title: 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 which l-nicotine is taken up by bacteria that are able to grow on it is unknown. Nicotine degradation by Arthrobacter nicotinovorans, a Gram-positive soil bacterium, is linked to the presence of the catabolic megaplasmid pAO1. l-[(14)C]Nicotine uptake assays with A. nicotinovorans showed transport of nicotine across the cell membrane to be energy-independent and saturable with a K(m) of 6.2+/-0.1 microM and a V(max) of 0.70+/-0.08 micromol 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 an A. nicotinovorans strain unable to degrade nicotine (pAO1(-)) showed no nicotine import. However, when the nicotine dehydrogenase genes were expressed in this strain, import of l-[(14)C]nicotine took place. A. nicotinovorans pAO1(-) and Escherichia coli were 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 by d-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.
authors.affiliation: Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, Stefan-Meier-Str. 17, D-79104 Freiburg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Ganas
authors.affiliation: Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, Stefan-Meier-Str. 17, D-79104 Freiburg, Germany.
authors.forename: Roderich
authors.initials: R
authors.lastname: Brandsch
mesh_terms: D001173:Arthrobacter / Q000378:metabolism* / Q000648:ultrastructure; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D055459:Facilitated Diffusion; D026901:Membrane Transport Proteins / Q000378:metabolism; D009538:Nicotine / Q000031:analogs & derivatives* / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity
chemical_list: C502317:6-hydroxynicotine; D026901:Membrane Transport Proteins; D009538:Nicotine; D000587:Oxidoreductases Acting on CH-NH Group Donors; C037584:6-hydroxy-L-nicotine oxidase