Regulation of the kduID operon of Bacillus subtilis by the KdgR repressor and the ccpA gene: identification of two KdgR-binding sites within the kdgR-kduI intergenic region

• Published in: Microbiology (Society for General Microbiology) Vol. 153; no. 3; pp. 701 - 710
• Main Authors: Lin, Jer-Sheng, Shaw, Gwo-Chyuan
• Format: Journal Article
• Language: English
• Published: England Soc General Microbiol 01.03.2007
• Subjects: Artificial Gene Fusion; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - physiology; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Blotting, Northern; DNA Footprinting; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA, Intergenic; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; Genes, Reporter; Glucose - metabolism; Hexuronic Acids - metabolism; Molecular Sequence Data; Operator Regions, Genetic; Protein Binding; Repressor Proteins - metabolism; RNA, Bacterial - analysis; RNA, Messenger - analysis; Transcription, Genetic
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2006/002253-0

Institute of Biochemistry and Molecular Biology, School of Life Science, National Yang-Ming University, Taipei, Taiwan Correspondence Gwo-Chyuan Shaw gcshaw{at}ym.edu.tw Transcription of the Bacillus subtilis kdgRKAT operon, which comprises genes involved in the late stage of galacturonate utilization, is known to be negatively regulated by the KdgR repressor. In this study, Northern analysis was carried out to demonstrate that the kdgR gene also negatively regulates the kduID operon, encoding ketodeoxyuronate isomerase and ketodeoxygluconate reductase. It has also been demonstrated that expression of the kduID operon can be induced by galacturonate and is subject to catabolite repression by glucose. The ccpA gene was found to be involved in this catabolite repression. Primer extension analysis identified a A -like promoter sequence preceding kduI . Gel mobility shift assays and DNase I footprinting analyses indicated that KdgR is capable of binding specifically to two sites within the kdgR–kduI intergenic region in vitro . Reporter gene analysis revealed that these two KdgR-binding sites function in vivo . One site is centred 33.5 bp upstream of the translational start site of kdgR and can serve as an operator for controlling expression of the kdgRKAT operon. The other is centred 57.5 bp upstream of the translational start site of kduI and can serve as an operator for controlling expression of the kduID operon. Possible physiological significance of this regulation is discussed. Abbreviations: DKI, 5-ketodeoxyuronate; DKII, 2,5-diketo-3-deoxygluconate; KDG, 2-keto-3-deoxygluconate