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. ⌬leuB and ⌬trpA 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.Less than 25 years ago, the archaea were virtually unknown. We now recognize that these organisms represent one of the fundamental domains of life (32) and constitute a significant fraction of the total biomass (9). Acceptance of the distinct status of the archaea has been largely due to genome sequencing projects (6). The data from the sequenced genomes have revealed that in spite of their prokaryotic morphology, the archaea have numerous similarities with eukaryotes (5), particularly in enzymes involved in core processes such as transcription (2), translation (10), and DNA replication (23). As the archaeal transcription and replication systems are considerably less complex than those found in eukaryotes, they are more amenable to analysis. Notwithstanding this sequence similarity, the archaea have a unique identity (12), which is best exemplified by novel enzymes such as the Holliday junction resolvase Hjc (19).In order to realize the true potential of the archaea, both as stripped-down models to dissect more complex eukaryotic systems and as a source of novel enzymes, it is essential to harness the power of genetics to underpin advances in biochemistry and genomics. Haloferax volcanii is an obligate halophile (25) that is genetically stable and grows aerobically in both complex and minimal media (24). It has some of the best genetic tools among the archaea, including a transformation system (8), reporter genes (17), shuttle vectors with antibiotic resistance (14,16,22), auxotrophic markers (26), and a recently developed gene knockout system based on the pyrE2 gene (3) (Fig. 1A), which encodes orotate phosphoribosyl transferase and is involved in uracil biosynthesis.Since the number of selectable genetic markers in H. volcanii is still limited, we wished to supplement it with additional markers. We describe here the isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively. Deletion of leuB, a homologue of the widely used Saccharomyces cerevisiae LEU2 gene (1), confers leucine auxotrophy in minimal medium. Deletion of the trpA gene (20) co...

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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 ⌬pyrE2 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.

Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature the tryptophan-inducible p.tnaA promoter and a 6؋His tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis. The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.

Haloarcula marismortui is an archaebacterium that flourishes in the world's saltiest body of water, the Dead Sea. The cytosol of this organism is a supersaturated salt solution in which proteins are soluble and active. The crystal structure of a 2Fe-2S ferredoxin from H. marismortui determined at 1.9 A is similar to those of plant-type 2Fe-2S ferredoxins of known structure, with two important distinctions. The entire surface of the protein is coated with acidic residues except for the vicinity of the iron-sulphur cluster, and there is an insertion of two amphipathic helices near the N-terminus. These form a separate hyperacidic domain whose postulated function to provide extra surface carboxylates for solvation. These data and the fact that bound surface water molecules have on the average 40% more hydrogen bonds than in a typical non-halophilic protein crystal structure support the notion that haloadaptation involves better water binding capacity.