelA is a strong synthetase and SpoT is bifunctional, with a strong hydrolase and a weak synthetase, each of which might be regulated differently in a stress dependent manner. The strong RelA synthetase is activated when the availability of codon-specified charged tRNA bound to the ribosomal A site when it fails to keep up with the demands of protein synthesis13,14,15. The weak SpoT (p)ppGpp synthetase is activated while the strong (p)ppGpp hydrolase is inhibited in response to other stress conditions and through other mechanisms. Under some conditions, proteins such as ACP or Rsd can bind to SpoT, which also change the balance between hydrolysis and synthesis16,17. In Gram positives, synthesis and hydrolysis reflect a more complex balance between a single RelA SpoT homolog (RSH) protein with strong synthesis and hydrolysis activities as well as smaller hydrolases and/or synthetases12.
The (p)ppGpp nucleotides were first discovered as unusual 32P labeled spots that appeared on autoradiograms of thin-layer chromatograms (TLC) during a stringent response induced by amino acid starvation3. More detailed labeling protocols have been reviewed 18. The protocol described here (Figure 1) is a modification of these protocols that allows monitoring growth of multiple samples on microtiter plates. This facilitates multiple biological and technical estimates of (p)ppGpp abundance changes and was initially developed for studies&#160; of diauxic shifts19. Labelling of (p)ppGpp with 32P and detection by TLC also allows measurements of (p)ppGpp degradation rates. Alternative methods have been developed to determine (p)ppGpp levels such as mass spectrometry, &#160;HPLC20, fluorescent chemosensors21,22, and GFP gene fusions to promoters affected by ppGpp23,24. Fluorescent chemosensors currently have a limited use due small spectral shifts after binding ppGpp as well as problems distinguishing between ppGpp and pppGpp21. This method is efficient to detect (p)ppGpp in vitro, but not in cellular extracts. Methods involving HPLC have been improved20 but require expensive equipment and are not well adapted to high through-put. Finally, GFP fusions can give an estimate of ppGpp dependent activation or inhibition, but do not measure ppGpp itself. While each of these alternative methods are valuable, they require expensive equipment or substantial hands-on time, or they are otherwise not amenable to multiple kinetic sampling and subsequent processing. With the method described here, 96 samples can be applied to six TLC plates in about 20 min (18 samples per plate), resolved by TLC development in less than a couple of hours, with quantitative data obtained after several hours or overnight, depending on labeling intensity.

<table>
	<tbody>
		<tr>
			<td>(NH4)6(MO7)24</td>
			<td>Fisher Scientifics</td>
			<td>A-674</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>Denville scientific inc.</td>
			<td>E3218</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>J.T.Baker</td>
			<td>1-1309</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>RPI</td>
			<td>C61000-25.0</td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl2</td>
			<td>Fisher Scientifics</td>
			<td>C-371</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>J.T.Baker</td>
			<td>1843</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4</td>
			<td>Fisher Scientifics</td>
			<td>I-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Fisher Biotech</td>
			<td>BP1215-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Macron</td>
			<td>4912-12</td>
			<td></td>
		</tr>
		<tr>
			<td>H3BO4</td>
			<td>Macron</td>
			<td>2549-04</td>
			<td></td>
		</tr>
		<tr>
			<td>H3PO4</td>
			<td>J.T.Baker</td>
			<td>0260-02</td>
			<td></td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>Sigma</td>
			<td>P9458-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Biotech</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Sigma</td>
			<td>V-6504</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Scientifics</td>
			<td>FL-06-0303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader Synergy HT</td>
			<td>Biotek</td>
			<td>Synergy HT</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma</td>
			<td>M-9522</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Mallinckrodt</td>
			<td>7892</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>J.T.Baker</td>
			<td>3624-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Mallinckrodt</td>
			<td>7917</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma</td>
			<td>A0171-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>P-32 radionuclide, orthophosphoric acid in 1 mL water (5 mCi)</td>
			<td>Perkin Elmer</td>
			<td>NEX053005MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage phosphor screen</td>
			<td>Kodak</td>
			<td>So230</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Sigma</td>
			<td>T-4625</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC PEI Cellulose F</td>
			<td>Merk-Millipore</td>
			<td>1.05579.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>RPI</td>
			<td>T2400-500.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon 9400 imager</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Fisher Scientifics</td>
			<td>Z-68</td>
			<td></td>
		</tr>
	</tbody>
</table>

using microtiter dish radiolabeling for multiple in vivo measurements of escherichia coli (p)ppgpp followed by thin layer chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

In E. coli K-12 strains, the addition of valine provokes an endogenous starvation of isoleucine, which results an increase of ppGpp levels after 5 min3. Cells grown in MOPS containing all amino acids except for ILV were labeled with 32P as indicated in Figure 1. Once labelled, 6 &#181;L of 10 mg/mL L-valine (100 &#181;g/mL final concentration) was added to produce isoleucine starvation. Samples were taken 0 and 5 min after the addition of va...

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Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

This method measures the levels of the global regulator ppGpp within different growth conditions and stress situations. PpGpp can be found in gram positive and negative bacteria, as well as in chloroplast. PpGpp has a rapid onset in seconds, which lead to a fast second relation once stress is produced.Simultaneously, it has a short half-life, up to 30 seconds. Therefore, there is a need to monitor many culture at time intervals that may vary from 10 seconds to hours, including stressed transitions. We also radiolabel bacterial cultures in microtiter dishes facilitates high throughput samplings that allow multiple technical and biological replicates.Make sure to follow the proper safety measures when working with radioactive materials. To begin this procedure, prepare all media as outlined in the text protocol. Grow overnight cultures in MOPS media with three millimolar sodium phosphate.Add 550 microliters of MOPS media, contining 0.2 micromolar sodium phosphate carrier and 30 microliters of each bacterial inoculum to the wells of a 24 well plate. This will result in an initial inoculum with an OD600 of 0.1. Add fresh media to one well as a sterility control.Place the plate in a shaking incubator at 37 degrees celsius, shaking at 900 RPM for 30 minutes. Growth can be monitored using a plate reader with a replicate plate in parallel in media lacking p32 using a plate reader while incubating under the same conditions. Next, add 100 microcuries of p32 orthphosphate to each well of the plate.Grow the cultures in a shaking incubator for one to two doublings to allow external label to largely equilibrate with intercellular nucleotide pools. For proper visualization, none of the active material was used in this video. In a real experiment, proper shielding is required as well as constant monitoring for contamination with a survey meter.First, induce stress using a method appropriate for the sort of stress studied. Next, add 20 microliters of each labeled cell sample to a separate 0.2 milliliter PCR tube, containing 20 microliters of ice cold six molar formic acid. Immediately place the samples on dry ice.Enhance cellular extraction efficiency by three cycles of freezing and thawing. Just before spotting PEI cellulose thin layer chromatograms, centrifuge the samples for one minute at maximum speed to pellet cell debris. First, set out a 20 by 20 centimeter PEI cellulose TLC plate.Use scissors to remove the top five centimeters. And use a soft pencil to mark an origin line one centimeter from the edge. Apply a five microliter droplet of each sample to the PEI surface.Before the spot can dry, transfer the plate to a tank containing a layer of 1.5 molar monopotassium phosphate that is shallow enough to not touch the origin sample spot. Cover the tank with an airtight seal, and allow liquid ascent to the top of the 15 centimeter trimmed sheet. After this, remove the fully developed chromatogram and air dry it at room temperature.Cut and discard the top portion of the chromatogram containing the free P32 into radioactive waste. Use a phosphor screen to expose the audioradiographic films overnight. The next day, develop the film and use a phosphorimager to capture and quantitate the phosphor's green signal.Then, use ImageJ software to quantitate the radioactive spots. After provoking stress or starvation for time sufficient to allow ppGpp accumulation, use an appropriate method to reverse the stress. Take samples every 20 to 30 seconds for up to two minutes and process the samples as previously described.Once the TLC is developed, and the levels of ppGpp are obtained during the time course, plot the residual ppGpp content on a semilogarithmic plot versus time. In this study, cells grown in MOPS containing all amino acids except for ILV are labeled with P32. Once labeled, L valine is added to produce isoleucine starvation.After five minutes, a two and two and a half old increase in the levels of tetra and pentaphosphate of guanosine occurred. A negative control of a cell free labeled sample is used to detect possible compounds that are not orthophosphate in the P32 source. Reversal of isoleucine starvation can be achieved by chloramphenicol, an inhibitor of protein synthesis, which reduces the consumption of charged isoleucine tRNA, and in turn, restores high ratios of charged to uncharged tRNA.Activation of the strong rela-mediated ppGpp synthetase is abolished, which allows a measure of ppGpp degradation unperturbed by residual synthesis. In this case, ppGpp decays with a half-life of approximately 64 seconds. It is important to let the cells grow for a couple of generations to achieve a uniform labeling before inducing any stress or starvation.Because ppGpp is widely spread among bacteria, this method can be applied to other organisms. Modification of the TLC development method could allow the proper separation of all the regulatory nucleotides, such as ppApp. P52 is a better emitter isotope, so it is important to use proper shielding and to properly discard any residue.

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