Brazil G M, Kenefick L, Callanan M, Haro A, de Lorenzo V, Dowling D N, O'Gara F
Department of Microbiology, University College, Cork, Ireland.
Appl Environ Microbiol. 1995 May;61(5):1946-52. doi: 10.1128/aem.61.5.1946-1952.1995.
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.
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。对一种基因改造菌株荧光假单胞菌F113pcb进行了详细表征，发现其在生长速率和次生代谢产物产生等重要参数方面未发生改变。通过基因改造微生物利用联苯作为唯一碳源的能力，证实了F113pcb中异源bph基因的表达。在实验室实验中，导入的性状保持稳定，在非选择性培养基中进行大量传代培养后，未发现bph阴性分离株。bph性状在非选择性根际微宇宙中也很稳定。在非无菌土壤微宇宙中对甜菜幼苗根进行定殖实验，评估了改造后的F113pcb的根际定殖能力。F113pcb能够像标记的野生型菌株一样有效地定殖，且未观察到竞争力下降。当将携带bph'lacZ报告基因融合体的F113pcb接种到甜菜种子上时，发现F113pcb中bph基因在原位表达。这表明bph基因产物在原位条件下也可能存在。这些实验表明，适应根际环境的微生物可以进行基因操作以代谢新化合物，而不影响其生态能力。在根际中可以检测到导入基因的表达，这表明将根际作为用于土壤中污染物生物修复的自我维持生物膜进行操作具有很大潜力。根际细菌如荧光假单胞菌属在生态上适应在根际环境中定殖和竞争。扩展这类假单胞菌降解污染物的代谢功能可能是一种有用的生物修复策略。
