Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes

Abstract

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.

FIG. 1.

Gene knockout system based on the pyrE2 gene. (A) A plasmid carrying the pyrE2 marker and flanking sequences of the gene to be deleted is used to transform a ΔpyrE2 H. volcanii strain to uracil prototrophy. Here, the crossover used to integrate the plasmid (pop-in) has occurred to the left of the deletion. Subsequent loss of the plasmid by intrachromosomal crossing over can occur on the left of the deletion, restoring the gene to wild type, or on the right of the deletion, resulting in the desired mutant. In either case the cell is rendered auxotrophic for uracil and is therefore resistant to 5-FOA by virtue of its inability to convert this compound to the toxic analog 5-fluorouracil. (B) The gene is replaced with the trpA marker, and the plasmid is used to transform a ΔpyrE2 ΔtrpA H. volcanii strain to prototrophy for uracil and tryptophan. Loss of the plasmid by crossing over on the right of the deletion, resulting in a trpA-marked mutant, can be selected in one step.

FIG. 2.

Construction of leuB deletion plasmids. The genomic leuB clone pTA44 contains a 4,162-bp genomic DNA BssHII fragment cloned in pBluescript II. Sequences flanking leuB were amplified from pTA44 and cloned in pBluescript II to generate the ΔleuB construct pTA65. The mevinolin resistance fragment from pMDS99 (31) was inserted into pTA65 to generate pTA70. Alternatively, the ΔleuB construct from pTA65 was cloned in the pyrE2-marked gene knockout plasmid pGB70 (3), generating pTA73. Only relevant sites are shown; full plasmid maps are available on request.

FIG. 3.

Deletion of leuB gene. (A) Plasmids pTA70 and pTA73 were constructed as described in the legend to Fig. 2. Integration of pTA70 into the chromosome of DS70 by homologous recombination upstream of leuB resulted in strain H23. Loss of the plasmid by intrachromosomal recombination (Fig. 1) resulted in the ΔleuB strain H37. Integration of pTA73 (in H26) by recombination downstream of leuB yielded strain H60, and loss of the plasmid resulted in the ΔleuB strain H66. Integration and deletion events were monitored by digestion with MluI (M), resulting in the fragments indicated. (B) Southern blot analysis of ΔleuB strain construction. Genomic DNA were prepared from the strains indicated, digested with MluI, and probed with the flanking regions of leuB.

FIG. 4.

Deletion of trpA gene. (A) Construction of trpA deletion plasmids. The genomic trpA clone pTA49 contains a 3,676-bp genomic DNA Sau3AI fragment cloned in pBluescript II. Sequences flanking trpA were amplified from pTA49 and cloned in pBluescript II to generate the ΔtrpA plasmid pTA92. The mevinolin resistance fragment from pMDS99 (31) was inserted into pTA92 to generate pTA93 (data not shown; similar to pTA70 in Fig. 2). Alternatively, the ΔtrpA construct from pTA92 was cloned in pGB70 (3), generating pTA95 (data not shown; similar to pTA73 in Fig. 2). Plasmid maps are available on request. (B) Deletion of the trpA gene in strains H77 and H53 was analyzed by MluI digestion and Southern blot hybridization by using flanking regions of trpA as a probe (similar to the leuB deletion in Fig. 3). Integration of pTA93 into the chromosome of DS70 by recombination upstream of trpA gave a novel 5.8-kb MluI fragment, producing strain H43. Loss of the plasmid, which deleted trpA (1.8-kb MluI fragment instead of 2.5-kb MluI fragment) resulted in ΔtrpA strain H77. Integration of pTA95 into H26 by recombination downstream of trpA gave a novel 5.5-kb MluI fragment, producing strain H47, and loss of the plasmid resulted in ΔtrpA strain H53.

FIG. 5.

Plasmid vectors marked with pyrE2, leuB, trpA, and hdrB. (A) Gene knockout plasmids. The pyrE2, leuB, trpA, and hdrB genes were placed under control of the ferredoxin promoter and cloned in pBluescript II to generate pTA131, pTA132, pTA133, and pTA192, respectively. (B) Shuttle vectors. The pHV2 replication origin from pWL102 (7, 22) was cloned in pTA131, pTA132, pTA133, and pTA192 to generate pTA230, pTA231, pTA232, and pTA233, respectively. Some sites in the polylinker are not unique in the shuttle vectors; full plasmid maps are available on request.

FIG. 6.

Deletion of lhr gene. (A) Construction of lhr deletion plasmid. The genomic lhr clone pTA150 was used to amplify the flanking regions of lhr, which were inserted into pTA131 to generate the Δlhr construct pTA166. A fragment containing the trpA gene under control of the ferredoxin promoter (p.fdx) was inserted at the site of the lhr deletion (NcoI), generating the Δlhr::trpA plasmid pTA172. (B) Twelve 5-FOA-resistant (Ura⁻) derivatives of pop-in strains H107 to H109 were grown on rich agar, transferred to a nylon filter, and probed with the lhr coding sequence. In the case of H109, the four Ura⁻ clones with lhr deleted had previously been shown to be prototrophic for tryptophan (Trp⁺), whereas the remaining eight clones in which lhr was not deleted were Trp⁻. (C) Deletion of the lhr gene was analyzed by BspEI digestion and Southern blot hybridization by using the downstream flanking region of lhr as a probe. Integration of pTA166 into the chromosomes of H26 and H53 gave a novel 4.8-kb BspEI fragment, producing strains H107 and H108, respectively, and loss of pTA166, which deleted lhr (4.5-kb BspEI fragment instead of 7.3-kb BspEI fragment), resulted in Δlhr strains H120 and H121, respectively. Integration of pTA172 in the chromosome of H53 gave a novel 4.6-kb BspEI fragment, producing strain H109, and loss of pTA172, which deleted lhr (4.3-kb BspEI fragment), resulted in Δlhr::trpA strain H122.