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.