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

In part due to the existence of simple methods for its cultivation and genetic manipulation, Haloferax volcanii is a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for an in silico prediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion in H. volcanii uracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT), which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I), which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family), which is involved in N4-acetylcytidine (ac4C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions.