Improving the genetic system for Halorubrum lacusprofundi to allow in-frame deletions

L Johanna Gebhard; Iain G Duggin; Susanne Erdmann

doi:10.3389/fmicb.2023.1095621

Improving the genetic system for Halorubrum lacusprofundi to allow in-frame deletions

Front Microbiol. 2023 Mar 31:14:1095621. doi: 10.3389/fmicb.2023.1095621. eCollection 2023.

Authors

L Johanna Gebhard¹, Iain G Duggin², Susanne Erdmann¹

Affiliations

¹ Archaeal Virology, Max Planck Institute for Marine Microbiology, Bremen, Germany.
² The Australian Institute for Microbiology and Infection, University of Technology Sydney, Sydney, NSW, Australia.

Abstract

Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, Antarctica. Hrr. lacusprofundi is commonly used to study adaptation to cold environments and thereby a potential source for biotechnological products. Additionally, in contrast to other haloarchaeal model organisms, Hrr. lacusprofundi is also susceptible to a range of different viruses and virus-like elements, making it a great model to study virus-host interactions in a cold-adapted organism. A genetic system has previously been reported for Hrr. lacusprofundi; however, it does not allow in-frame deletions and multiple gene knockouts. Here, we report the successful generation of uracil auxotrophic (pyrE2) mutants of two strains of Hrr. lacusprofundi. Subsequently, we attempted to generate knockout mutants using the auxotrophic marker for selection. However, surprisingly, only the combination of the auxotrophic marker and antibiotic selection allowed the timely and clean in-frame deletion of a target gene. Finally, we show that vectors established for the model organism Haloferax volcanii are deployable for genetic manipulation of Hrr. lacusprofundi, allowing the use of the portfolio of genetic tools available for H. volcanii in Hrr. lacusprofundi.

Keywords: Halorubrum lacusprofundi; archaea; auxotrophic mutant; cold adaptation; genetic system; haloarchaea.