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 NH4Cl, 2.6 mM K2SO4, 5 &#181;M CaCl2, 10.56 mM MgCl2, and 500 mM NaCl. Add the different components in the order written to avoid precipitation. Adjust to pH 7.4 with KOH and sterilize by filtration.</li>
		<li>For 100x micronutrients solution, use 3 &#181;M (NH4)6(MO7)24, 0.4 mM H3BO4, 30 &#181;M CoCl2, 10 &#181;M CuSO4, 80 &#181;M MnCl2, and 10 &#181;M ZnSO4. Sterilize by autoclaving.</li>
		<li>For glucose (20%) and sodium phosphate (300 mM) solutions, sterilize as separate 100x stocks by autoclaving. Sterilize 100x stocks of thiamine and amino acid mixtures by filtration. Prepare fresh media from sterile stock solutions for each experiment.</li>
	</ol>
	</li>
</ol>
2. Labelling with 32P
<ol>
	<li>Grow overnight cultures in MOPS media with 3 mM sodium phosphate.</li>
	<li>In a 24 well plate, add 550 &#181;L of MOPS media containing 0.2 mM sodium phosphate carrier and 30 &#181;L of each bacterial inoculum (dilution 1/20). This will produce an initial inoculum of OD600nm 0.1. Use one well with fresh media as a sterility control.</li>
	<li>Place the plate on a shaking incubator at 37 &#176;C shaking at 900 rpm for 30 min. Cultures are heated from below and shaking provides aeration. Attach the plate firmly with tape to avoid tilting or spills. Growth can be monitored with a replicate plate in parallel in media lacking 32P using a plate reader and incubating under the same conditions.</li>
	<li>Add 100 &#181;Ci to each well of 32P orthophosphate (20 &#181;L from a 5 mCi/mL stock). 
	NOTE: The amount of radioactivity can also be adjusted to meet experimental needs. For low basal levels 100 &#181;Ci with 0.2 mM carrier phosphate works well, but for measuring (p)ppGpp levels that are expected to become equivalent to GTP pools, label can be dropped to 25 or 50 &#181;Ci. Lowering carrier phosphate below 0.2 mM is not recommended to avoid phosphate levels becoming limiting for growth. 
	CAUTION: 32P is a &#946; emitting isotope so use proper shielding specified for safety. All radioactive material must be properly disposed taking all the possible precautions.</li>
	<li>Grow in shaking incubator for 1 to 2 doublings (1 h or more) to allow external label to largely equilibrate with intracellular nucleotide pools before inducing stress.</li>
</ol>
3. Induction of Stress or Starvation
<ol>
	<li>Induce stress using a method of your choosing, depending on the sort of stress studied, e.g. adding metabolic pathway inhibitors, changing temperature, exhausting required nutrients, shifting osmolarity shifts, inducing oxidative stress, adding toxins, etc.
	<ol>
		<li>For example, add 100 &#181;g/mL L-valine to induce isoleucine starvation in E. coli K-12 strains. Increased levels of ppGpp will be observed after 5 minutes of induction.</li>
	</ol>
	</li>
</ol>
4. Sampling and ppGpp Extraction
<ol>
	<li>Add 20 &#181;L of each labelled cell sample to a 0.2 mL PCR tube containing 20 &#181;L of ice cold 6 M Formic acid.</li>
	<li>Immediately place the samples in dry ice.</li>
	<li>Enhance cellular extraction efficiency by 3 cycles of freezing and thawing.</li>
	<li>Just before spotting PEI cellulose thin layer chromatograms, centrifuge samples for 1 min at maximum speed to pellet cell debris to avoid spotting it on chromatograms.</li>
</ol>
5. Thin-layer Chromatography
<ol>
	<li>With a soft pencil, mark an origin line 1 cm from the edge of the 20 cm x 20 cm PEI-Cellulose TLC plate that has had 5 cm removed from the top with scissors. Apply 5 &#181;L as a droplet in the PEI surface for each sample. Begin ascending development without allowing this spot to dry, which improve resolution by minimizing streaking.</li>
	<li>Do ascending development in a tank with a layer of 1.5 M KH2PO4 (pH 3.4) solution shallow enough so that liquid does not touch the origin sample spot. Cover the tank with an air-tight seal and allow liquid ascent to the top of the trimmed sheet, 15 cm. To achieve pH 3.4 for a 1.5 M KH2PO4 solution, it is necessary adjust the pH by addition of H3PO4.</li>
	<li>Remove the fully developed chromatogram and air dry at room temperature.</li>
	<li>Cut and discard the top (pH front) portion of the chromatogram containing the free 32P into radioactive waste. This portion is represented in yellow color in Figure 2 and easily visualized under UV light. If measuring only (p)ppGpp nucleotides that migrate slower than GTP, it is recommended to run a UV-visible GTP standard and then cut and discard a larger upper portion (anything above GTP, as shown in Figure 2).</li>
	<li>Expose autoradiographic films overnight with a phosphor screen.</li>
	<li>Develop the film. Capture and quantitate the phosphor screen signal with a phosphoimager.</li>
</ol>
6. Quantitation of (p)ppGpp
<ol>
	<li>Quantitate radioactive spots with Image J26,27. 
	NOTE: The amount of (p)ppGpp can be normalized to total amount of G nucleotides observed in each sample (Figure 2). If the amount of background is homogeneous, a single blank (box 4 from Figure 2) can be subtracted to correct for background. Total G is the sum of GTP + ppGpp + pppGpp detected. This normalization assumes that GMP and GDP levels are negligible, which is true for E. coli. The ratio of a given nucleotide to total G provides an important way to correct for variations of applied sample volume.</li>
</ol>
7. Measurement of the Rate of ppGpp Decay
NOTE: A modification of this protocol allows measurements of ppGpp decay rates.
<ol>
	<li>After provoking stress or starvation for a time sufficient to allow ppGpp accumulation (step 3), reverse the stress in order to block continued ppGpp synthesis, which allows detection of hydrolytic rates. The procedure for reversal will depend on the stress. For example, add 200 &#181;g/mL of chloramphenicol to reverse any amino acid starvation.</li>
	<li>Decay rates are usually rapid but may vary, so take samples every 20-30 s up to 2 min. Process samples as in step 4.</li>
	<li>Once the TLC is developed (as in step 5) and the levels of ppGpp obtained during the time course, plot residual ppGpp content on a semi-logarithmic plot vs time to allow visualization of zero order rates of decay, as a straight line, and estimation of the ppGpp half-life.</li>
</ol>

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The authors have nothing to disclose.

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>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;6&lt;/sub&gt;(MO&lt;sub&gt;7&lt;/sub&gt;)&lt;sub&gt;24&lt;/sub&gt;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>CoCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientifics</td><td>C-371</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T.Baker</td><td>1843</td><td></td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;</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>H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;4&lt;/sub&gt;</td><td>Macron</td><td>2549-04</td><td></td></tr><tr><td>H&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T.Baker</td><td>0260-02</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9458-250G</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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>MnCl&lt;sub&gt;2&lt;/sub&gt;</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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Mallinckrodt</td><td>7917</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</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>ZnSO&lt;sub&gt;4&lt;/sub&gt;</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...

Watch this Scientific Journal Video about Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography at JoVE.com

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 ...

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Research

JoVE Journal

Biology

6.0K Views.  Eunice Kennedy Shriver NICHD, NIH. Deze methode meet de niveaus van de wereldwijde regulator ppGpp binnen verschillende groeiomstandigheden en stresssituaties. PpGpp kan worden gevonden in gram positieve en negatieve bacteriën, evenals in chloroplast. PpGpp heeft een snelle start in seconden, die leiden tot een snelle tweede relatie zodra stress wordt geproduceerd.Tegelijkertijd heeft het een korte halflevenstijd, tot 30 seconden. Daarom is het nodig om veel cultuur te controleren met tijdsintervallen die kunnen varië...

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

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Environment

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Immunology and Infection

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Chemistry

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

Biochemistry

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

Bioengineering

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Source: Pokhrel, A., et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
This ...

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

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