SummaryThis work describes the identification and characterization of SurR, Pyrococcus furiosus sulphur (S 0 ) response regulator. SurR was captured from cell extract using promoter DNA of a hydrogenase operon that is downregulated in the primary response of P. furiosus to S

SummaryWe present structural and biochemical evidence for a redox switch in the archaeal transcriptional regulator SurR of Pyrococcus furiosus, a hyperthermophilic anaerobe. P. furiosus produces H2 during fermentation, but undergoes a metabolic shift to produce H2S when elemental sulfur (S 0 ) becomes available. Changes in gene expression occur within minutes of S 0 addition, and the majority of these S 0 -responsive genes are regulatory targets of SurR, a key regulator involved in primary S 0 response. SurR was shown in vitro to have dual functionality, activating transcription of some of these genes, notably the hydrogenase operons, and repressing others, including a geneencoding sulfur reductase. This work demonstrates via biochemical and structural evidence that the activity of SurR is modulated by cysteine residues in a CxxC motif that constitutes a redox switch. Oxidation of the switch with S 0 inhibits sequence-specific DNA binding by SurR, leading to deactivation of genes related to H2 production and derepression of genes involved in S 0 metabolism.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally near 100°C and undergoes a heat shock response at 105°C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA ؉ ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcription of fragmented chromosomal DNA in the presence or absence of the regulator and hybridization of in vitro RNA to P. furiosus whole-genome microarrays. Our results confirmed the phr, the hsp20, and the aaa ؉ ATPase genes as targets of Phr and also identified six additional open reading frames, PF0624, PF1042, PF1291, PF1292, PF1488, and PF1616, as Phr-responsive genes, which include that encoding di-myo-inositol phosphate synthase. Transcription of the identified novel genes was inhibited by Phr in standard transcription assays, and the novel consensus sequence 5-TTTAnnn ACnnnnnGTnAnnAAAA-3 (uppercase letters denote a high conservation of the bases) was inferred from our data as the Phr recognition motif. Mutational evidence for the significance of this sequence as Phr recognition was provided in DNA-binding experiments.