Novel mechanisms of transcriptional regulation of vitamin and co‐factor metabolism in Archaea

Abstract

Water-soluble B-type vitamins are essential components of the cellular metabolism in all three kingdoms of life. In contrast to Bacteria, little is known about the metabolic pathways of vitamin biosynthesis and salvage in Archaea. Bacteria utilize several types of vitamin-responsive RNA riboswitches and transcriptional factors to control expression of their vitamin biosynthesis and transport genes, however no such regulatory mechanisms were found in Archaea. We used the comparative genomics approach to identify genes involved in the biosynthesis and salvage of four B-type vitamins (B1, B2, B3 and B12) in complete genomes of Archaea. For each vitamin metabolic pathway, we identified unique DNA motifs and reconstructed the putative regulons sharing the conserved DNA sites in diverse archaeal lineages including Sulfolobales, Thermoproteales, Desulfurococcales, Archaeoglobales, Thermoplasmatales, Thermococcales, Halobacteriales, Methanomicrobia and Thaumarchaeota. Candidate transcription factors for direct control of the identified novel regulons were identified by genome context analysis. Three novel transcriptional regulators, RbkR, ThiR and NdkR, were predicted to control the riboflavin, thiamine and NAD/niacin biosynthesis and transport genes, respectively. ThiR regulators are composed of a DNA-binding helix-turn-helix (HTH) domain and a ThiN-like domain that is similar to archaeal thiamin phosphate synthase. NdkR regulators also contains two domains, a NAD kinase domain and a DNA-binding domain, however the kinase domain in NdkR proteins is degenerated. RbkR proteins represent a fusion of a DNA-binding HTH domain and a catalytic domain that is orthologous to CTP-dependent riboflavin kinase from Methanocaldococcus jannaschii. The identified DNA binding motifs of novel regulators were experimentally validated by in vitro DNA-binding assays with the purified recombinant transcription factors RbkR and ThiR from several archaeal species. The CTP-dependent riboflavin kinase activity of bifunctional RbkR proteins was confirmed by in vitro enzymatic assay. We have solved the crystal structures of the apo and DNA operator-bound forms of RbkR from Thermoplasma acidophilum and compared them with the previously determined structure of riboflavin kinase from M. jannaschii. The majority of CDP- and FMN-binding residues in the kinase protein are well conserved in RbkR proteins. In the structure of the RbkR-DNA dimeric complex, we found residues involved in specific recognition of a palindromic DNA site. However, both the candidate RbkR-binding DNA motifs and contacting residues in HTH domains showed significant variability between archaeal lineages. Further experimental investigations of divergent RbkR memners are required to achieve a better understanding of their DNA-specificity determinants. In summary, we have identified novel regulators for transcriptional control of vitamin metabolism genes in Archaea. The reconstructed regulons contains novel candidate vitamin uptake transporters. These discoveries contribute to our understanding of metabolic and regulatory networks involved in vitamin homeostasis in Archaea. Support or Funding Information This research was supported by the Genomic Science Program (GSP), Office of Biological and Environmental Research (OBER), and U.S. Department of Energy (DOE) and is a contribution of the Pacific Northwest National Laboratory (PNNL) Foundational Scientific Focus Area. Figure 1Open in figure viewerPowerPoint Transcriptional regulation of riboflavin metabolism genes by a novel bifunctional repressor/kinase protein in Pyrobaculum yellowstonensis.