Ganas Petra, Brandsch Roderich
Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, Stefan-Meier-Str. 17, D-79104 Freiburg, Germany.
Microbiology (Reading). 2009 Jun;155(Pt 6):1866-1877. doi: 10.1099/mic.0.028688-0. Epub 2009 May 14.
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.
能够在L-尼古丁上生长的细菌摄取该物质的机制尚不清楚。革兰氏阳性土壤细菌嗜烟节杆菌对尼古丁的降解与分解代谢大质粒pAO1的存在有关。用嗜烟节杆菌进行的L-[(14)C]尼古丁摄取试验表明，尼古丁跨细胞膜的转运不依赖能量，且具有饱和性，米氏常数（K(m)）为6.2±0.1微摩尔，最大反应速度（V(max)）为0.70±0.08微摩尔·分钟(-1)·（毫克蛋白）(-1)。这符合由尼古丁浓度梯度驱动的易化扩散机制。尼古丁的摄取与其细胞内降解相关联，一株无法降解尼古丁的嗜烟节杆菌菌株（pAO1(-)）未显示出尼古丁的摄入。然而，当该菌株中表达尼古丁脱氢酶基因时，L-[(14)C]尼古丁发生了摄入。嗜烟节杆菌pAO1(-)和大肠杆菌也无法摄入6-羟基-L-尼古丁，但6-羟基-L-尼古丁氧化酶基因的表达使这两种细菌都能摄取该化合物。L-尼古丁的摄取受到D-尼古丁、6-羟基-L-尼古丁和2-氨基-L-尼古丁的抑制，这可能表明这些尼古丁衍生物是通过一种共同的通透酶进行转运的。将尼古丁摄取与与氨基酸转运体具有相似性的pAO1基因进行关联的尝试未成功。与血脑屏障的情况不同，这些细菌跨细胞膜的尼古丁转运不是通过被动扩散或主动转运，而是通过易化扩散。
