Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system

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 micro M. 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.

FIG. 1.

Schematic representation of the ORFs on the two strands of the pAO1 DNA. Dark blue, ORFs related to nicotine utilization; light blue, ORFs related to carbohydrate; violet, ORFs related to amino acid and sarcosine; red, ORFs related to IS elements and composite transposons; yellow, ORFs related to replication; green, ORFs related to conjugation; brown, conserved hypothetical ORFs; white, ORFs with no known function.

FIG. 2.

l-[¹⁴C]nicotine uptake by A. nicotinovorans carrying pAO1 and 6HLNO activity. l-[¹⁴C]nicotine (1.5 μCi) was added to bacterial cultures, and nicotine uptake and 6HLNO activity (dashed line), were determined at the indicated time points as described in Materials and Methods. Diamonds, A. nicotinovorans carrying pAO1; triangles, A. nicotinovorans not carrying pAO1; squares, E. coli XL1-Blue. Results are means ± standard deviations calculated from at least three independent experiments. The inset shows the l-[¹⁴C]nicotine concentration-dependent uptake determined as outlined in Materials and Methods.

FIG. 3.

Formation of nicotine blue and l-[¹⁴C]nicotine uptake in the presence of l- and d-amino acids. (A) To A. nicotinovorans/pAO1 stationary-phase bacteria, a 100 μM concentration of each of the following amino acids was added: bar 2, l-Glu; bar 3, l-Ser; bar 4, l-Arg; bar 5, l-Lys; bar 6, l-Ala; bar 7, l-His; bar 8, d-Pro; bar 9, d-Lys; bar 10, d-Arg; bar 11, d-His; bar 12, d-Ala; bar 13, d-Ser; bar 14, D, l-Phe; and bar 15, l-Pro. The cultures were induced with 50 μM l-nicotine, and nicotine blue formation was monitored after 3 h. Bar 1, nicotine blue formation in the presence of nicotine only. (B) Concentration-dependent effects of l-Pro and d-Arg on the formation of nicotine blue. (C) l-[¹⁴C]nicotine (1.5 μCi) was added to the A. nicotinovorans cultures in the absence of amino acids (crosses) or in the presence of 1 mM l-Pro (squares) or 1 mM d-Arg (triangles), and the time-dependent accumulation of radioactivity by the bacteria was determined as described in Materials and Methods.

FIG. 4.

G+C contents of the ORFs of pAO1. Solid line, mean G+C content of pAO1 DNA (59.7%); broken line from Tn554 to IS1473, average G+C content of 56.6%; broken line between IS elements, average G+C content of 60.6%. The average G+C content of remaining ORFs is 62%. ORF93, 6HLNO. A and B indicate the two ORFs of the transposases.