Combined use of different Gfp reporters for monitoring single-cell activities of a genetically modified PCB degrader in the rhizosphere of alfalfa
Combined use of different Gfp reporters for monitoring single-cell activities of a genetically modified PCB degrader in the rhizosphere of alfalfa
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DOI：
10.1016/j.femsec.2004.01.002
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发表时间：
2004-05-01
影响因子：
4.2
通讯作者：
Ramos, C
中科院分区：
文献类型：
--
作者：
Boldt, TS;Sorensen, J;Ramos, C
利用大肠杆菌rrnBP1核糖体启动子与编码不同稳定性绿色荧光蛋白(GFP)基因的融合，对紫花苜蓿根部定植的荧光假单胞菌F113的单细胞定位和活性进行了研究。监测系统允许无损地原位检测在存在和不存在3-氯联苯(PCB-2)的情况下生长的整个根系上的F113 rifpcb细胞。根尖和侧根萌发部位是快速生长细胞的热点。此外，利用另一种绿色荧光蛋白与恶臭假单胞菌代谢途径Pin启动子(TOL质粒)的融合，构建了一株降解氯联苯的荧光假单胞菌F113ifpcb报告菌株。该启动子受多氯联苯-2降解产物3-氯苯甲酸酯的强烈诱导，在体外进行了表达测试，随后利用荧光假单胞菌F113报告基因在紫花苜蓿根上进行了体内监测。一小部分但不同的导入细菌激活了PM启动子，因此似乎在紫花苜蓿根际感受到了PCB-2降解产物。降解细胞在设计上与感受性细胞完全相同，它们位于根表面的不同微菌落中或根表皮细胞之间的细胞间隙中。然而，在含有快速生长细胞的根部区域没有观察到降解多氯联苯的细胞，这表明多氯联苯的降解与高细胞活性无关。(C)2004年欧洲微生物学会联合会。爱思唯尔出版，版权所有。
Single-cell localization and activity of Pseudomonas,fluorescens F113, colonizing alfalfa roots, were monitored using fusions of the Escherichia coli rrnBP1 ribosomal promoter and gfp genes encoding green fluorescent protein (Gfp) of different stability. The monitoring systems permitted non-destructive in situ detection of F113rifpcb cells on the entire root system grown in both the presence and absence of 3-chlorobiphenyl (PCB-2). The root tip and sites of lateral root emergence were found to be hotspots for fast-growing cells. In addition, a reporter strain of P. fluorescens F113rifpcb for degradation of chlorinated biphenyl was constructed, using another gfp fusion with the meta-pathway Pin promoter from Pseudomonas putida (TOL plasmid). Expression of this promoter, which is strongly induced by the PCB-2 degradation product, 3-chlorobenzoate, was tested in vitro and subsequently monitored in vivo on alfalfa roots using the P. fluorescens F113rifpcb reporter. A small but distinct fraction of the introduced bacteria activated the Pm promoter and thus appeared to sense a PCB-2 degradation product in the alfalfa rhizosphere. The degrading cells, which by design were identical to the sensing cells, were located in distinct microcolonies on the root surface or in intercellular crevices between the root epidermal cells. However, PCB-degrading cells were not observed in the root areas containing fast-growing cells, indicating that PCB degradation was not linked to high cellular activity. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.