Uptake of l-nicotine and of 6-hydroxy-l-nicotine by Arthrobacter nicotinovorans and by Escherichia coli is mediated by facilitated diffusion and not by passive diffusion or active transport

Abstract

The mechanism by whichl-nicotine is taken up by bacteria that are able to grow on it is unknown. Nicotine degradation byArthrobacter nicotinovorans, a Gram-positive soil bacterium, is linked to the presence of the catabolic megaplasmid pAO1.l-[14C]Nicotine uptake assays withA. nicotinovoransshowed transport of nicotine across the cell membrane to be energy-independent and saturable with aKmof 6.2±0.1 μM and aVmaxof 0.70±0.08 μmol min−1(mg protein)−1. This is in accord with a mechanism of facilitated diffusion, driven by the nicotine concentration gradient. Nicotine uptake was coupled to its intracellular degradation, and anA. nicotinovoransstrain unable to degrade nicotine (pAO1−) showed no nicotine import. However, when the nicotine dehydrogenase genes were expressed in this strain, import ofl-[14C]nicotine took place.A. nicotinovoranspAO1−andEscherichia coliwere also unable to import 6-hydroxy-l-nicotine, but expression of the 6-hydroxy-l-nicotine oxidase gene allowed both bacteria to take up this compound.l-Nicotine uptake was inhibited byd-nicotine, 6-hydroxy-l-nicotine and 2-amino-l-nicotine, which may indicate transport of these nicotine derivatives by a common permease. Attempts to correlate nicotine uptake with pAO1 genes possessing similarity to amino acid transporters failed. In contrast to the situation at the blood–brain barrier, nicotine transport across the cell membrane by these bacteria was not by passive diffusion or active transport but by facilitated diffusion.