The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases

Abstract

SummaryThe gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DhdrA, DhdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.

A Gateway platform for functional genomics in Haloferax volcanii: deletion of three tRNA modification genes

“…We have shown elsewhere that this strain displays a growth rate comparable to H26 in supplemented medium (Phillips et al 2008). The folE2 gene can therefore be used as a positive auxotrophic selectable marker adding to the few already available (Ortenberg et al 2000, Bitan-Banin et al 2003, Allers et al 2004.…”

“…Haloferax volcanii is one of the rare archaea with both active folate and G + pathways (Gupta 1984, Gupta 1986, Ortenberg et al 2000; however, no folE gene has been identified in its genome. Instead, H. volcanii possesses a folE2 gene (HVO_2348) encoding a newly discovered GTP cyclohydrolase enzyme IB that is involved in archaeosine biosynthesis (El Yacoubi et al 2006, Phillips et al 2008.…”

Maturation of the 5S rRNA 5′ end is catalyzed in vitro by the endonuclease tRNase Z in the archaeon H. volcanii

“…A 685-bp PCR product, comprising the hdrB coding sequence (Ortenberg et al 2000), was inserted at the XbaI-HindIII sites directly downstream of the pyrE2 gene in pGB70 (BitanBanin et al 2003). To generate pTA409, the 1253-bp pyrE2ThdrB operon was inserted at the PsiI site in pBluescript II, and a 948-bp BmgBI-AciI fragment containing the ori-pHV1/4 DNA replication origin of H. volcanii plasmids pHV1 and pHV4 (Norais et al 2007) was inserted at the PciI site.…”

Development of a Gene Knockout System for the Halophilic ArchaeonHaloferax volcaniiby Use of thepyrEGene

“…H. volcanii is a genetically stable prototroph that has become a model organism for molecular genetic studies of the archaea (6,21,25). The presence in H. volcanii of an efficient transformation system (5), several shuttle vectors (7,10), and selectable markers (8,15) has made a wide variety of molecular genetic studies possible. However, a key tool for genetic analysis, namely, the availability of an efficient gene knockout system, has been lacking.…”