The vhuU gene encoding a small subunit of a selenium-containing [NiFe]-hydrogenase in Methanococcus voltae appears to be essential for the cell

Abstract

We developed a general method for the site-specific deletion of gene sequences to obtain new selectable markers in the archaeon Methanococcus voltae. Using a deletion in the hisA gene, a vector was integrated into the chromosome by homologous recombination, thereby reconstituting histidine prototrophy. The vector contained the beta-glucuronidase gene uidA of Escherichia coli as a reporter under the control of an M. voltae promoter that normally drives the expression of a selenium-free [NiFe]-hydrogenase after selenium deprivation. This construct has allowed us to check whether the selenium supply was sufficiently low to induce the transcription of the genes encoding the selenium-free hydrogenases. We tried to introduce a chromosomal deletion of the vhuU gene of the archaeon M. voltae by gene replacement and by keeping the cells under selenium deprivation. The gene vhuU encodes the very small, selenocysteine-containing subunit that is part of the primary reaction center of the Vhu hydrogenase. All transformants bearing the deletion also contained the vhuU wild-type gene. Therefore, the vhuU gene appears to be essential for the cell even under conditions that lead to the induction of the selenium-free homologue Vhc of the Vhu hydrogenase.

Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation

“…Most methanogens have only a single methanogenic pathway, which is believed to be essential for viability because all known methanogens are obligate methanogens. This belief is supported by the inability to construct mutants lacking a key hydrogenase in the hydrogenotrophic methanogen Methanococcus voltae (38). In contrast, Methanosarcina species are metabolically diverse, being able to use a number of substrates for methanogenesis via four distinct methanogenic pathways (see above).…”

Insertional inactivation of the flaH gene in the archaeon Methanococcus voltae results in non-flagellated cells

“…The ®nd-ing of tandem copies of the plasmid DNA in the targeted aH locus was not completely unexpected. Similar cases of duplicate tandem copies of insertional vectors have been reported in M. voltae and its close relative M. maripaludis (Sandbeck and Leigh 1991;Pfeier et al 1998). Transformants with tandem copies of plasmid DNA in the chromosome bene®t from having a second copy of the pac expression cassette, which in turn confers additional resistance to puromycin on the cell.…”

Overview of the genetic tools in the Archaea

“…Initial integration shuttle vectors using the pac gene were constructed and successfully used to transform M. voltae (Gernhardt et al, 1990; Patel et al, 1994) and Methanococcus maripaludis (Sandbeck and Leigh, 1991; Tumbula et al, 1994). Selection based on histidine auxotrophy/prototrophy using the hisA gene as a marker has also been demonstrated (Pfeifer et al, 1998). Counterselection methods have been developed (Moore and Leigh, 2005) based on the observation that M. maripaludis cells displaying growth were sensitive to the base analogs 6-azauracil and 8-azahypoxanthine (Bowen and Whitman, 1987; Ladapo and Whitman, 1990; Bowen et al, 1996; Kim and Whitman, 1999).…”