P009 Recruitment of Orf44, an ad hoc transcriptional repressor, into a putative partition cassette in the archaeon Sulfolobus

Sulfolobus NOB8H2 is a strain isolated from acidic hot springs at Noboribetsu, Japan. This strain harbours a 41 kb plasmid, pNOB8. The plasmid includes a putative partition cassette. This cassette consists of three genes: orf44, a parB-like gene and a parA-like gene. The protein encoded by orf44 shares sequence homology with bacterial transcriptional repressors of the ArsR/ SmtB family. Orf44 is characterized by a highly basic pI. These features suggested that Orf44 might be a DNA-binding protein. Orf44 was purified as a recombinant protein in E. coli. Gel filtration, analytical ultracentrifugation, SEC-MAL and chemical cross-linking experiments indicated that Orf44 is predominantly dimeric. Circular dichroism spectroscopy revealed that the protein is extremely thermostable. Electrophoretic mobility shift assays and DNase I footprinting studies showed that Orf44 is a DNA- binding protein contacting a palindromic sequence upstream of its own gene. The inverted repeat is positioned between the putative TATA box and the translation start site of orf44. This operator-like location suggests that Orf44 may repress the transcription of its own gene and that of parB and parA by binding to this box and disrupting the assembly of the transcription machinery at the promoter.