Sequence of the 165-Kilobase Catabolic Plasmid pAO1 from Arthrobacter nicotinovorans and Identification of a pAO1-Dependent Nicotine Uptake System

Abstract

ABSTRACT The 165-kb catabolic plasmid pAO1 enables the gram-positive soil bacterium Arthrobacter nicotinovorans to grow on the tobacco alkaloid l -nicotine. The 165,137-nucleotide sequence, with an overall G+C content of 59.7%, revealed, besides genes and open reading frames (ORFs) for nicotine degradation, a complete set of ORFs for enzymes essential for the biosynthesis of the molybdenum dinucleotide cofactor, as well as ORFs related to uptake and utilization of carbohydrates, sarcosine, and amino acids. Of the 165 ORFs, approximately 50% were related to metabolic functions. pAO1 conferred to A. nicotinovorans the ability to take up l -[ 14 C]nicotine from the medium, with an K m of 5.6 ± 2.2 μM. ORFs of putative nicotine transporters formed a cluster with the gene of the d -nicotine-specific 6-hydroxy- d -nicotine oxidase. ORFs related to replication, chromosome partitioning, and natural transformation functions ( dprA ) were identified on pAO1. Few ORFs showed similarity to known conjugation-promoting proteins, but pAO1 could be transferred by conjugation to a pAO1-negative strain at a rate of 10 −2 to 10 −3 per donor. ORFs with no known function represented approximately 35% of the pAO1 sequence. The positions of insertion sequence elements and composite transposons, corroborated by the G+C content of the pAO1 sequence, suggest a modular composition of the plasmid.