Strains, growth conditions and transformation of Sulfolobus strains

All Sulfolobus strains were derived from the original isolate S. islandicus REY15A (41) (Supplementary Table S1). Genetic host E233S1 and the Δcmr-β mutant were reported previously (42,43). S. islandicus MF1 was constructed with the E233S1 strain in two steps using a CRISPR-assisted gene deletion/mutagenesis procedure recently developed in our laboratory (Supplementary Figure S2) (44); (i) the genetic region encompassing the two cassettes of type I-A cas genes and the two CRISPR arrays was deleted and (ii) the promoter of csa5 and the coding sequence of cas6 were fused together, yielding an active cas6 gene. Four cmr-2α mutants were used in this work, two of which, cmr-2α-HD-M1 and -M2 were reported previously (44) whereas cmr-2αPalm-M1 and -M2 strains were constructed as for the two cmr-2αHD mutants (Supplementary Table S1). Sulfolobus strains were grown in SCV medium (basic salts and 0.2% sucrose, 0.2% casa amino acids, 1% vitamin solution) at 78°C. If appropriate, uracil was supplemented to 20 μg/ml. Transformation was performed by electroporation as previously described (42).