Buffered solution: 0.5/1/1.5/2 M NaCl; 27 mM KCl; 50 mM Tris-HCl pH8. After autoclaving, it was topped up with 15% sucrose (filter-sterilized) and stored at room temperature (RT).

Unbuffered solution: 0.5/1/1.5/2 M NaCl; 27 mM KCl; Adjust to pH~7. After autoclaving, it was topped up with 15% sucrose (filter-sterilized) and stored at RT.

60% PEG₆₀₀: 60% (v/v) PEG₆₀₀ and 40% (v/v) unbuffered solution. PEG₆₀₀ was stored at −80 °C for up to 6 months and then thawed at 65 °C.

This protocol was adapted from Orsini et al. (2020). Nmn. pharaonis was cultured in modified DSM 205 medium with 70 μg/ml of bacitracin at 37 °C and 110 rpm. After 72 h (OD₆₀₀ 0.2–0.3, ~1–1.5 × 108 cells/ml), the culture was centrifuged for 15 min at 3,900 × g. The pellet was resuspended in buffered solution (2 M) at half the culture volume, containing 0.3 mg/ml pronase E. The cells were then incubated for 48 h at 42 °C. The combination of bacitracin and pronase E enables the removal of the S-layer (a pseudo-crystalline proteinaceous layer that constitutes the archaeal cell wall) and the formation of spheroplasts. After verifying the spheroplastic appearance of the cells by microscopy, 3 ml of spheroplasts were collected by centrifugation (3 min at 10,000 × g) and resuspended in 150 μl of buffered solution (2 M). After adding 50 mM EDTA pH 8 and incubating for 10 min at RT, 5 μg of DNA was added (as used in Derntl et al., 2015) and incubated for 5 min at RT. The cells were then mixed with 150 μl of 60% PEG₆₀₀ [in unbuffered solution (2 M)] and incubated for 30 min at RT. The cells were washed twice with 1 ml of NVM⁺ medium, then centrifuged for 3 min at 10,000 × g. After resuspension of the pellet in 1 ml of NVM⁺ medium, the cells were regenerated at 37 °C and 110 rpm for 48 h. After verification of cell regeneration by microscopy, 100 μl of cells were plated onto NVM⁺ agar plates containing 10 μg/ml of novobiocin. The plates were incubated at 42 °C until colonies appeared. Transformation efficiency was calculated by counting all colonies appearing from the first detectable transformants (about 3 weeks) up to the last colonies observed after 5 weeks of incubation.

The transformations were performed using cultures of Nmn. pharaonis in modified DSM 205 medium. To this end, 3 ml of a culture at the end of its exponential phase (OD₆₀₀ 0.3–0.4, ~1.5–2 × 108 cells/ml) was centrifuged for 3 min at 10,000 x g and the pellet was resuspended in 150 μl of buffered solution (0.5–2 M). After adding 50 mM EDTA pH 8 and incubating for 10 min at RT, 5 μg of DNA was added and incubated for 5 min at RT. The cells were then mixed with 150 μl of 60% PEG₆₀₀ in unbuffered solution (0.5–2 M) and incubated for 30 min at RT. The mixture was then introduced into a 1 ml syringe sealed with a hermetic rubber stopper and placed inside a steel tube. Pressure was applied using a manual hydraulic pump to 10–60 MPa (Figure 1). For the 0 MPa condition, samples were not placed into a syringe or steel tube. The steel tubes had an internal diameter 38 mm, a maximum operating temperature of 250 °C and a maximum pressure of 90 MPa. The target pressure was reached at a rate of 60 MPa per minute. Pressure was released instantly (a few milliseconds), by opening the high pressure valve. Following treatment, cells were washed twice with 1 ml of NVM⁺ medium and centrifuged for 3 min at 10,000 × g. After resuspension of the pellet in 1 ml of NVM⁺ medium, the cells were regenerated at 37 °C and 110 rpm for 48 h. Subsequently, 100 μl of cells were plated onto NVM⁺ agar plates containing 5 or 10 μg/ml of novobiocin. The plates were incubated at 42 °C until colonies appeared. Transformation efficiency was calculated by counting all colonies appearing from the first detectable transformants (which could appear as early as 1 week, depending on the condition) up to the last colonies observed after 5 weeks of incubation.

The transformations were carried out using cultures of Nmn. pharaonis in modified DSM 205 medium. To this end, 3 ml of a culture at the end of its exponential phase (OD₆₀₀ 0.3–0.4, ~1.5–2 × 10⁸ cells/ml) was centrifuged for 3 min at 10,000 × g and the pellet was resuspended in 150 μl of betaine solution (4.3 M betaine in 50 mM Tris-HCl pH 8.7). Next, 5 μg of DNA was added and incubated for 10 min at RT. A pressure of 35 MPa was then applied as described previously (Figure 1). The cells were then washed twice with 1 ml of NVM⁺ medium and centrifuged for 3 min at 10,000 × g. After resuspension of the pellet in 1 ml of NVM⁺ medium, the cells were regenerated at 37 °C and 110 rpm for 48 h. Subsequently, 100 μl of cells were plated onto NVM⁺ agar plates with 10 μg/ml of novobiocin. The plates were incubated at 42 °C until the appearance of transformants. Transformation efficiency was calculated by counting all colonies appearing from the first detectable transformants (about 3 weeks) up to the last colonies observed after 5 weeks of incubation.

Biophysical methods for cellular transformation rely on a delicate balance between inducing sufficient membrane disruption to allow the entry of exogenous DNA and preserving cell viability (Huang et al., 2024). To identify the optimal pressure required to achieve this balance, we tested a range of hydrostatic pressures from 0 to 60 MPa. For each condition, we evaluated both cell survival/regrowth and transformation efficiency (calculated based on all colonies appearing up to 5 weeks of incubation).

As shown in Table 1, applying pressure ranging from 0 to 60 MPa did not significantly affect cell survival prior to regeneration. For most pressures conditions, survival rates remained between 64.4 and 100%, except at 20 MPa, where survival dropped significantly to 25.1% (Table 1). Considerable variability between replicates was also observed for some pressure (Table 1). After regeneration, cell recovery reached 100% across all pressure conditions (Table 1).

As shown in Table 1, cell transformation induced by hydrostatic pressure in the presence of the pRo-5 vector led to the appearance of novobiocin-resistant colonies across the entire range of pressures tested. For pressures ≥10 MPa, transformation efficiencies ranged from 1 to 2.6 log₁₀ CFU/μg DNA, with the highest efficiencies observed at 35 and 40 MPa at 5 μg/ml of novobiocin and 20 and 35 MPa at 10 μg/ml of novobiocin (Table 1).

At 5 μg/ml of novobiocin, application of pressure led to a significant increase in transformation efficiency compared with the non-pressurized condition (Table 1). Nevertheless, in the absence of pressure (0 MPa), transformants were detected, but at much lower frequencies for both novobiocin concentrations (Table 1).

Compared to the standard PEG-mediated spheroplast transformation, the hydrostatic pressure-based protocol increased transformation efficiency by 5.5-fold at 10 μg/ml of novobiocin (2.2 vs. 0.4 log₁₀ CFU/μg DNA for the hydrostatic pressure-based protocol at 35 MPa and the standard protocol, respectively) and by 6.5-fold at 5 μg/ml of novobiocin (2.6 vs. 0.4 log₁₀ CFU/μg DNA for the hydrostatic pressure-based protocol at 35 MPa and the standard protocol, respectively; Table 1).

These results suggest that applying hydrostatic pressure ≥10 MPa probably induces a transient increase in membrane permeability, thereby facilitating DNA uptake. Since the magnitude of the applied pressure applied did not significantly affect transformation efficiency, a pressure of 35 MPa was selected for subsequent experiments, as it provided the best combination of cell survival/regrowth and transformation efficiency.

In the PEG-mediated spheroplast transformation protocol, false positives (typically 1–2 true transformants out of ≤ 10 colonies) were observed at 5 μg/ml novobiocin, whereas none were detected at 10 μg/ml. Therefore, the hydrostatic pressure-based protocol was tested at both concentrations to determine the most suitable condition. Only a few false positives ( ≤ 1% of the clones obtained) were detected for both novobiocin concentrations, with similar frequencies. Due to the higher number of transformants at 5 μg/ml of novobiocin (2.6 log₁₀ CFU/μg DNA, which is 6.5-fold higher than the PEG-mediated spheroplast transformation protocol and 1.2-fold higher than the hydrostatic pressure-based protocol at 10 μg/ml of novobiocin; Table 1), this concentration was selected for the final protocol.

In conclusion, the hydrostatic pressure-based protocol (35 MPa with 5 μg/ml novobiocin) enabled successful transformation and increased efficiency up to 6.5-fold compared to the PEG-mediated spheroplast transformation protocol. A pressure of 35 MPa was therefore used for all of the following results.

Nmn. pharaonis requires at least 2 M NaCl to maintain cellular integrity (Bonnaud et al., 2024) and this concentration is therefore used as the standard concentration in transformation solutions (Orsini et al., 2020). Here, we investigated whether a further reduction in NaCl concentration (in both buffered and unbuffered solution used for the DNA introduction step) could improve transformation efficiency under the hydrostatic pressure-based protocol by weakening the cell membrane without compromising viability. Four different NaCl concentrations (0.5/1/1.5/2) were tested, and for each condition, both cell survival/regrowth and transformation efficiency (calculated based on all colonies appearing up to 5 weeks of incubation) were assessed.

Cell survival and regrowth remained high under most conditions (Figure 2A). At the lowest concentration (0.5 M), a non-significant decrease in survival was observed prior to regeneration, with an average survival rate of 67.8% (± 26.9%; Figure 2A). However, following regeneration, 100% of regrowth was observed (Figure 2A). This suggests that although the osmotic downshift to 0.5 M causes substantial initial cell death, the regeneration time allows the surviving population to re-establish a normal phenotype. At 1.5 M, a non-significant decrease is also observed before regeneration, with an average survival rate of 72.4% (±25.8%).

Even with a decrease in survival under some conditions, transformants were obtained at all tested concentrations (Figure 2A). Overall transformation efficiency across all NaCl concentration was around 2.4 log₁₀ CFU/μg DNA at 5 μg/ml of novobiocin and 1.9 log₁₀ CFU/μg DNA at 10 μg/ml of novobiocin, with no significant differences between concentrations of salt (Figure 2A). The highest transformation measured values were at 2 M NaCl (2.6 log₁₀ CFU/μg DNA at 5 μg/ml of novobiocin and 2.2 log₁₀ CFU/μg DNA at 10 μg/ml of novobiocin; Figure 2A). Looking at the standard deviations shows that the intervals overlap, indicating that salt concentration has no significant impact on the transformation efficiency (Figure 2A).

The timing of colony appearance varied with NaCl concentration (Figure 2B). Over the first 3 weeks, transformation efficiency was higher at 1 M, with colonies appearing as early as 1 week and reaching an average of 6.8 CFU/μg DNA. In contrast, at 2 M, the first transformants were observed only after 4 weeks (>1 clone), with an average efficiency of 0.5 CFU/μg DNA (Figure 2B). These results indicate that, although overall transformation efficiency over 5 weeks is similar across NaCl concentrations, a transient reduction to 1 M NaCl facilitates earlier clone emergence and higher efficiency during the initial weeks of incubation.

In conclusion, the temporary reduction of the NaCl concentration to 1 M allows the overall efficiency of the transformation to be maintained while promoting faster emergence of transformants and higher efficiency during the first few weeks. This adjustment saves up to 21 days on the total duration of the protocol (first transformants at 7 days at 1 M vs. 28 days at 2 M).

The PEG-mediated spheroplast transformation method (Orsini et al., 2020) relies partly on PEG₆₀₀, which allows cells to become permeable and facilitates the entry of DNA. To assess whether this compound is essential for the successful generation of transformants under hydrostatic pressure-based protocol conditions, we evaluated the efficiency of the protocol with and without PEG₆₀₀. All transformations were performed under “high-salt” condition (2 M NaCl), i.e., the standard condition. Cells had greater difficulty recovering without PEG₆₀₀. Omission of PEG₆₀₀ reduced post-regeneration growth from 100% (with PEG₆₀₀) to 66.1% (±37.2) but this difference was not statistically significant due to the high variability among replicates.

Regarding transformation efficiency, only one of the four replicates yielded transformants of the same order of magnitude as those obtained with PEG₆₀₀ (2.5 log₁₀ CFU/μg DNA without PEG₆₀₀ vs. 2.6 log₁₀ CFU/μg DNA with PEG₆₀₀). Since this step is very simple and transformation efficiency shows considerable variability between replicates in absence of PEG₆₀₀, we therefore chose to keep proceeding using PEG₆₀₀ in the final protocol.

The study by Huang et al. (2024) indicated that the duration of the applied pressure does not appear to influence transformation efficiency. We therefore investigated the effect of multiple pressurization/depressurization cycles. Since treatment at 35 MPa did not affect cell survival (Table 1), we aimed to determine whether applying several cycles could enhance transformation efficiency without substantially compromising cell viability.

Before regeneration, cell survival is maintained at 100% for up to two pressurization/depressurization cycles (Table 2). In the third cycle, a marked drop in cell viability is observed (46.7%). However, after four cycles, survival returns to 100% (Table 2). After regeneration, no difference is observed between the different pressure cycles (Table 2). Thus, Nmn. pharaonis shows good resistance to repeated cycles of successive pressurization/depressurization, with no significant alteration in survival. Regarding transformation efficiency, no difference was observed between one and multiple cycles (Table 2).

Similar to electroporation, high-pressure-mediated transformation relies theoretically on the transient formation of pores in the cell membrane, followed by their closure and the restoration of membrane integrity (Huang et al., 2024). Although “poration” is transient, we wanted to determine whether a regeneration step was still necessary to obtain transformants.

Regardless of salt concentration, cells show high survival rates of 100% (1 M/2 M at 35 MPa) before and after regeneration (Table 1 and Figure 2). However, no transformant was obtained prior to regeneration. Thus, although “poration” is transient, the recovery step appears to be necessary after high-pressure treatment in order to obtain transformants. This regeneration time is undoubtedly necessary for the cell to restore normal metabolism and express the inserted antibiotic resistance gene.