The experimentally used agar plates were set-up as followed:

1. Nitrogen free Jensen’s medium
2. Jensen’s medium supplemented with nitrogen (adding Fe-[III]-ammonium citrate and potassium glutamate)
3. Liquid Jensen’s medium mixed with Fmoc-FF
4. Jensen’s medium supplemented with Fmoc-FF after the plates were dried

Type one plates serve as negative control, since B. subtilis (wild type) should not be able to grow in the absence of nitrogen. On the second plates, we added two nitrogen sources (Fe-[III]-ammonium citrate and potassium glutamate) allowing B. subtilis to grow. With plates of type three and four we intended to test if B. subtilis is capable of using the Fmoc-FF. We could only detect growth on type two plates, demonstrating that B. subtilis can neither grow with our provided nitrogen (type 1 plates) nor use Fmoc-FF as nitrogen source (type three and four plates).

Tolerance of B. subtilis against alkaline pH

In the course of Peptidosome generation, B. subtilis will be exposed to alkine conditions. Therefore we performed a pre-test to check the tolerance of B. subtilis towards high pH. Cultures were treated with four different NaOH molarities to adjust the final pH in the wells to 7, 8, 9 or 10. NaOH was added after one hour of cultivation (Figure 11, highlighted by the red dot). The experiment was carried out in a plate reader to follow the change of the optical density (OD₆₀₀) over a time period of 14 hours.

Figure 11, shows the growth of B. subtilis under various pH conditions. No significant changes in growth can be observed for pH 7 to 9. However, under pH 10 conditions, B. subtilis stops growing and shows a slight decrease in optical density. To check if the bacteria were still viable after the high pH treatment, we transferred bacteria at the end of the assay (after 14 hours) to agar plates and could obtain colony forming units. Thus, demonstrating that B. subtilis was able to recover and continue growing normally. Overall, we could clearly show that B. subtilis can survive the process of Peptidosome generation in regards to the pH conditions.

B. subtilis in Peptidosomes

After we have proven that B. subtilis can survive the designed encapsulation process, and that the organism cannot use the building block Fmoc-FF as a nitrogen source, experiments to entrap the bacteria were started.

For this we resuspended an appropriate amount of bacteria in the Fmoc-FF solution. Afterwards droplets of this were placed on an ultrahydrophobic membrane and exposed to CO₂ for 10 minutes, followed by a transfer to water or LB medium.

You can find the detailed protocols here.

Well Scan experiment using B. subtilis reporter strains

For the first experiments of encapsulation, we used the two B. subtilis reporter strains: TMB4131 W168 lacA::erm P_veg-sfGFP and TMB3090 W168 sacA::cat P_veg-luxABCDE. The first strain constitutively expresses sfGFP, which we used to detect the presence of bacteria in the Peptidosomes by following the fluorescence signal. The latter strain expresses luciferase in a constitutive way, which makes a detection of a luminescence signal possible. Having both of these well-evaluated readouts at hand, we wanted to demonstrate their applicability, with bacteria encapsulated in Peptidosomes. We performed a plate reader assay using the well-scan-mode. In this mode, the whole well is scanned to detect the exact position of a signal, either fluorescence or luminescence. Its absence is displayed in a map with a green color, while the position of the fluorescence/luminescence source appears as red. Please check out the according protocol for details.

In Figure 12 examples of the well scans of different samples are displayed.

The well scan maps appear completely green when fluorescence/luminescence signals are absent, i.e. in water and empty Peptidosomes. Wells containing a sample of the day culture and lyophilized eGFP dissolved in water show a red colour in the whole map. However, a localized red spot over a green background is observed when the source of fluorescence/luminescence is contained: the cells are trapped inside the Peptidosomes. This also shows, that Peptidosomes containing cells hinder the bacteria from diffusing and rather keeps them contained within the structure, demonstrated by the locally limited red signal. We were able to reproduce these results over a longer time period under shaking, highlighting the robustness of Peptidosomes.

Fluorescence microscopy with B. subtilis

Next, we used Fluorescence microscopy to analyse a B. subtilis strain encapsulated in Peptdiosomes, which constitutively expresses sfGFP (Figure 13). We could demonstrate, that the fluorescence signal was restricted to the Peptidosome - standing in line with the results obtained in the well scan experiments. This was the final proof that the bacteria are encapsulated in the Peptidosomes and are fully trapped.

The Growth of B. subtilis in Peptidosomes

After we checked the functionality of reporters in Peptidosomes, we were also interested to observe growth of B. subtilis while encapsulated. Since the Peptidosomes should be liquid-filled and allow the exchange of molecules, B. subtilis should be able to grow. To demonstrate this, we loaded Peptidosome with a known amount of bacteria and plated them on agar plates after different time periods of incubation at 37°C. These plates were then incubated over night and we documented the colony numbers on the next day (Table 1). We expected to observe an increase of the colony forming units in correlation of longer incubation times. Peptidosomes plated after 0 hours represent the starting bacterial concentration, as these Peptidosomes were plated on agar plates immediately after their generation.

As observed on the results, the number of colonies in the plates increased after 3.5 h of incubation, when compared to the starting bacterial concentration (0 hours). After 7 hours it was not possible to count the amount of colonies, cause B. subtilis formed a lawn. This clearly showed that B. subtilis was fully capable to grow inside of the Peptidosome cages. Thus, Peptidosomes are indeed suitable for the establishment of bacterial cultures.

Cryo-Scanning Electron Microscopy

To fully investigate how B. subtilis is encapsulated in the Peptidosomes, we performed Cryo-Scanning Electron Microscopy (Cryo-SEM). By doing so, we should get inside if the surface of the Peptidosome is also covered by bacterial cells.

As one can clearly see in the images above, some cells are completely or only partially integrated into the membrane of the Peptidosome (Figure 14, B). These cells, which are found on the outer surface of the membrane, if released, can result in bacterial growth outside of the Peptidosome.

To address this issue, we designed a second growth experiment, in which we also plated the supernatant of each Peptidosome to check the amounts of the "released" bacteria.

During this experiment two additional counter measurements were implemented, the first one consisted in adding “washing” step, were the Peptidosomes were transferred twice to fresh media before their incubation. We hypothesized that the bacteria on the outer membrane would be released and left behind in the course of the transfers. The second treatment that we tested consisted in pre-incubating of the Peptidosome in LB broth adjusted to an acid pH, this should guarantee an increased closing of the membrane by triggering the self-assembly of the any unreacted Fmoc-FF solution still present in the Peptidosome membrane.

By introducing the treatments, the number of colonies was dramatically reduced from several hundreds to an average of 0.7 for the washing steps and 1.7 for the low pH treatment (Table 2). From here, we concluded that the washing steps are important to reduce the number of cells released in the supernatant, whereas the treatment with acid broth does not add a significant effect on top of this. By introducing the washing steps and also reducing the concentration of cells in the Peptidosomes, the amount of bacteria present on the outer surface of the membrane was dramatically decreased. We also confirmed this by performing a second round of cryo-SEM, shown below.

In this second round of cryo-SEM the number of detectable bacteria that lie or are integrated in the membrane is significantly lower. Only a few individual cells are visible and the surface is less wrinkled. (Figure 15 A), B))

Only on the Peptidosome with the low pH treatment, a cell that was not integrated in the membrane was observed (Figure 15, D).

The results of the growth experiment with the additional treatments were confirmed microscopically. There was no significant improvement in the Peptidosomes treated with acid LB medium compared to the sole integration of washing steps. This confirms the reduced number of colonies in the supernatant of the growth experiment when carrying out the additional treatment methods (Table 2 and Figure 15).

The one loosely attached cell might be a problem for some applications where the surrounding medium is expected to be free of bacteria, therefore, the manufacturing technique of Peptidosomes should be adapted accordingly. An adequate method for this could be introducing the cells by microinjection, since there is not a chance that bacteria can be attached to the outer part of the membrane.

3. Surface Decoration

The properties of self-assembled Fmoc-FF surfaces allow tiny objects, such as Dynabeads, to be incorporated into the shell of Peptidosomes. Incorporating Dynabeads enables the control of the Peptidosomes' movements in a magnetic field, thereby providing a powerful delivery tool, which can be used in various applications.

Moreover, the surface of Dynabeads themselves can be decorated with a His-Tag. For the “proof of principle” experiments His-tagged GFP was selected allowing for an easy imaging procedure. First, the Invitrogen protocol was optimized for our goals and tested for Dynabead decoration with histidine-tagged GFP (Figure 16).

To prove the surface of the Dynabeads enveloped to the Peptidosome membrane availability for the decoration the labeling procedure was applied after the Peptidosome formation in both binding/washing buffer and LB media (Figure 17).

Here, we clearly demonstrated the possibility of Peptidosome surface decoration with histidine-tagged GFP. Hereby, the GFP serves as a proof of principle that can be exchanged with any other protein of interest and thus used for various applications where enzymatic activity of the surface of Peptidosome or Peptidosome immobilization is required. The surface decoration protocol is compatible with almost any goals due to its flexibility, as the decoration procedure can be performed before and after Peptidosome generation.

Conclusion

Our team successfully introduced a new immobilization system, the Peptidosome. We established a robust protocol for creating the Peptidosomes and to we are able to encapsulate bacteria that can live and grow inside the Peptidosome. In addition, we managed to functionalize the cages by decorating the surface. We can proudly announce that the Peptidosomes are the new fundamental approach for generating and applying encapsulated bacteria.