Interaction of the regulatory protein NicR1 with the promoter region of the pAO1-encoded 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans.
The D,L-nicotine catabolism of the Gram-positive soil bacterium Arthrobacter oxidans is linked to the presence within the cells of the 160 kb catabolic plasmid pAO1. pAO1-cured cells lost the catabolic enzymes and reintroduction of pAO1 by electroporation into cured cells reestablished the nic+ phenotype. DNA band shift assays with extracts from cured and pAO1+ cells suggested that pAO1 encodes the regulatory protein NicR1. Footprint analysis revealed that two homologous palindromes (IR1 and IR2), present in the 5'-regulatory region of the 6-HDNO gene, were protected from DNase I digestion. Binding of NicR1 to the palindromes is symmetrical, co-operative, and stronger to IR1 containing the 6-HDNO gene promoter than to IR2. Site-directed mutagenesis revealed that steric constraints and sequence requirements for NicR1-binding are located exclusively in the palindromic sequences. Deletions and insertions in the interpalindromic region and in the 6-HDNO promoter -10 sequence had no effect on the binding characteristics of NicR1 to the 6-HDNO regulatory region. Acting as a repressor, NicR1 prevents binding of the E. coli RNA-polymerase to the consensus sigma 70 promoter in vitro. However, the interaction of NicR1 with the 6-HDNO promoter region in extracts of nicotine-induced cells from various growth stages did not differ from that observed with extracts of nicotine-uninduced cells.
Bernauer
H
H
Biomchemisches Institut der Universität, Freiburg, Germany.
Mauch
L
L
Brandsch
R
R
D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009538:Nicotine / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D012097:Repressor Proteins; D012808:Sigma Factor; D009538:Nicotine; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase