Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry.Haloferax volcaniioffers 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 ofH. volcanii leuBandtrpAgenes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. ΔleuBand ΔtrpAmutants 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 theleuBortrpAgene under control of a constitutive promoter. The use of these selectable markers in deletion of thelhrgene ofH. volcaniiis described.

Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general. We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb). The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

The tree of life is split into three main branches: eukaryotes, bacteria, and archaea. Our knowledge of eukaryotic and bacteria cell biology has been

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.