Intracellular redox equilibrium and growth phase affect the performance of luciferase-based biosensors
Intracellular redox equilibrium and growth phase affect the performance of luciferase-based biosensors
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DOI：
10.1016/j.jbiotec.2006.06.019
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发表时间：
2007-01-01
影响因子：
4.1
通讯作者：
Karp, Matti
中科院分区：
文献类型：
--
作者：
Galluzzi, Lorenzo;Karp, Matti
在过去的二十年中，细菌荧光素酶操纵子的光发射已经被各种各样地利用。方便的诱导型启动子的使用已经为用于高通量筛选和环境监测的基于全细胞的测定授予了显著程度的特异性。然而，反复报告了无法解释的非特异性反应。在这里，我们表明，细胞内的生化平衡的损害干扰发光大肠杆菌和金黄色葡萄球菌菌株携带的勒克斯操纵子组成或诱导控制。化合物如甲氧苄啶和甲氨蝶呤，通过间接诱导NADPH积累，增强发光。相反，驱动细胞向氧化状态的分子，如二甲基亚砜，抑制发光。这些发现符合公认的生物发光的生化途径，其中NADPH和还原当量是产生荧光素酶底物所必需的，尽管它们不直接参与发光反应。此外，我们研究了诱导时间对诱导型报告系统的生物发光响应的影响，并证明了所发射的光与进行诱导的生长阶段之间的相关性。我们的研究结果提供了一些非特异性的反应记录到目前为止，在全细胞为基础的发光生物传感器的解释，并强调这种报告系统的内在局限性。(c)2006 Elsevier B.V.保留所有权利。
Light emission from the bacterial luciferase operon has been variously exploited during last two decades. The use of convenient inducible promoters has granted significant degrees of specificity to whole cell-based assays for high-throughput screening and environmental monitoring. Nevertheless, unexplained unspecific responses have been repeatedly reported. Here, we show that the impairment of the intracellular biochemical equilibrium interferes with the luminescence produced by Escherichia coli and Staphylococcus aureus strains carrying the lux operon under constitutive or inducible control. Compounds as trimethoprim and methotrexate, by indirectly inducing NADPH accumulation, enhance light emission. Conversely, molecules driving the cell toward an oxidized state, as dimethyl sulfoxide, inhibit luminescence. These findings fit into the accepted biochemical pathway for bioluminescence, where NADPH and reducing equivalents are necessary for the production of luciferase substrates, although they do not directly take part into the light-emitting reaction. Moreover, we investigated the influence of induction timing upon the bioluminescence response from inducible reporter systems and demonstrated a correlation between the emitted light and the growth phase at which induction is performed. Our results provide explanations for some unspecific responses recorded so far in whole cell-based luminescent biosensors and emphasize the intrinsic limitations of this kind of reporting system. (c) 2006 Elsevier B.V. All rights reserved.