Hydrostatic pressure-enabled transformation in Natronomonas pharaonis: breaking barriers in haloalkaliphilic Archaea...

Haloalkaliphilic Archaea that thrive in hypersaline and hyperalkaline environments represent valuable models for fundamental research as well as promising resources for biotechnological applications. However, fully exploiting their potential is hindered by the difficulty of their genetic manipulation, due to the limited availability of genetic tools and the use of a transformation protocol that remain slow and weakly efficient. This limits both industrial exploitation and the study of these organisms. In this study, we describe the development of a new, faster, and more efficient transformation method. It relies on the application of hydrostatic pressure, followed by a rapid return to atmospheric pressure, which generates transient pores in the cell membrane, facilitating the uptake of exogenous DNA. The results demonstrate that applying a pressure of 35 MPa in the presence of 1 M NaCl allows the rapid and efficient generation of transformants in Natronomonas pharaonis. This protocol enhances transformation efficiency 6.5-fold while cutting the time required to obtain transformants by 17 days, in contrast to Polyethylene Glycol (PEG)-mediated spheroplast transformation. This new transformation method greatly facilitates the genetic manipulation of Nmn. pharaonis (and surely other haloalkaliphilic Archaea that are difficult to transform), thereby opening up new industrial and research applications.