Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene

Abstract

So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A DeltapyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.

FIG. 1.

Restriction map of H. volcanii gene knockout plasmid pGB70. Hbt. sal., Halobacterium salinarum.

FIG. 2.

Multiple alignment of the E. coli PyrE, S. cerevisiae Ura5 (S.cer), H. volcanii PyrE1 (H.vol1), and H. volcanii PyrE2 (H.vol2) proteins.

FIG. 3.

Schematic diagram of disruption of the pyrE1 gene in strain WR445 and disruption of the pyrE2 gene in strain WR340 and Southern blot analyses of the mutant strains. (A) Plasmids pGB53 and pGB68 were constructed as described in Materials and Methods. The plasmids were integrated into the chromosome by homologous recombination between the chromosomal sequence A and the plasmid sequence A′, and novobiocin-resistant recombinants were selected. Following relief of selection, recombination events could result in either reconstitution of the wild-type allele or deletion of the chromosomal pyrE gene. pyrE1′ is the first 330 bp of the pyrE1 gene. (B) Analysis of pyrE1 mutant. Total DNA was prepared from WR445 (parental strain) and WR473 (ΔpyrE1), digested with the EcoRI and PstI restriction enzymes, and analyzed by Southern blotting by using fragment A as a probe. For analysis of the ΔpyrE2 mutant, total DNA was prepared from WR430 (parental strain) and WR480 (ΔpyrE2), digested with the ClaI and PstI restriction enzymes, and analyzed by Southern blotting by using fragment B as a probe.

FIG. 4.

Partial restriction map of the cmi4 gene and its flanking sequences (A) and PCR analysis (B) and Southern blot analysis (C) of cells that underwent excision of the integrated plasmid and could grow on media containing 5-FOA. (A) The long solid arrow represents the cmi4 gene. The locations of PCR primers 415 us cmi4 and ds cmi4-Rev are indicated by short arrows, and fragment C was used as a probe for Southern blot analysis. (B) PCR were performed with DNA isolated from 16 colonies that grew on media containing 5-FOA by using primers 415 us cmi4 and ds cmi4-Rev. Lanes M contained molecular weight markers. (C) Total DNA was prepared from WR341 (parental strain), one cmi4 deletion strain (as determined by PCR) (lane 1), and a cmi4 reconstituted strain (as determined by PCR) (lane 3). The DNA was digested with the BsiWI and ClaI restriction enzymes and analyzed by Southern blotting by using fragment C as a probe.