ThiN as a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea.

Thiamine pyrophosphate (TPP), the active form of vitamin B1, is a cofactor required for catalyzing key reactions in carbohydrate and amino acid biosynthesis. TPP can be acquired through environmental salvage or de novo biosynthesis. Two separate pathways are used for the de novo biosynthesis of thiamine: one produces a sulfur-containing thiazole ring and the other a pyrimidine moiety, which are then joined and phosphorylated to form TPP. While the synthesis and regulation of thiamine are well characterized in certain bacteria, plants and fungi, these pathways are comparatively understudied in archaea. Recent work reveals archaeal thiamine metabolism to be chimeric, combining eukaryotic THI4-dependent thiazole formation with bacterial ThiC-mediated pyrimidine synthesis. Moreover, archaeal thiamine synthesis can be regulated by a ThiN-domain-dependent transcriptional repression mechanism distinct from riboswitch-based control. This chapter describes the experimental techniques that have been utilized for defining catalytic enzymes and transcriptional regulators of thiamine biosynthesis in archaea, using Haloferax volcanii as a model organism. Approaches include targeted gene deletion, complementation, site-directed mutagenesis (SDM), 3D-structural modeling, DNA regulatory motif identification, transcriptional reporter assays, and bioinformatic comparisons providing a versatile toolbox for functional and regulatory analysis of thiamine metabolism in extremophilic microorganisms.