Miyata Naoyuki, Iwahori Keisuke, Foght Julia M, Gray Murray R
Institute for Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Appl Environ Microbiol. 2004 Jan;70(1):363-9. doi: 10.1128/AEM.70.1.363-369.2004.
The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-(14)C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state (14)C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K(t)) of 26 +/- 3 nM (mean +/- standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated (14)C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K(d)) of 41 +/- 21 nM (mean +/- standard deviation). Given the low values of K(t) and K(d), Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.
对多环烃降解菌分枝杆菌属菌株RJGII - 135摄取菲的机制进行了研究，使用在菲（诱导进行菲代谢）和乙酸盐（未诱导）上生长的培养物。将洗涤后的细胞悬浮于[9-(14)C]菲的水溶液中，然后通过过滤收集细胞。未诱导细胞在孵育的最初15秒内达到低水平稳态(14)C浓度。这种即时摄取不显示饱和动力学，且不受主动运输抑制剂氰化物和羰基氰化物间氯苯腙的影响。这些结果表明菲通过被动扩散迅速进入细胞。然而，诱导细胞在几分钟内显示出累积摄取。初始摄取速率遵循饱和动力学，表观亲和常数（K(t)）为26±3 nM（平均值±标准差）。诱导细胞对菲的摄取受到抑制剂的强烈抑制。对细胞相关的(14)C标记化合物的分析表明，摄取过程中的同时代谢迅速，且在所测试的底物浓度下不饱和，这表明观察到的可饱和摄取反映的是膜运输而非细胞内代谢。这些结果与诱导细胞中存在一种可饱和的、能量依赖的菲运输机制一致。此外，累积摄取的动力学数据表明，基于其表观解离常数（K(d)）为41±21 nM（平均值±标准差）的饱和情况，菲与诱导细胞特异性结合。鉴于K(t)和K(d)的值较低，分枝杆菌属菌株RJGII - 135可能使用高亲和力运输系统从水相中摄取菲。
