The laboratory of GSCh is supported in part by grant IN201222 from Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica (PAPIIT), Direcci&#243;n General de Asuntos del Personal Acad&#233;mico -UNAM.

This procedure is schematized in Supplementary Figure 1. The reagents and equipment used for the study are listed in the Table of Materials.
1. Detection of mRL and dRL in culture supernatants of P. aeruginosa using TLC
<ol>
	<li>Start with the centrifuged broth of the P. aeruginosa strain of interest, cultivated in liquid medium for 24 h (to reach the stationary phase of growth where RL is produced). Typically, these cultures contain 1 x 109 bacteria per mL.</li>
	<li>Adjust the pH of the culture to 2 using concentrated HCl.</li>
	<li>Put 5 mL of the acidified culture supernatant in a 50 mL polypropylene tube and add 5 mL of a 2:1 chloroform: methanol mixture.</li>
	<li>Agitate the tube by inversion three times, each time for 10 s, and leave the tube without inversion for 2 min between each agitation.</li>
	<li>Leave the tube without agitation for approximately 3 h until the two phases are separated, or centrifuge the tube for 10 min at 3,000 x g at 4 &#176;C.</li>
	<li>Transfer the organic phase (bottom layer) to a clean tube and leave the tube in an extraction hood until dryness occurs.</li>
	<li>Repeat the process starting from step 1.3, putting the organic phase from the second chloroform: methanol extraction, into the tube that was used in the first extraction.</li>
	<li>Evaporate the organic phase until 1 mL or 1.5 mL are left. Transfer this volume to a 1.5 mL centrifuge tube and evaporate the solvent to dryness overnight.</li>
	<li>Add 50 &#181;L of methanol to the dried tube to resuspend RL.</li>
	<li>Perform thin-layer chromatography on silica gel plates. 
	NOTE: The size of the TLC plate should be cut according to the number of samples that will be analyzed. Each sample should be applied at 1.5 cm, and the point of application should be 1 cm from the edge of the plate (draw a horizontal line with a pencil).</li>
	<li>Apply 5 &#181;L of each sample using a 10 &#181;L micropipette.</li>
	<li>The TLC liquid phase consists of a 65:15:2 mixture of chloroform: methanol: acetic acid. To prepare 35 mL of this mixture, mix 26 mL of chloroform, 6 mL of methanol, and 0.8 mL of a 20% stock solution of acetic acid. Mix these solvents and place them in the closed TLC chamber for at least 10 min before beginning the chromatography, so the atmosphere is saturated with the volatile solvents.</li>
	<li>Place the TLC plate into the chamber, avoiding contact with the point where the samples were applied.</li>
	<li>Close the chamber and leave the TLC until the solvent reaches 1 cm before the edge of the plate. At this point, remove the plate and let it dry (a flow of air can be applied to accelerate the process).</li>
	<li>Prepare a solution of &#945;-naphthol by dissolving 5 g of this compound in 33 mL of ethanol. Once dissolved, add 127.5 mL of ethanol, 12.6 mL of water, and 20.5 mL of cold H2SO4.</li>
	<li>To detect the presence of mRL and dRL, spray the &#945;-naphthol solution onto the plate in the extraction hood and place the sprayed plate in an oven at 85 &#176;C for several minutes until the pinkish mark of RL is apparent.</li>
	<li>Use a picture of the TLC to calculate the proportion of mono- and di-RL present in each sample using software that detects the density of each spot.</li>
</ol>
2. Quantification of the total amount of RL measuring the rhamnose equivalents present in the biosurfactant
<ol>
	<li>Place 1.2 mL of a stationary phase culture (grown for 24 h) in a 1.5 mL centrifuge tube and centrifuge for 3 min at 3,000 x g at 4 &#176;C. Collect the supernatant in a clean tube.</li>
	<li>Transfer 333 &#181;L to a clean tube (perform this step in triplicate) and add 1 mL of ether.</li>
	<li>Vigorously mix in a vortex for 30 s. Repeat this procedure with one tube at a time.</li>
	<li>Centrifuge for 2 min at 3,000 x g at 4 &#176;C. Collect the organic phase (upper phase), transfer it to a clean tube, and leave it open in the extraction hood until dryness occurs.</li>
	<li>Repeat the extraction with ether as described in step 2.2. Repeat steps 2.3 and 2.4.</li>
	<li>Once completely dried, add 1 mL of water to each tube. Leave the tubes for 12 h to allow the RL to hydrate, then agitate vigorously in the vortex.</li>
	<li>Place a clean flask in ice for 5 min (as it is an exothermic reaction). Prepare a solution of 60% H2SO4 (protect the flask from light).</li>
	<li>In a clean tube, prepare a 1.6% orcinol solution in distilled water. To prepare the orcinol reagent, mix 4.4 mL of the 60% H2SO4 solution with 0.6 mL of the 1.6% orcinol solution.</li>
	<li>Calculate the final volume of the orcinol reagent needed. Add 900 &#181;L of this reagent to each sample and to each concentration of the calibration curve, using different rhamnose concentrations (typically, 9 concentrations of rhamnose are used in the range of 1 &#181;g/mL to 9 &#181;g/mL), and one with 100 &#181;L of water, ensuring that all are performed in triplicate.</li>
	<li>Add 100 &#181;L of each RL sample to 900 &#181;L of the orcinol reagent in a glass tube and mix the two solutions.</li>
	<li>Incubate for 30 min in a water bath preheated to 80 &#176;C.</li>
	<li>Allow the tubes to cool to room temperature.</li>
	<li>Read the absorbance of the samples and calibration curve at 421 nm using a quartz cell.</li>
	<li>Calculate the concentration of rhamnose in each sample by interpolating the absorbance on the calibration curve and considering the volume used for the determination.</li>
	<li>To calculate the &#181;M concentration, divide the concentration obtained in &#181;g/mL by 182.2 (the molecular weight of rhamnose) and multiply by 1000.</li>
</ol>

Comparative Analysis of Rhamnolipid Production in Pseudomonas aeruginosa Strains

<ol><li>Moradali, M. F., Ghods, S., Rehm, B. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+lifestyle%3A+A+paradigm+for+adaptation%2C+survival%2C+and+persistence%5BTitle%5D)+AND+%22Frontiers+in+Cell+Infection+and+Microbiology%22%5BJournal%5D)">Pseudomonas aeruginosa lifestyle: A paradigm for adaptation, survival, and persistence.</a> Frontiers in Cell Infection and Microbiology. 7, 1-29 (2017).</li>
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<li>Filbig, M., et al. Metabolic and process engineering on the edge-Rhamnolipids are a true challenge: A review. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFilbig%2C+M+%22Biosurfactants.+Foundations+and+Frontiers+in+Enzymology%22">Biosurfactants. Foundations and Frontiers in Enzymology</a>. Soberón-Chávez, G., et al. , Academic Press. New York. Chapter 8 157-181 (2023).</li>
<li>Noll, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limits+for+sustainable+biosurfactant+production%3A+Techno-economic+and+environmental+assessment+of+a+rhamnolipid+production+process%5BTitle%5D)+AND+%22Bioresource+Technology%22%5BJournal%5D)">Limits for sustainable biosurfactant production: Techno-economic and environmental assessment of a rhamnolipid production process.</a> Bioresource Technology. 25, 101767(2024).</li>
<li>García-Reyes, S., Cocotl-Yañez, M., González-Valdez, A., Servín-González, L., Soberón Chávez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+PqsR-independent+quorum-sensing+response+of+Pseudomonas+aeruginosa+ATCC+9027+outlier-strain+reveals+new+insights+on+the+PqsE+effect+on+RhlR+activity%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">The PqsR-independent quorum-sensing response of Pseudomonas aeruginosa ATCC 9027 outlier-strain reveals new insights on the PqsE effect on RhlR activity.</a> Molecular Microbiology. 116 (4), 1113-1123 (2021).</li>
<li>Grosso-Becerra, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+ATCC+9027+is+a+non-virulent+strain+suitable+for+mono-rhamnolipids+production%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production.</a> Applied Microbiology and Biotechnology. 100 (23), 9995-10004 (2016).</li>
<li>Gutiérrez-Gómez, U., Soto-Aceves, M. P., Servín-González, L., Soberón-Chávez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overproduction+of+rhamnolipids+in+Pseudomonas+aeruginosa+PA14+by+redirection+of+the+carbon+flux+from+polyhydroxyalcanoate+synthesis+and+overexpression+of+the+rhlAB-R+operon%5BTitle%5D)+AND+%22Biotechnology+Letters%22%5BJournal%5D)">Overproduction of rhamnolipids in Pseudomonas aeruginosa PA14 by redirection of the carbon flux from polyhydroxyalcanoate synthesis and overexpression of the rhlAB-R operon.</a> Biotechnology Letters. 40 (11), 1561-1566 (2018).</li>
<li>Zibek, S., Soberón-Chávez, G. Overview on glycosylated lipids produced by bacteria and fungi: Rhamno-, Sophoro-, Mannosylerythritol and Cellobiose Lipids. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aZibek%2C+S+%22Biosurfactants+for+a+Biobased.+Advances+in+Biochemical+Engineering%2FBiotechnology%22">Biosurfactants for a Biobased. Advances in Biochemical Engineering/Biotechnology</a>. Hausmann, R., Henkel, M. , Springer. Berlin, Heidelberg. Chapter 4 181(2022).</li>
<li>Twigg, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+biosurfactant+research%3A+time+to+improve+the+rigour+in+the+reporting+of+synthesis%2C+functional+characterization+and+process+development%5BTitle%5D)+AND+%22Microbial+Biotechnology%22%5BJournal%5D)">Microbial biosurfactant research: time to improve the rigour in the reporting of synthesis, functional characterization and process development.</a> Microbial Biotechnology. 14 (1), 147-170 (2021).</li>
<li>Matsuyama, T., Sogawa, M., Yano, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+colony+thin+layer+chromatography+and+rapid+characterization+of+Serratia+marscescens+wetting+agents%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Direct colony thin layer chromatography and rapid characterization of Serratia marscescens wetting agents.</a> Applied and Environmental Microbiology. 53 (5), 1186-1188 (1987).</li>
<li>Chandrasekaran, E. V., Bemiller, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constituent+analyses+of+glycosaminoglycans%5BTitle%5D)+AND+%22Methods+on+Carbohydrate+Chemistry%22%5BJournal%5D)">Constituent analyses of glycosaminoglycans.</a> Methods on Carbohydrate Chemistry. 8, 89-96 (1980).</li>
<li>Behrens, B., Engelen, J., Tiso, T., Blank, L. M., Hayen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+rhamnolipids+by+liquid+chromatography%2Fmass+spectrometry+after+solid-phase+extraction%5BTitle%5D)+AND+%22Analytic+and+Bioanalytic+Chemistry%22%5BJournal%5D)">Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction.</a> Analytic and Bioanalytic Chemistry. 408 (10), 2505-2514 (2016).</li>
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</ol>

The authors have no conflict of interest to disclose.

The most accurate method for detecting and quantifying RL is HPLC coupled with mass spectrometry (MS)7,8,27; however, it requires specialized and expensive equipment that may not be accessible to many researchers. The methods described here can be routinely performed with basic laboratory materials and equipment to detect and estimate RL concentrations, but they have some limitations, particularly their inaccuracy in determining...

Pseudomonas aeruginosa is an environmental bacterium and an opportunistic pathogen of great concern due to its production of virulence-associated traits and its high antibiotic resistance1,2. A characteristic secondary metabolite produced by this bacterium is the biosurfactant RL, which is produced in a coordinated manner with several virulence-associated traits such as the phenazine pyocyanin, an antibiotic with redox activity, and the protease elastase3. The tensio-active and emulsification properties of RL have been exploited in different industrial applications and are currently commercialized4.
Most P. aeruginosa strains, belonging to phylogroups 1 and 2, produce two types of RL: mRL, which consists of one rhamnose moiety linked to a fatty acid dimer mainly of 10 carbons, and dRL, which contains an additional rhamnose moiety linked to the first rhamnose4 (see Figure 1). However, it has been reported that two minor P. aeruginosa phylogroups (groups 3 and 5) only produce mRL5,6. The two types of RL contain a mixture of fatty acid dimers, which, as mentioned, are mainly C10-C10, but smaller proportions of molecules containing C12-C10, C12-C12, and C10-C12:1 dimers are also produced. The characterization of the RL congeners produced by different strains using HPLC MS/MS has been reported7,8. The methods described in this work can only differentiate between mRL and dRL but cannot be used for the characterization of the RL congeners.
P. aeruginosa and some Burkholderia species are natural producers of RL9, but the former bacterium is the most efficient producer. However, commercially used RL is currently produced in Pseudomonas putida KT2440 derivatives expressing P. aeruginosa genes to avoid the use of this opportunistic pathogen10,11. The detection and quantification of RL produced by P. aeruginosa are of great importance for studying the molecular mechanisms involved in the expression of virulence-related traits12, in the characterization of strains belonging to clades 3 or 513, and for constructing P. aeruginosa derivatives that overproduce these biosurfactants while having reduced virulence14. The production of biosurfactants by different microorganisms has been detected based on some general characteristics of these compounds, such as the collapse drop method or emulsification index15, but these methods are neither accurate nor specific16.
Here, we describe the protocol to detect mRL and dRL using the liquid extraction of total RL from the culture supernatants of different P. aeruginosa-type strains and the separation of both types of RL using TLC. In this method, the RL extracted from the culture supernatant is separated by their differential solubility in the solvents used for TLC, causing differential migration on the silica gel plate. Thus, mRL have a more rapid migration than dRL, and they can be detected as separate spots when the plates are dried and stained with &#945;-naphthol.
The method described here for detecting mRL and dRL by TLC is based on a previously published article17, which is easy to perform and does not require expensive equipment. This method has been useful for detecting RL in various P. aeruginosa isolates13 using appropriate controls, such as a P. aeruginosa-derived mutant unable to produce RL. However, it is not the preferred method for characterizing novel biosurfactants produced by bacteria other than Pseudomonas aeruginosa due to its lack of specificity.
Additionally, a method for quantifying the rhamnose equivalents of total RL extracted from a P. aeruginosa culture supernatant is presented. This method quantifies these biosurfactants based on the reaction of orcinol with reductive sugars, resulting in a product that can be measured spectrophotometrically at 421 nm, as previously described18. Since the reaction with orcinol is not specific to rhamnose, it is important to perform this method with RL extracted from the culture supernatant that does not contain significant amounts of other sugar-containing molecules, such as lipopolysaccharides (LPS). An acidified chloroform/methanol mixture is used here for liquid extraction of RL18, but ethyl acetate can also be used, and solid-phase extraction (SPE) yields very good results19. The orcinol method described here does not require sophisticated equipment and can provide reliable results if performed with special care in preparing the analyzed samples, as discussed. To ensure proper sample preparation, it is important to include a Pseudomonas aeruginosa rhlA mutant unable to produce RL20 and to perform three biological and three technical replicates for each determination.
There has been significant controversy in the literature16,21regarding RL determination by the orcinol method, with some studies suggesting that RL production is overestimated and that the assay lacks specificity for rhamnose, potentially detecting other sugars. However, we demonstrate here that the methods described can be accurate and specific under the appropriate conditions. Furthermore, for comparison with the procedures outlined in this article, we utilize UPLC-MS/MS detection of a dRL standard and demonstrate that similar results are obtained with the orcinol method. The detailed protocol for quantifying RL using this method is included in Supplementary File 1.
These protocols are exemplified using the type strains PAO1 (phylogroup 1), PA14 (phylogroup 2), and PA7 (phylogroup 3). These strains were chosen because they are well-characterized and produce different RL profiles.

<table><tbody><tr><td>1-NAPHTHOL</td><td>SIGMA-ALDRICH</td><td>70442</td><td></td></tr><tr><td>ACETIC ACID</td><td>J.T. BAKER</td><td>9508-02</td><td></td></tr><tr><td>CENTRIFUGE</td><td></td><td></td><td>For centrifuging tubes 1.5 mL and&amp;nbsp; 50 mL</td></tr><tr><td>CHLOROFORM</td><td>J.T. BAKER</td><td>9180-02</td><td></td></tr><tr><td>DRYING OVEN</td><td></td><td></td><td></td></tr><tr><td>ETHER</td><td>J.T. BAKER</td><td>9244-02</td><td></td></tr><tr><td>GLASS PIPETTE</td><td>SIGMA-ALDRICH</td><td>CLS706510</td><td></td></tr><tr><td>HYDROCHLORIC ACID</td><td>J.T. BAKER</td><td>5622-02</td><td></td></tr><tr><td>LB</td><td></td><td></td><td></td></tr><tr><td>L-RHAMNOSE MONOHYDRATE</td><td>SIGMA-ALDRICH</td><td>R-3875</td><td></td></tr><tr><td>METHANOL</td><td>J.T. BAKER</td><td>9049-02</td><td></td></tr><tr><td>ORCINOL MONOHYDRATE</td><td>SIGMA-ALDRICH</td><td>O1875</td><td></td></tr><tr><td>PPGAS Broth</td><td></td><td></td><td>Tris HCL (0.12M), Potassium Chloride ( 0.02M) Ammonium Chloride (0.02M),&amp;nbsp; Peptone (1%), pH 7.4&amp;nbsp;&amp;nbsp; Autoclaved. Add&amp;nbsp; Glucose (5%) and Magnesium Sulfate (0.0016M)</td></tr><tr><td>QUARTZ CELL (CUVETTE)</td><td>SIGMA-ALDRICH</td><td>Z276669</td><td></td></tr><tr><td>RECTANGULAR TLC DEVELOPING TANK</td><td>FISHER SCIENTIFIC</td><td>K4161801020</td><td></td></tr><tr><td>RHAMNOLIPIDS&amp;nbsp;</td><td>SIGMA-ALDRICH</td><td>R-90</td><td></td></tr><tr><td>SPECTROPHOTOMETER</td><td></td><td></td><td>VIS</td></tr><tr><td>SPRAYER</td><td>SIGMA-ALDRICH</td><td>Z529710-1EA</td><td></td></tr><tr><td>SULFURIC ACID</td><td>J.T. BAKER</td><td>9681-02</td><td></td></tr><tr><td>TES TUBES 5mL</td><td>CORNING</td><td>352002</td><td></td></tr><tr><td>TLC SILICA GEL 60 F254</td><td>MERCK</td><td>1.05554.0001</td><td></td></tr><tr><td>WATER BATH</td><td></td><td></td><td>&gt; 80 &amp;deg;C</td></tr></tbody></table>

detection and quantification of mono-rhamnolipids and di-rhamnolipids produced by pseudomonas aeruginosa

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This bacterium produces high levels of biosurfactants known as rhamnolipids (RL), which are molecules with significant biotechnological value but are also associated with virulence traits. In this respect, the detection and quantification of RL may be useful for both biotechnology applications and biomedical research projects. In this article, we demonstrate step-by-step the technique to detect the production of the two forms of RL produced by P. aeruginosa using thin-layer chromatography (TLC): mono-rhamnolipids (mRL), molecules constituted by a dimer of fatty acids (mainly C10-C10) linked to one rhamnose moiety, and di-rhamnolipids (dRL), molecules constituted by a similar fatty acid dimer linked to two rhamnose moieties. Additionally, we present a method to measure the total amount of RL based on the acid hydrolysis of these biosurfactants extracted from a P. aeruginosa culture supernatant and the subsequent detection of the concentration of rhamnose that reacts with orcinol. The combination of both techniques can be used to estimate the approximate concentration of mRL and dRL produced by a specific strain, as exemplified here with the type strains PAO1 (phylogroup 1), PA14 (phylogroup 2), and PA7 (phylogroup 3).

In this article, three different P. aeruginosa type strains were utilized to represent three phylogroups, each with varying RL production levels and proportions of mRL and dRL. These strains include PAO1 (a wound isolate from Australia, 195522), PA14 (a plant isolate from the USA, 197723), and PA7 (a clinical isolate from Argentina, 201024). As a negative control, the PAO1 rhlA mutant was employed, which is incapable of RL production. All st...

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Author Spotlight: Understanding Rhamnolipid Regulation in Pseudomonas aeruginosa

Pseudomonas aeruginosa&#160;produces the rhamnolipid biosurfactants. Thin-layer chromatography detects and determines the proportion of mono- and di-rhamnolipids produced by each strain. Quantification of total rhamnolipids involves assessing rhamnose equivalents present in these biosurfactants extracted from the culture supernatants using the orcinol method.

Detection and Quantification of Mono-Rhamnolipids and Di-Rhamnolipids Produced by Pseudomonas aeruginosa

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This ...

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Biochemistry

1.2K Views.  Universidad Nacional Autónoma de México. Pseudomonas aeruginosa produces the rhamnolipid biosurfactants. Thin-layer chromatography detects and determines the proportion of mono- and di-rhamnolipids produced by each strain. Quantification of total rhamnolipids involves assessing rhamnose equivalents present in these biosurfactants extracted from the culture supernatants using the orcinol method.

Video: Author Spotlight: Understanding Rhamnolipid Regulation in Pseudomonas aeruginosa

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Immunology and Infection

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is ...

Assessment of Siderophore Production from Pseudomonas aeruginosa Isolates

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.
The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Author Spotlight: Quantifying Siderophores and Pyochelin for Infection Control 

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

Detection and Quantification of Mono-Rhamnolipids and Di-Rhamnolipids Produced by Pseudomonas aeruginosa

Summary

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