quantification of rhamnolipids present in the biosurfactant of pseudomonas aeruginosa

Comparative Analysis of Rhamnolipid Production in Pseudomonas aeruginosa Strains

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.

Author Spotlight: Understanding Rhamnolipid Regulation in Pseudomonas aeruginosa

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

The aim of our research is to understand the regulatory mechanisms involved the expression of the different virulence factors produced by the opportunistic pathogen, Pseudomonas aeruginosa, that include the biosurfactants rhamnolipids. Rhamnolipids are non-toxic and environmental-friendly, and have several biotechnological applications, so they are now in the market. We have reported the rhamnolipids biosynthetic root and the regulation of their expression in different isolates that are atypical, and refractory to alternative therapeutic approaches based on antivirulence strategies.In addition, we have reported the construction of strains that produce high levels of rhamnolipids and are non-virulent. The technique describe, enable the detection and quantification of rhamnolipids by Pseudomonas aeruginosa. This protocol is accurate and reproducible and do not need expensive equipment.

Quantification of Rhamnolipids Present in the Biosurfactant of Pseudomonas aeruginosa

To begin pipette 1.2 milliliters of a 24-hour culture of Pseudomonas aeruginosa into a 1.5 milliliter centrifuge tube. Centrifuge the tube at 3000 G for four degrees Celsius for three minutes. Collect the supernatant in a clean tube.Pipette 333 microliters of the supernatant into a clean tube. Then add one milliliter of ether into the tube. Mix the solution in a vortex for 30 seconds.After all the tubes have been vortexed, centrifuge them at 3000 G at four degrees Celsius for two minutes. Then collect the organic phase and transfer it into a clean tube tube. Place the solution in an extraction hood until it is dried.Next place a clean flask covered with foil paper in ice for five minutes to prepare a 60%sulfuric acid solution. Use this sulfuric acid solution to prepare the orcinol reagent. Add 100 microliters of each Rhamnolipid sample to a glass tube and add 900 microliters of orcinol to the samples.After mixing the solutions together, incubate the sample in a water bath at 80 degrees Celsius for 30 minutes. When the tube cools to room temperature, transfer a sample to a quart cell and place it in the spectrophotometer to read the absorbance at 421 nanometers. The strain PA14 produces the highest amount of rhamnose equivalents while strain PA7 produces the lowest amount.

Research

JoVE Journal

Biochemistry

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