Directed Evolution Towards Detergent Tolerance

Sandra Rizk

Microbial Diversity course 2018 Directors G. O’ Toole, R. Whitaker

Abstract Sodium Dodecyl sulfate (SDS) is an anionic detergent that is used in biology mainly for protein extraction due to its known ability to dissolve membranes1. Moreover, some bacteria are able to catabolize it and use it as a carbon source which can be utilized in biotechnological applications to remove pollutants from water or soil2. For my miniproject, I aimed to answer the question of whether bacteria can be evolved to tolerate SDS, and which mutations does it have to undergo in order to withstand that stress. Rhodococcus strain isolated from corals was used in this study, and grown under different SDS concentrations, up to 1% SDS in media. Genome sequencing of the WT and mutated strain was attempted to answer the question of whether the mutation is affecting the cell envelope or a metabolic pathway that allows it to catabolize the detergent. Introduction SDS is an anionic detergent that can bind to biological membranes at high and low concentrations, the effect of which varies from changes in membrane permeability at very low concentrations to severe damage at higher concentrations such as membrane lysis and fusion1. The bacterial isolate used in this study was isolated from a coral sample provided by the marine resource center (MRC, MBL, Woods Hole, Ma) from an unbleached coral grown at 6°C, and unbleached. Complete genome sequencing has revealed that the isolated bacteria belongs to the Rhodococcus sp. Which has been shown to be part of the coral actinobacteria community3. Rhodococcus strains have been shown to have bioremediation potential, and solvent tolerance4. Which explains its ability to tolerate 1% SDS as shown in this miniproject. The evolved strain has potential to be used in biotechnological applications to clean the water from pollutants containing SDS. Materials and Methods: Media and Culturing conditions: The media used in the experiment is seawater complete (SWC) as described in the lab manual of the microbial diversity course 2018, chapter 2. 0.01%, 0.1%, and 1% SDS were added to the media from a 10% autoclaved SDS stock solution. SDS was added to the media after autoclaving, and the same method was used to prepare agar plates. The cells were grown in liquid culture at 30°C overnight at 150 rpm. Freezer stocks were prepared in SWC and 10% glycerol. Directed evolution experiment: Cells already showed sensitivity to 0.1%, and 1% SDS as measured by taking OD readings (Figure 2.). Inoculum from the cells that could grow on 0.01% SDS were used to start cultures at 0.1%, and 1% SDS-SWC media. Cells were passaged every ~48 hours (until confluency), till they were able to grow faster (around the 5th passage). Then colony

forming unit (CFU) spot assay was carried out on SWC agar plates at different concentrations of SDS. Colonies were picked (from each concentration) and they were grown on SWC media without SDS, and then spotted again on plates with SDS to make sure that SDS tolerance is due to a mutation not just a phenotype. Cells were then frozen in the form of glycerol stocks at MBL, and 9 colonies were sent for genome sequencing. CFU spot assay: Cells were diluted from 100 to 10-9, and 5 µl of the dilution was spotted on dry agar plates and let grown overnight. Cell numbers in the strain book: WT (56), cells that grow on 0.01% SDS (159, 160, 161), cells that grow on 0.1% SDS (162, 163, 164), cells that grow on 1% SDS (165, 166, 167). The sample names are: “2018.SR.18.8. #strainbook” SEM: Materials: 2.5% glutaraldehyde in phosphate buffer, 50%, 75%, 85%, 95%, and 100% Ethanol. Osmium tetroxide (provided by the facility), 0.1 M potassium phosphate buffer pH 7.2 and sterile filtered (1x PBS worked better for aggregates), 0.2µm Whatman® Anodisc inorganic filter membrane. Sample preparation: Grow cells overnight in 12 well plate: WT, 1% SDS on SWC, 1% SDS on 0.1% SDS-SWC, 1% SDS on 1% SDS- SWC. Collect 500µl of cells, spin it down at 5000 rpm for 3 minutes, Resuspend cells in 500µl phosphate buffer (1st wash). Spin down at 5000 rpm for 3 minutes, wash with phosphate buffer, spin down at 5000 rpm for 3 minutes, resuspend in 2.5% Glutaraldehyde for 2 hours (1-4 hours). Wash 3 times for 20 minutes each with potassium phosphate buffer (stopping point, leave at 4°C), Filter 20µl of cells (fixed with glutaraldehyde) on a 0.2µm Anodisc Whatman filter as shown in Figure 1. the “bottom syringe” is used to exert negative pressure, sucking the liquid through the filter, thus attaching the bacteria on to the filter. The top syringe is used by removing the plunger and adding the cells/buffer for washes through it, the tubing is necessary for attaching the bottom syringe, the filter outlet has the filter on-top-of-which the filter Figure 1. Membrane filtration setup

for SEM

Is placed. I then used forceps and placed the filter into 1ml (drop on a small petri dish) of 1% Osmium tetroxide for 1 hour on ice, then washed the filters 3 times with potassium phosphate buffer, 10 minutes each. Then I did Ethanol dehydration using 50%, 70%, 95% EtOH for 10 minutes each, on ice then 100% EtOH 3 x 15 minutes each on ice. The samples were then placed in critical point dryer (to dry without surface tension), afterwards the dry membrane filters carrying the samples were stuck to the Aluminium SEM stubs using tape, and imaged5,6. Crystal Violet assay: 100µl of cell suspension was transferred to 4x 96-well plates, each were tested for biofilm formation at different time points (6 hours, 12 hour, 24 hours, and 48 hours). The cells were washed 5 times from the plate with water, then 120µl 0.1% crystal violet dye was added to each well and incubated for 15 minutes. Afterwards the crystal violet was discarded, and the plate was washed 5 times with water, and let dry. This assay can be analyzed qualitatively with the naked eye for biofilm formation, or 95% EtOH can be added to the wells, and OD 600 can be measure to get a quantitative result7. Results and Discussion: Directed Evolution: At the beginning the WT showed sensitivity to 0.1% and 1% SDS as shown in (figure 2). Which decreased with the number of passages. On passages 2, and 3 crystal structures were

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0 5 10 15 20 25 30

OD

Growth on SDS

WT 0.01% SDS 0.1% SDS 1% SDS

Figure 2. Pilot growth experiment to test the sensitivity of the WT cells to different concentrations of SDS.

Figure 3. P2 shows pictures of the WT and the cells grown with 1% SDS at the second passage, crystals can be seen in the 1% SDS cells. At P7 (7th passage) the cells that could grow on 1% SDS look similar to the WT and no crystals can be observed.

observed in the media (figure 3. P2), especially at higher concentrations of SDS. This is suspected to be due to an interaction between the lysed cells, the high salt concentration of the SWC media and the SDS. This phenotype of crystal formation was no longer observed as the cells grew faster and less lysis was observed towards the end of the experiment. Cells that were tolerant to 1% SDS were grown overnight on SWC media without SDS, then CFU spot assay was carried out on SWC agar plates with different concentrations of SDS, to make sure that a mutation is present not just an artifact (figure 4). Throughout the experiment, cells had a slightly different morphology (they curled more) when observed under the light microscope, however, as their fitness increased their WT morphology was restored (figure 3). Crystal Violet assay: Biofilm formation started to show after 24 hours in cells incubated with media containing 1% SDS (results not shown). This experiment needs to be repeated and a better way of visualizing the results needs to be figured out. SEM imaging: SEM was proposed as a method to observe if there are any changes in the membrane structure or general cell appearance between the WT cells and the mutant cells that could tolerate 1% SDS. The results may suggest changes in the overall cell size, however, the sample and cell numbers observed with SEM were too low to make any confident conclusions. Sample preparation needs to be further optimized, for example by adding more cells, and more images need to be

Figure 4. (1), (2), (3) Cells tolerant to 1% SDS, grown on 0.01%, 0.1% and 1% SDS plates respectively. Columns I. CFU spot assay of cells tolerant to 1% SDS from liquid culture. II, III. Restreaking cells that grew. IV. CFU spot assay after growing mutant cells on media without SDS to confirm that the tolerance is due to a mutation.

taken to make sure that the observed phenotype is not an artifact.

Future outlook: Due to time constraints, I attempted genome sequencing of the mutated strains by the end of the course, and as soon as I get the results back I will compare it to the genome of the WT and find out which genes were mutated. This tolerant bacterial strain can be used in biotechnological applications to clean out pollutants from the water and soil. The question of whether SDS can be utilized by the bacteria as a carbon source should be answered, and whether it has undergone modifications in its envelope should be determined. I plan to continue working on the project to answer the suggested questions. Moreover, technique optimization should be attempted especially the crystal violet assay and the SEM sample preparation.

Figure 5. A) WT cells. B) Cells tolerant to 1% SDS grown on 0.1% SDS. C) Cells tolerant to 1% SDS grown on 1% SDS.

References:

1. Helenius, A., & Simons, K. A. I. (1975). Solubilization of membranes by detergents. Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes, 415(1), 29-79.

2. Jovčić, B., BEGOvIć, J. E. L. E. N. A., Lozo, J., Topisirović, L., & Kojić, M. (2009). Dynamics of sodium dodecyl sulfate utilization andantibiotic susceptibility of strain Pseudomonas sp. ATCC19151. Archives of Biological Sciences, 61(2), 159-164.

3. Mahmoud, H. M., & Kalendar, A. A. (2016). Coral-associated actinobacteria: diversity, abundance, and biotechnological potentials. Frontiers in microbiology, 7, 204.

4. Kuyukina, M. S., & Ivshina, I. B. (2010). Application of Rhodococcus in bioremediation of contaminated environments. In Biology of Rhodococcus (pp. 231-262). Springer, Berlin, Heidelberg.

5. Fischer ER, Hansen BT, Nair V, Hoyt FH, Dorward DW. Scanning Electron Microscopy. Current Protocols in Microbiology. 2012;CHAPTER:Unit2B.2. doi:10.1002/9780471729259.mc02b02s25.

6. Golding, C. G., Lamboo, L. L., Beniac, D. R., & Booth, T. F. (2016). The scanning electron microscope in microbiology and diagnosis of infectious disease. Scientific reports, 6, 26516.

7. O'Toole, G. A. (2011). Microtiter dish biofilm formation assay. Journal of visualized experiments: JoVE, (47).