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

Abstract

SummaryIn 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 N 4 -acetylcytidine (ac 4 C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions.

Zinc-Independent Folate Biosynthesis: Genetic, Biochemical, and Structural Investigations Reveal New Metal Dependence for GTP Cyclohydrolase IB

“…A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table 1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13,44).…”

Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

“…The plasmids were subsequently transformed into Hfx. volcanii strain H26, and the deletion strains were generated by the pyrE2- based “pop-in/pop-out” deletion method [47, 48], with standard media preparations [46] except that the media was supplemented with 100  μ M agmatine for deletion of HVO_1958 or with or without 1 mM putrescine for the deletion of HVO_2299 . Deletion of HVO_1958 was validated by PCR methods using the primers ext f and ext r (Table S3), generating strain VDC3253 (Table S2).…”

“…H26 was transformed with pIKB298 and the pop-in was generated [47, 48] with standard media preparation. Ten isolated colonies of the pop-in were pooled together and grown in liquid to generate competent cells [47].…”

A 5-formyltetrahydrofolate cycloligase paralog from all domains of life: comparative genomic and experimental evidence for a cryptic role in thiamin metabolism

“…Cells were grown at 44°C (unless otherwise specified) in Hv-YPC, Hv-CA, or Hv-Min media (Dyal-Smith 2008). The deletion construct was designed to delete >80% of the COG0212 ORF ( HVO_1928 ) by homologous recombination (El Yacoubi et al 2009). A region from 1 kb before the start codon to the first 114 nucleotides of the ORF was amplified by PCR with primers Hv2Rev and Hv1Fwd, which includes a Kpn I site (Table S1).…”