Genomic manipulations in alkaliphilic haloarchaea demonstrated by a gene disruption in Natrialba magadii.

Alkaliphilic haloarchaea, a distinct physiological group from the closely related neutrophilic haloarchaea, represent an underutilized resource for basic research and industrial applications. In contrast to the neutrophilic haloarchaea, no reports on genomic manipulations in haloalkaliphiles have been published until now. Genomic manipulations via homologous recombination are useful for basic research. In this study, we demonstrate the possibility for this strategy in alkaliphilic haloarchaea for the first time. In a previous study, we developed a PEG-mediated transformation technique for alkaliphilic haloarchaea that was deployed in this study to deliver a gene disruption cassette into the model organism Natrialba magadii. The gene encoding for the well-studied Natrialba extracellular protease was successfully disrupted by a recombination marker gene, demonstrating a proof of principle for the usability of homologous recombination for genomic manipulations in alkaliphilic haloarchaea. Since halo(alkali)philic Archaea are polyploid, a selection process was applied in order to obtain a mutant strain containing exclusively disrupted genes. The resulting strain exhibited no proteolytic activity measurable by an azo-casein assay. Complementation was able to restore proteolytic activity. The expression pattern of the Natrialba extracellular protease was different in the complemented strain.