Chromosomally located gene fusions constructed in Acinetobacter sp ADP1 for the detection of salicylate
Chromosomally located gene fusions constructed in Acinetobacter sp ADP1 for the detection of salicylate
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DOI：
10.1111/j.1462-5822.2005.00821.x
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发表时间：
2005-09-01
影响因子：
5.1
通讯作者：
Whiteley, AS
中科院分区：
文献类型：
--
作者：
Huang, WE;Wang, H;Whiteley, AS
不动杆菌属。ADP1是一种常见的土壤相关细菌，具有很高的天然能力，使其能够有效地将外源DNA片段整合到其染色体中。这一特性被用来改造水杨酸诱导的不动杆菌luxCDABE和绿色荧光蛋白(GFP)变异体。ADP1。具体地说，不动杆菌属。ADPWH_lux具有较高的灵敏度(最小检出量为0.5~1 mU水杨酸盐)，且标记基因的更新速度较快，适合于水杨酸浓度的全细胞发光检测。相比之下，GFP蛋白的成熟和周转时间较长，使不动杆菌成为sp.ADPWH_GFP变体更适合于涉及水杨酸盐存在的全细胞成像的应用。通过在降解萘的培养物中检测水杨酸的产生，证明了LuxCDABE变异体的敏感性。使用ADPWH_lux的分析专门绘制了由萘生成水杨酸盐的动力学图，并与高效液相色谱(HPLC)观察到的数据相似。然而，ADPWH_lux在检测萘代谢的前24 h水杨酸的生成方面比高效液相色谱法显示出更高的灵敏度。这些数据表明，工程不动杆菌变异体在实验室和现场研究中具有很大的潜力用于水杨酸盐检测策略，特别是在需要构建结构的遗传稳定性来进行现场监测的情况下。
Acinetobacter sp. ADP1 is a common soil-associated bacterium with high natural competency, allowing it to efficiently integrate foreign DNA fragments into its chromosome. This property was exploited to engineer salicylate-inducible luxCDABE and green fluorescent protein (GFP) variants of Acinetobacter sp. ADP1. Specifically, Acinetobacter sp. ADPWH_lux displayed the higher sensitivity when comparing the two variants (minimum detection c. 0.5-1 mu M salicylate) and a faster turnover of the lux marker gene, making it suitable for whole-cell luminescence assays of salicylate concentration. In contrast, the longer maturation and turnover times of the GFP protein make the Acinetobacter sp. ADPWH_gfp variant more suited to applications involving whole-cell imaging of the presence of salicylate. The sensitivity of the luxCDABE variant was demonstrated by assaying salicylate production in naphthalene-degrading cultures. Assays using ADPWH_lux specifically mapped the kinetics of salicylate production from naphthalene and were similar to that observed by high-performance liquid chromatography (HPLC) data. However, ADPWH_lux exhibited the higher sensitivity, when compared with HPLC, for detecting salicylate production during the first 24 h of naphthalene metabolism. These data demonstrate that the engineered Acinetobacter variants have significant potential for salicylate detection strategies in laboratory and field studies, especially in scenarios where genetic stability of the construct is required for in situ monitoring.