Dual regulation of a polyethylene glycol degradative operon by AraC-type and GalR-type regulators in Sphingopyxis macrogoltabida strain 103

Abstract

The genes for polyethylene glycol (PEG) catabolism (pegB,C,D,AandE) inSphingopyxis macrogoltabidastrain 103 were shown to form a PEG-inducible operon. ThepegRgene, encoding an AraC-type regulator in the downstream area of the operon, is transcribed in the reverse direction. The transcription start sites of the operon were mapped, and three putativeσ70-type promoter sites were identified in thepegB,pegAandpegRpromoters. A promoter activity assay showed that thepegBpromoter was induced by PEG and oligomeric ethylene glycols, whereas thepegAandpegRpromoters were induced by PEG. Deletion analysis of thepegBpromoter indicated that the region containing the activator-binding motif of an AraC/XylS-type regulator was required for transcription of thepegBCDAEoperon. Gel retardation assays demonstrated the specific binding of PegR to thepegBpromoter. Transcriptional fusion studies ofpegRwithpegAandpegBpromoters suggested that PegR regulates the expression of thepegBCDAEoperon positively through its binding to thepegBpromoter, but PegR does not bind to thepegApromoter. Two specific binding proteins for thepegApromoter were purified and identified as a GalR-type regulator and an H2A histone fragment (histone-like protein, HU). The binding motif of a GalR/LacI-type regulator was found in thepegAandpegRpromoters. These results suggested the dual regulation of thepegBCDAEoperon through thepegBpromoter by an AraC-type regulator, PegR (PEG-independent), and through thepegAandpegRpromoters by a GalR/LacI-type regulator together with HU (PEG-dependent).