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Testing for different buffer conditions

In order to test the feasibility of our project, we wanted to simulate the periplasmic conditions of our chassis, S. cerevisiae and investigated the growth rates of different bacteria which we considered as promising endosymbionts. This was done by measuring the growth rates of those on purified yeast lysate with a microplate reader (Fig. 1) and by performing flow cytometry (Fig. 2).

Figure 1. Growth curves for different microorganisms on yeast lysate, their corresponding medium and 5% SDS, PBS and tethering buffer as control. E. Coli and PCC 6803 show growth on yeast lysate, while M. extorquens did not appear to grow well in yeast lysate. Initial difficulties with BG11 media resulted in suboptimal growth of PCC 6803. Differences in optical density are a result of varying generation times.

Figure 2. Viability of E. coli growing on yeast lysate Cells quantified with flow cytometry and data normalized to the forward scatter given in arbitrary units. E. coli growing shows elongated phenotype compared to growth on LB.

These experiments showed promising results since several microorganisms were able to grow at rates similar to our controls in corresponding media. This led us to the improvement of the buffer conditions for the PEG mediated fusion of the cells.

All steps of the PEG fusion protocol are performed in liquid media. Therefore, it is crucial to achieve a maximal efficiency for spheroplast formation and regeneration for establishing a dependency and a production pathway.

Figure 3. Comparison of different buffers used for yeast spheroplast conversion. The relative cell number normalized to a control with yeast grown on YPD is shown. Measurements were performed doing fluorescence microscopy and counting calcofluor-white stained as well as unstained cells.

Due to the osmotic instability of yeast spheroplasts, buffers containing 1M sorbitol showed to be most suitable to minimize cell lysis. This applies for the spheroplasting medium as well, which is composed out of 1x YNB, 2% glucose, 1x amino acids, 50 mM Hepes-KOH, pH 7.2 and 1 M sorbitol. This buffer was chosen for further procedures since it showed to be the most stable, even though the initial spheroplasting efficiency is lower than in 1M sorbitol.

Figure 4. Measurement of spheroplasting efficiency using different amounts of Zymolyase in spheroplasting medium. The percentage of conversion into spheroplasts was calculated by comparison to samples from the same charge before adding Zymolyase.

However, the results from microscopy do not take cell lysis into account. Therefore, we also measured the optical density of yeast cells during zymolyase digestion in distilled water (Fig. 4). Since spheroplasts will undergo lysis in water, a decrease of the OD shows efficient digestion of the cell wall. By comparing the percentage of decrease in optical density to the ratio of intact cells and spheroplasts the percentage of lysed cells can be obtained.

These measurements show an efficiency for spheroplast conversion of about 50 % with a minimum of 5U Zymolyase for 5mL spheroplasts in 1M sorbitol with about 50% cell lysis. We decided to use the spheroplasting medium for for further experiments, since no fluctuation regarding the detectable cell number could be obtained.

Similar experiments have been performed to check the regeneration efficiency of spheroplasts after PEG treatment. We figured that the spheroplasting medium does not meet the requirements for regeneration due to a lack of essential nutrients. The most obvious approach, using 1M sorbitol for osmotic stability in YPD instead of water, showed promising results (Fig. 5). Unfortunately, we were unable to obtain suitable microscopy images for cell counting. This was a result of occurring cell aggregates after adding PEG to the cell suspension. Hence, division and therefore growth of regenerated cells cannot be taken into account.

Figure 5. Efficiency of spheroplast regeneration in different liquid media. Percentage of cells was calculated through comparison with samples from the same charge after resuspension in H₂O. Increasing cell number after 17h can occur due to cell division.

As expected, the regeneration medium containing 1M sorbitol in YPD showed to be most effective, since it contains all essential nutrients for yeast growth as well as an osmotically stabilizing concentration of sorbitol.

Optimization and verification of PEG-mediated cell fusion

An optimization of buffer conditions enabled us to test several variations of the originally described PEG protocols for bacterial uptake into yeast spheroplasts [1,2].

As initial setting we performed an uptake as described first [1], using calcofluor-white stained S. cerevisiae and E. coli carrying pFAB3677, a plasmid, which encodes for the mTangerine reporter protein (Fig. 6, Fig. 7).

Figure 6. Fluorescence microscopy images of S. cerevisiae and E. coli after applying the initial PEG protocol. (A) Calcofluor-white staining of yeast cells. (B) Brightfield image. (C) RFP fluorescence of E. coli MG1655. (D) Overlay image of all three channels.

Figure 7. Enlarged overlay image of S. cerevisiae and E. coli after applying the initial PEG protocol. Combined are calcofluor-white stained yeast (blue), RFP expressing E. coli MG1655 (red) and the brightfield image after PEG treatment. The arrow indicates a possible positive event of bacterial uptake.

These results were rather unsatisfying because the location of the bacteria cannot be distinguished. It is not clear whether they are inside of yeast or attached to the surface. In order to determine this, we switched from wild type CEN.PK-117 to a modified yeast constitutively expression mTurquoise into the cytoplasm.

This gave better results (not shown), even though the exact localization of the bacteria still remained unclear. Additionally, the cells showed to be extremely unstable which made fixation for proper microscope images rather difficult.

To obtain better microscopy images for showing a clear uptake of the bacteria, we optimized the PEG protocol further, starting with an optimization described previously [2]. Microscopy with this new attempt showed an improvement for fluorescent microscopy images (Fig. 8, Fig. 9), yet with still unclear localization. Although this has not been certain evidence, the qualitative improvement helped us define a final and optimized uptake protocol (see supplementary information). Even though only minor changes have been made to the already described optimized protocol, improvements towards the regeneration with respect to further applications regarding dependency and production were achieved.

Figure 8. Microscope images of S. cerevisiae and E. coli after applying the improved PEG protocol. (A) Brightfield image. (B) RFP fluorescence of E. coli MG1655. (C) mTurquoise fluorescence of S. cerevisiae.

Figure 9. Microscope overlay of improved PEG method. Overlay image of brightfield, mTurquoise (green) and RFP (red). Arrows indicate possible positive events for bacterial uptake.

To obtain certain evidence regarding the uptake and location we performed confocal microscopy. Through this we were able to make a 3D model by adding a third dimension through high resolution z-stacks (Fig. 10, Fig. 11).

Figure 10. Montage of z-stacks taken through confocal microscopy. Yeast cells shown in cyan and E. coli in red. Crossover of yellow lines indicate the focus point from which cross section of the (A) yz-axis, (B) xy-axis and (C) xz-axis were obtained. Scale bar = 5µm.

Figure 11. Gif-animated image of taken up E. coli (red) into yeast cells (cyan) after applying the finalized PEG protocol using PEG 3,350. This was obtained through interpolation of the z-stacks showed in figure 10. Scale bar = 5µm. Note that the scale bar is not appropriate for the z-axis.

This clearly proves the uptake of bacteria into yeast spheroplasts. Comparable models could be obtained for different bacteria using PEGs of different molecular weights, respectively (Fig. 12, Fig. 13). Even though this proves the uptake, the optimization of uptake conditions was determined by trial-and-error without the possibility of having data for exact quantification of protocol variations.

Figure 12. Gif-animated image of taken up E. coli (red) into yeast cells (cyan) after applying the finalized PEG protocol using PEG 6,000. Scale bar = 5µm. Note that the scale bar is not appropriate for the z-axis.

Figure 13. Gif-animated image of taken up PCC 7002 (red) into yeast cells (cyan) after applying the finalized PEG protocol using PEG 3,350. Scale bar = 5µm. Note that the scale bar is not appropriate for the z-axis.

To obtain data regarding the quantity of uptake we performed flow cytometry directly after the applying the fusion protocol. As a control, besides E. coli and normal yeast as well as spheroplasts, we performed the PEG fusion protocol omitting the cell wall digestion. Unfortunately, the flow cytometer could not distinguish between taken up bacteria and those that are attached to the host cell surface through the adhesive properties of PEG (Fig. 14). Hence, an exact quantification of the protocol still remains unclear.

Figure 14. Plotted flow cytometry data using (A) a co-culture control and (B) a sample after PEG treatment. Double positive events are displayed in Q2 for both (A) and (B). A comparison of both shows no relevant differences and therefore does not allow any conclusion regarding the quantity of bacterial uptake.

Literature

[1] Yoshida, Naoto, and Misa Sato. "Plasmid uptake by bacteria: a comparison of methods and efficiencies." Applied microbiology and biotechnology 83.5 (2009): 791-798.
[2] Guerra-Tschuschke, I., I. Martin, and M. T. Gonzalez. "Polyethylene glycol-induced internalization of bacteria into fungal protoplasts: electron microscopic study and optimization of experimental conditions." Applied and environmental microbiology 57.5 (1991): 1516-1522.

Supplementary information

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