With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment. 1. Introduction Since the identification of the three-kingdom paradigm over 30 years ago [1], the quest for model archaeal organisms, representing each of the main groups, has been ongoing. To date, of the culturable Archaea, Sulfolobus sp. remain amongst the most intensely investigated and have become the subject of modern postgenomics experimental techniques [2, 3], yet in vivo studies in these organisms have lagged, as robust genetic methodologies are lacking (although this is now being addressed [4, 5]). The ability of thermophiles, such as Thermococcus kodakaraensis and Pyroccocus furiosus, to thrive at in excess of 95？C is of great interest (particularly to industrials). However, whilst T. kodakaraensis has a well-developed genetic system, optimal growth at high temperatures coupled with anaerobic growth requirements puts these organisms out of reach of nonspecialist laboratories [6–8]. The methanogens received an early lead with the first available archaeal genome sequence, Methanococcus jannaschii [9], and genetic manipulation techniques have been described in the closely related Methanococcus maripaludis [10, 11]. Yet, as strict anaerobes, their manipulation is also still restricted to specialists. Thanks to the endeavours of a growing community of investigators (see http://www.haloferax.org/), Haloferax volcanii is filling the role of model archeaon and model halophile. Its relatively short generation time of <4 hours [12, 13], ease of growth under laboratory conditions, genome-sequence availability [14], and development of genetic tools [15–17] (including
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