H. volcanii Genetic Manipulation Protocol

The growth and genetic manipulation of H. volcanii were performed as previously described (12 (link), 68 (link)). The primers used for the knockout plasmids based on pTA131 are described in Table S1. Cells were grown in rich YPC medium with Bacto yeast extract, peptone (Oxoid) and Bacto Casamino acids (BD) or in selective CA medium containing only Bacto Casamino acids.
To express the proteins, plasmids based on pTA1228 (69 (link)) were constructed for this study (see Table S2), harboring the pyrE2 cassette. In addition, these plasmids contained mCherry and GFP genes and in-frame restriction sites to enable the expression of N-terminal and C-terminal fluorescent fusion proteins under the control of the tryptophan promoter (see Table S2). Salt-stable GFP and mCherry genes were kindly provided by Duggin et al. (52 (link)).