Interaction of the regulatory protein NicR1 with the promoter region of the pAO1‐encoded 6‐hydroxy‐D‐nicotine oxidase gene of Arthrobacter oxidans

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Abstract
Summary: 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 σ70 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.