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

• Published in: Microbiology (Society for General Microbiology) Vol. 155; no. 6; pp. 1866 - 1877
• Main Authors: Ganas, Petra, Brandsch, Roderich
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.06.2009; Society for General Microbiology
• Subjects: Arthrobacter - metabolism; Arthrobacter - ultrastructure; Arthrobacter nicotinovorans; Bacteriology; Biological and medical sciences; Biological Transport, Active; Cell Membrane - metabolism; Escherichia coli; Escherichia coli - metabolism; Escherichia coli - ultrastructure; Facilitated Diffusion; Fundamental and applied biological sciences. Psychology; Membrane Transport Proteins - metabolism; Microbiology; Miscellaneous; Nicotine - analogs & derivatives; Nicotine - metabolism; Oxidoreductases Acting on CH-NH Group Donors - metabolism; Plasmids - genetics; Substrate Specificity; Alkaloid; Escherichia coli; Bacteria; Actinomycetes; Arthrobacter; Uptake; Enterobacteriaceae; Nicotine
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.028688-0

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, Stefan-Meier-Str. 17, D-79104 Freiburg, Germany 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 µM and a V max of 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 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. Correspondence Roderich Brandsch roderich.brandsch{at}biochemie.uni-freiburg.de Abbreviations: DPI, diphenyleneiodonium; 6HLN, 6-hydroxy- L -nicotine; 6HLNO, 6-hydroxy- L -nicotine oxidase; 6HPON, 6-hydroxy-pseudooxynicotine; NDH, nicotine dehydrogenase