title: Transcriptional regulation of the vanillate utilization genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-like repressor.
abstract: Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced PvanABK in C. glutamicum, while only vanillate induced the activity of PvanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the PvanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (KD) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (Kd) of (267 ± 23) × 10(-6) s(-1), with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at PvanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered PvanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N4)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator.
authors.affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany kambiz.morabbi@iig.uni-stuttgart.de.
authors.forename: Kambiz
authors.initials: K
authors.lastname: Morabbi Heravi
authors.affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Lange
authors.affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Hildegard
authors.initials: H
authors.lastname: Watzlawick
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Bielefeld, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
authors.affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Altenbuchner
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D019729:Carbon-Oxygen Ligases / Q000235:genetics / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014641:Vanillic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; C071437:VanA ligase, Bacteria; C073902:VanR protein, bacteria; D019729:Carbon-Oxygen Ligases; D014641:Vanillic Acid