1887

Abstract

The genes for polyethylene glycol (PEG) catabolism (, , , and ) in strain 103 were shown to form a PEG-inducible operon. The gene, 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 putative -type promoter sites were identified in the , and promoters. A promoter activity assay showed that the promoter was induced by PEG and oligomeric ethylene glycols, whereas the and promoters were induced by PEG. Deletion analysis of the promoter indicated that the region containing the activator-binding motif of an AraC/XylS-type regulator was required for transcription of the operon. Gel retardation assays demonstrated the specific binding of PegR to the promoter. Transcriptional fusion studies of with and promoters suggested that PegR regulates the expression of the operon positively through its binding to the promoter, but PegR does not bind to the promoter. Two specific binding proteins for the promoter 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 the and promoters. These results suggested the dual regulation of the operon through the promoter by an AraC-type regulator, PegR (PEG-independent), and through the and promoters by a GalR/LacI-type regulator together with HU (PEG-dependent).

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2006-10-01
2019-11-19
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Bacterial strains and plasmids used in this study. [PDF](67 kb) Primers used in this study. [PDF](29 kb) Determination of transcription start sites of , and . The nucleotide sequences of the , the and the promoter regions are shown in (a), (b) and (c), respectively. The transcription start sites, which were determined by 5'-RACE, are indicated by the angled arrow for and and the dashed angled arrow for . In (b), the angled arrow shows the pegA transcription start site determined by primer extension analysis (d). The sequence is numbered relative to the transcription start site from 5'-RACE results (the bold G nucleotide). The putative translation start site, RBS, -10 region, -35 promoter sequence, and histone-like protein HU binding site (hbs) are underlined. Boxes indicate the putative consensus transcriptional regulator binding regions. (d) Primer extension analysis of . The products of reverse-transcription (lane 1, PEG-4000-grown cells and lane 2, glucose-grown cells) were coelectrophoresed with a DNA-sequencing ladder (A, G, C, and T) of the pCR-PpegA. The expanded views of the nucleotide sequence around the transcription initiation sites (t1 and t2) are shown. [PDF](75 kb) MALDI-TOF MS spectrum of the trypsin-digested pegA promoter-binding proteins. (a) The 20 kDa protein and (b) The 40 kDa protein. [PDF](44 kb)

PDF

Bacterial strains and plasmids used in this study. [PDF](67 kb) Primers used in this study. [PDF](29 kb) Determination of transcription start sites of , and . The nucleotide sequences of the , the and the promoter regions are shown in (a), (b) and (c), respectively. The transcription start sites, which were determined by 5'-RACE, are indicated by the angled arrow for and and the dashed angled arrow for . In (b), the angled arrow shows the pegA transcription start site determined by primer extension analysis (d). The sequence is numbered relative to the transcription start site from 5'-RACE results (the bold G nucleotide). The putative translation start site, RBS, -10 region, -35 promoter sequence, and histone-like protein HU binding site (hbs) are underlined. Boxes indicate the putative consensus transcriptional regulator binding regions. (d) Primer extension analysis of . The products of reverse-transcription (lane 1, PEG-4000-grown cells and lane 2, glucose-grown cells) were coelectrophoresed with a DNA-sequencing ladder (A, G, C, and T) of the pCR-PpegA. The expanded views of the nucleotide sequence around the transcription initiation sites (t1 and t2) are shown. [PDF](75 kb) MALDI-TOF MS spectrum of the trypsin-digested pegA promoter-binding proteins. (a) The 20 kDa protein and (b) The 40 kDa protein. [PDF](44 kb)

PDF

Bacterial strains and plasmids used in this study. [PDF](67 kb) Primers used in this study. [PDF](29 kb) Determination of transcription start sites of , and . The nucleotide sequences of the , the and the promoter regions are shown in (a), (b) and (c), respectively. The transcription start sites, which were determined by 5'-RACE, are indicated by the angled arrow for and and the dashed angled arrow for . In (b), the angled arrow shows the pegA transcription start site determined by primer extension analysis (d). The sequence is numbered relative to the transcription start site from 5'-RACE results (the bold G nucleotide). The putative translation start site, RBS, -10 region, -35 promoter sequence, and histone-like protein HU binding site (hbs) are underlined. Boxes indicate the putative consensus transcriptional regulator binding regions. (d) Primer extension analysis of . The products of reverse-transcription (lane 1, PEG-4000-grown cells and lane 2, glucose-grown cells) were coelectrophoresed with a DNA-sequencing ladder (A, G, C, and T) of the pCR-PpegA. The expanded views of the nucleotide sequence around the transcription initiation sites (t1 and t2) are shown. [PDF](75 kb) MALDI-TOF MS spectrum of the trypsin-digested pegA promoter-binding proteins. (a) The 20 kDa protein and (b) The 40 kDa protein. [PDF](44 kb)

PDF

Bacterial strains and plasmids used in this study. [PDF](67 kb) Primers used in this study. [PDF](29 kb) Determination of transcription start sites of , and . The nucleotide sequences of the , the and the promoter regions are shown in (a), (b) and (c), respectively. The transcription start sites, which were determined by 5'-RACE, are indicated by the angled arrow for and and the dashed angled arrow for . In (b), the angled arrow shows the pegA transcription start site determined by primer extension analysis (d). The sequence is numbered relative to the transcription start site from 5'-RACE results (the bold G nucleotide). The putative translation start site, RBS, -10 region, -35 promoter sequence, and histone-like protein HU binding site (hbs) are underlined. Boxes indicate the putative consensus transcriptional regulator binding regions. (d) Primer extension analysis of . The products of reverse-transcription (lane 1, PEG-4000-grown cells and lane 2, glucose-grown cells) were coelectrophoresed with a DNA-sequencing ladder (A, G, C, and T) of the pCR-PpegA. The expanded views of the nucleotide sequence around the transcription initiation sites (t1 and t2) are shown. [PDF](75 kb) MALDI-TOF MS spectrum of the trypsin-digested pegA promoter-binding proteins. (a) The 20 kDa protein and (b) The 40 kDa protein. [PDF](44 kb)

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