Part:BBa_K2062006

rhamnosyltransferase 2 [Pseudomonas aeruginosa]

RhlC codes for rhamnosyltransferase 2, which is the last enzyme in the rhamnolipid production pathway. Its function is to convert mono-rhamnolipid to di-rhamnolipid by catalyzing an additional dTDP-L-rhamnose.(1) It was cloned from Pseudomonas aeruginosa PAO1 from an operon also containing a putative fosfomycin resistance protein and putative MFS transporter(2).

Rhamnolipids repel Aedes aegypti mosquitos

In order to quantify how effectively rhamnolipids repel mosquitoes, we conducted mosquito feeding and landing assays. Aedes aegypti, the species of mosquito observed to carry Zika virus, were grown from larval stage, and females were sorted at the pupae or adult stage. Since only females consume blood for reproduction, we were only interested in using them for the assays.

File:Mosquito-1.png

test

One day before experiment, 50 total mosquitos (with 30 females) were isolated in cages and starved from 23-25 hours. Each cage was then taken to a warm room (~30 °C), and the cage was covered with wet paper towels to preserve humidity. For each trial, our blood feeding system (Figure) was placed on top of the cage each with a cotton gauze soaked with either negative control water, 1 mg/mL mono-rhamnolipid solution, 1 mg/mL di-rhamnolipid solution, or positive control 25% DEET, and the mosquito activity was videotaped for 1 hour. Afterwards, the cage was taken to the cold room to paralyze the assayed mosquitoes, and mosquitoes that had consumed blood were counted. It is important to note that the age of female mosquitoes and the time of feeding played an important role in how mosquitoes behave. Typically, it is optimum to use female mosquitoes of age from 4-6 days for feeding assays as any mosquitoes older than this age range will be too old to reproduce, and thereby not needing to drink blood. Furthermore, their feeding is most active 4 hours before dusk. Some of our trials that didn’t meet these criteria did not result in any feeding, but we did observe significant difference in landing between the control and rhamnolipids. Our landing assay results showed that while DEET was the strongest mosquito repellent with no landings or fed mosquitos, 1 mg/mL mono and di-rhamnolipid still showed statistically significant repulsion as shown in the graph below.

P. putida, S. epidermidis, and rhamnolipids are compatible with human keratinocytes

Determination of rhamnolipid IC50

Keratinocytes, human skin cells, were grown for several days. When the cells were 80% confluent, they were seeded in 24 well plates at a density of 2.5105. The cells were weaned off of antibiotics the following day before they were treated with varying concentrations of rhamnolipids and the reagent MTS. The MTS assay reveals the cell viability of the cells. Using this information, the data was normalized and statistically analyzed to determine the keratinocyte IC50—or the concentration of rhamnolipid that induces 50% cell death. The IC50 was determined to be between 45.19 µ/mL and 65.52 µ/mL. Relating the results to rhamnolipid quantification, the concentration of rhamnolipid the construct produces should not cause significant cell death.

Keratinocyte cell viability bacteria assay

Keratinocytes were co-cultured with different strains of bacteria (Pseudomonas putida, Pseudomonas aeruginosa PAK, Staphylococcus aureus, Staphylococcus epidermidis, and mutant rhlAB P. putida). Half were cultured in plain DMEM with serum, and half were culture in DMEM with 1 mg/mL mixed mono- and di- rhamnolipids. After co-culturing, the keratinocytes were washed with PBS, exposed to gentamicin in an attempt to kill the bacteria, and incubated in MTS cell viability assay for up to 4 hours and viewed in a plate reader. MTS assay is colorimetric cell viability assay and reacts with NADPH-dependent dehydrogenase enzymes, which are only active in live (metabolically active) cells^{<a href="http://www.biovision.com/manuals/K300.pdf">6</a>}. For the MTS assay, pure media were used as a negative control (100% cell death), and keratinocyte culture with normal DMEM was used as a positive control (“0%” cell death, or the maximum number of cells that could be alive).

We originally tried to do plating experiments to see if keratinocytes internalized any bacteria, but were unable to completely kill off all the bacteria in the keratinocyte supernatant even at extremely high gentamicin concentrations and thus could not get an accurate read.

The results indicate that there is no consistent trend regarding the addition of rhamnolipid and cell viability. Rhamnolipids did not significantly increase or decrease cell viability regardless of the bacteria type as shown in the first figure since the error bars overlap. We hypothesized that the concentration of P. putida would not influence cell viability as it is an environmental strain not nearly as potent as other bacterial strains such as Pseudomonas aeruginosa PAK. As depicted in the second figure, all MOIs (ranging from 0 to 20) did not significantly influence the cell viability of the strain as shown by the overlapping error bars in the graph. These results overall indicate that our construct may not cause significant cell death once applied to the skin in an acute setting of a few hours.

Mutant rhlAB P. putida produces rhamnolipids

Transformation of P. putida KT2440

In order to avoid the virulence factors of Pseudomonas aeruginosa, bacterial strains with similar or shared metabolic pathways to the one above were chosen as potential candidates. The final candidates were Pseudomonas putida and Staphylococcus epidermidis. Although S. epidermidis doesn’t share the same exact pathway as P. aeruginosa, it is a naturally-occurring skin microbiome and only need two additional enzymes, RhlA and RhlB, to produce mono-rhamnolipids. Genes rhlA and rhlB necessary for mono-rhamnolipid synthesis were extracted from the P. aeruginosa P14 bacterial strain. These genes were cloned into the modified plasmid pNJ3.1 using standard cloning methods for transformation into the desired bacterial strains (Figure 2). The plasmid pC194 and a shuttle vector strain, S. aureus RN4220 (details on S. epidermidis transformation are discussed in the experiments and result section) were used for S. epidermidis transformations with the same basic design (Figure 3). The conversion of mono-rhamnolipids to di-rhamnolipids requires the additional gene rhlC, which was also extracted from P14 strain and cloned into the same pNJ3.1 vector (Figure 4).

Quantification of rhamnolipids

To confirm the presence of rhamnolipids produced by our mutant strains (P. putida, E. coli transformed with pNJ3.1_rhlAB), we explored three different methods: cetyl trimethylammonium bromide agar plating (CTAB), thin-layer chromatography (TLC), and supercritical fluid chromatography mass spectrometry (SFC-MS). For TLC and SFC-MS analysis, rhamnolipids were extracted from cell culture supernatant through liquid-liquid extraction with ethyl acetate and redissolved in methanol prior to measurement. Detailed protocols on the extraction is discussed under protocols.

Cetyl trimethylammonium bromide agar plate assay

Cetyl trimethylammonium bromide (CTAB) agar plates detect the presence of rhamnolipid by reacting with the sugar in rhamnolipids^{<a href="http://doi.org/10.1007/s10529-009-0049-7">7</a>}. When rhamnolipid is present, it forms blue halos around the compound, and the halo size usually correlates to the amount of rhamnolipids^{<a href="http://doi.org/10.1007/s10529-009-0049-7">7</a>}. We tested this method with 95% pure rhamnolipids (Sigma-Aldrich) by plating different concentrations of the compound dissolved in water onto SW agar plate*. Blue halos were present after incubating the plate for 24 hours at 37°C, but the limit of detection was too high (~1g/L). Furthermore, depending on the amount of CTAB used per plate, the size of halos varied, which made it difficult for us to use this method as a quantitative measurement.

Thin-layer chromatography

Thin-layer chromatography (TLC) was used as a more reliable method of detecting rhamnolipids. TLC is a very common separation technique used to isolate a desired compound from a mixture. It typically involves two different phases, stationary and mobile, in which the mobile phase flows through the stationary phase and carries the components of the mixture with it^{<a href="http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html">8</a>}. Separation of compounds is based on the affinity of the compound towards the stationary phase vs. the mobile phase, and depending on which phase the compounds prefer, they travel with the solvent at different rates^{<a href="http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html">8</a>}. We used silica gel as the stationary phase and solvent consisted of chloroform, methanol, and acetic acid in 65:15:2 % volume ratio as the mobile phase. Knowing that di-rhamnolipids have more hydroxyl groups, we predicted it to have a smaller retention factor than mono-rhamnolipids as they would prefer to stay on polar silica gel. To visualize the plate, the silica gel plate was stained with four different dyes: CAM, KMnO4, orcinol with 50% H2SO4, and orcinol with 10% H2SO4. Among the four staining methods, orcinol with 10% gave the best visibility. The chemical mechanism in which orcinol and sulfuric acid react with rhamnose to create a dye is illustrated in Figure 2.

We confirmed that TLC method shows two distinct bands for mono-rhamnolipids and di-rhamnolipids, and that it has a limit of detection lower than CTAB (approximately 0.5 mg/mL). Next, we tested our mutant P. putida and E. coli strains with promoters of different strengths. For positive controls, WT P. aeruginosa and mutant P. aeruginosa were used, and for a negative control, WT P. putida was tested. When the cells were grown in LB only media, none of the rhamnolipids was detected from P. putida or E. coli. However, when the cells were grown in LB supplemented with glucose, a faint band for mono-rhamnolipids was detected from mutant E. coli transformed with a high expression level promoter. Although our construct in P. putida didn’t show any clear band, mutant P. aeruginosa transformed with the same construct showed to produce a lot more mono-rhamnolipids compared to WT P. aeruginosa, which mainly produces di-rhamnolipids. This result confirms that our construct is working as expected, yet we need a detection method with higher sensitivity.

Supercritical fluid chromatography

In order to accurately measure the amount of rhamnolipids produced by our mutant strains, we used supercritical fluid chromatography (SFC-MS). First, a test run was executed with a mixture of mono-rhamnolipids and di-rhamnolipids at the concentration of 5 mg/mL by running the sample through the column packed with 2-PIC. From this test run, we have obtained the retention times of mono-rhamnolipids (rha-C₁₀-C₁₀: pseudomolecular ion of 503.56 m/z) and di-rhamnolipids (rha-rha-C₁₀-C₁₀: pseudomolecular ion of 649.8 m/z) to be approximately 3.974 min and 4.942 min respectively. Then, a calibration curve was constructed with 95% pure mono-rhamnolipids, and the limit of detection was found to be approximately 5 µ/mL. The mass fractions were obtained from electrospray ionization (ESI) negative mode.

From our TLC analysis, it was found that supplementing the LB media with glucose is crucial to the production of rhamnolipid. Therefore, for SFC-MS analysis, all the mutant strains (E. Coli_RhlAB, E. Coli_L1_RhlAB, and P. putida_L1_RhlAB) were grown in LB supplemented with glucose. From the SFC-MS data, it was found that mutant E. coli strain makes more mono-rhamnolipids than mutant P. putida. Furthermore, the promoter strength was confirmed as expected since the mutant E. coli strain transformed with a high expression level promoter H2 produced almost 6 times more rha-C₁₀-C₁₀.

In order to investigate the optimum growth conditions for rhamnolipid by the mutant P. putida strain, the amount of glucose added and the time of growth were varied. Using the calibration curve above, we were able to measure the accurate amount of rhamnolipids produced in each cell culture. From this data, we have concluded that P. putida produces the most mono-rhamnolipids when grown for 24 hours in the media LB supplemented with 50 g/L of glucose.

We have also tested the mutant strain of S. aureus RN4220, the strain that carries shuttle vector for S. epidermidis. Unfortunately, SFC-MS data didn't show any production of rhamnolipids from S. aureus strain.

In order to investigate the amount of di-rhamnolipids produced, we have tested our mutant strains of P. putida transformed with rhlC gene. It was grown under the same condition of 24 hours incubation in LB media supplemented by 50 g/L of glucose. Approximately 142 µ/mL of rha-C₁₀-C₁₀ and 3.524 µ/mL of rha-rha-C₁₀-C₁₀ were detected.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 622
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 205
Illegal NgoMIV site found at 393
Illegal NgoMIV site found at 931
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 664
Illegal BsaI.rc site found at 898