CONSTRUCTION OF A RHIZOSPHERE PSEUDOMONAD WITH POTENTIAL TO DEGRADE POLYCHLORINATED-BIPHENYLS AND DETECTION OF BPH GENE-EXPRESSION IN THE RHIZOSPHERE
CONSTRUCTION OF A RHIZOSPHERE PSEUDOMONAD WITH POTENTIAL TO DEGRADE POLYCHLORINATED-BIPHENYLS AND DETECTION OF BPH GENE-EXPRESSION IN THE RHIZOSPHERE
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DOI：
10.1128/aem.61.5.1946-1952.1995
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发表时间：
1995-05-01
影响因子：
4.4
通讯作者：
OGARA, F
中科院分区：
文献类型：
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作者：
BRAZIL, GM;KENEFICK, L;OGARA, F
基因工程转座子TnPCB含有编码联苯降解途径的基因（bph）。TnPCB被稳定地插入到两种不同根际假单胞菌的染色体中。一个转基因菌株，荧光假单胞菌F113pcb，详细的特点，并发现是不变的重要参数，如生长速度和生产的次级代谢产物。F113 pcb中异源bph基因的表达通过遗传修饰的微生物利用联苯作为唯一碳源的能力来证实。在实验室试验中，引入的性状保持稳定，在非选择性培养基中广泛继代后未发现bph阴性菌株。bph性状在非选择性根际微宇宙中也是稳定的。在非无菌土壤微宇宙中对甜菜幼苗根系进行定殖实验，评估了经修饰的F113 pcb的根球能力。F113pcb能够像标记的野生型菌株一样有效地定殖，并且没有观察到竞争力的降低。将带有bph'lacZ报告基因融合体的F113 pcb接种到甜菜种子上，发现F113 pcb中的bph基因原位表达。这表明bph基因产物也可能存在于原位条件下。这些实验表明，根际适应的微生物可以通过基因操作代谢新化合物，而不影响其生态能力。引入的基因的表达可以在根际中检测到，表明相当大的潜力操纵根际作为一个自我维持的生物膜的污染物在土壤中的生物修复。荧光球细菌如荧光假单胞菌属（Pseudomonas spp.）在生态上适应于在根际环境中定殖和竞争。扩大这种假单胞菌的代谢功能，降解污染物可能被证明是一个有用的生物修复策略。
The genetically engineered transposon TnPCB, contains genes (bph) encoding the biphenyl degradative pathway. TnPCB was stably inserted into the chromosome of two different rhizosphere pseudomonads. One genetically modified strain, Pseudomonas fluorescens F113pcb, was characterized in detail and found to be unaltered in important parameters such as growth rate and production of secondary metabolites. The expression of the heterologous bph genes in F113pcb was confirmed by the ability of the genetically modified microorganism to utilize biphenyl as a sole carbon source. The introduced trait remained stable in laboratory experiments, and no bph-negative isolates were found after extensive subculture in nonselective media. The bph trait was also stable in nonselective rhizosphere microcosms. Rhizosphere competence of the modified F113pcb was assessed in colonization experiments in nonsterile soil microcosms on sugar beet seedling roots. F113pcb was able to colonize as efficiently as a marked wild-type strain, and no decrease in competitiveness was observed. In situ expression of the bph genes in F113pcb was found when F113pcb bearing a bph'lacZ reporter fusion was inoculated onto sugar beet seeds. This indicates that the bph gene products may also be present under in situ conditions. These experiments demonstrated that rhizosphere-adapted microbes can be genetically manipulated to metabolize novel compounds without affecting their ecological competence. Expression of the introduced genes can be detected in the rhizosphere, indicating considerable potential for the manipulation of the rhizosphere as a self-sustaining biofilm for the bioremediation of pollutants in soil. Rhizosphere bacteria such as fluorescent Pseudomonas spp. are ecologically adapted to colonize and compete in the rhizosphere environment. Expanding the metabolic functions of such pseudomonads to degrade pollutants may prove to be a useful strategy for bioremediation.