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

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.

Figure 1

Successful pyrE2 knockout in Hrr. lacusprofundi ACAM34 strains. (A) Characterization of ACAM34_UNSWΔpyrE2 mutants. Left: PCR on the pyrE2 locus of the two knockout strains S1 and S2 (see also Supplementary Figure 2) and a wild-type ACAM34_UNSW control (C+). Right: Growth of ACAM34_UNSWΔpyrE2 on DBCM2- supplemented with uracil (top), and growth defect on DBCM2− without uracil (bottom). (B) Characterization of ACAM34_DSMZΔpyrE2. Left: PCR on the pyrE2 locus of the knockout mutant, ACAM34_DSMZ wild-type DNA served as the positive control (+C) (see also Supplementary Figure 5). Right: Growth of ACAM34_DSMZΔpyrE2 on DBCM2- supplemented with uracil (top), and growth defect on DBCM2- without uracil (bottom). (C) Schematic representation of the genomes including the wild-type strain ACAM34_UNSW with the position (bp) of pyrE2 and neighboring genes. ACAM34_DSMZ and ACAM34_UNSW deviate in the length of the annotated pyrE2 gene, the different position is marked in brackets for ACAM34_DSMZ. The primer binding sites for upstream (UF, UR) and downstream (DF, DR) flanking regions are represented as arrows. The deletion in the ACAM34_UNSWΔpyrE2 S1 and S2 clones is identical and highlighted in red. The deletion in ACAM34_DSMZΔpyrE2 is also highlighted in red.

Figure 2

Growth comparison of Hrr. lacusprofundi ACAM34_UNSW and ACAM34_UNSWΔpyrE2. The growth of wild-type (A) and mutant cells (C) in rich medium was observed by optical density measurements at 600 nm (OD_{600 nm}) over 102 h. Points represent the average values and each time point measured, including the standard deviation represented by error bars (n = 3). Light microscopy images of wild-type (B) and mutant cells (D) represent one of the three biological replicates (Supplementary Figure 4). Larger image at 400× magnification, black scale bar indicates 10 μm, inlet at 1,000× magnification, white scale bar indicates 2 μm.

Figure 3

Successful trpA knockout in Hrr. lacusprofundi ACAM34_UNSWΔpyrE2. (A) Recombination strategy used to generate ACAM34_UNSWΔpyrE2 ΔtrpA via double selection. A vector based on pTA131 (Allers et al., 2004) (with pyrE2 for uracil synthesis), was used to insert a pravastatin resistance gene (hmgA), as well as a non-functional copy of the trpA gene created by the fusion of upstream (US) and downstream (DS) fragments of the gene. Both the non-functional gene (represented by a triangle) and the functional wild-type trpA gene are marked in red. Crossover at the trpA genes leads to the integration of the plasmid into the genome (pop-in) using uracil depletion and the addition of pravastatin as selection pressure. Excision of the plasmid (pop-out) is enforced by the addition of 5-FOA and uracil, through which either the non-functional construct (left) or the wild-type gene (right) remains in the genome. (B) PCR on the trpA locus of the two knockout strains S1 and S2, with a wild-type ACAM34_UNSW control (C+) and a negative control (C−). (C) Operon encoding genes for tryptophan synthesis in the wild-type strain ACAM34_UNSW including the position (bp) of trpA and the primer binding sites for upstream (UF, UR) and downstream (DF, DR) flanking regions amplified for the deletion construct. Deletion in ACAM34_UNSWΔpyrE2ΔtrpA S1 and in ACAM34_UNSWΔpyrE2ΔtrpA S2 highlighted in red. (D) Growth of ACAM34_UNSWΔpyrE2ΔtrpA S1 (top) and S2 (bottom) on DBCM2 supplemented with uracil and tryptophan (left), growth defect on DBCM2 with uracil but without tryptophan (middle), growth defect on DBCM2 (right).