Lajoie C A, Layton A C, Sayler G S
Department of Microbiology, University of Tennessee, Knoxville 37932.
Appl Environ Microbiol. 1994 Aug;60(8):2826-33. doi: 10.1128/aem.60.8.2826-2833.1994.
Polychlorinated biphenyl (PCB)-degradative genes, under the control of a constitutive promoter, were cloned into a broad-host-range plasmid and a transposon. These constructs were inserted into a surfactant-utilizing strain, Pseudomonas putida IPL5, to create a field application vector (FAV) in which a surfactant-degrading organism cometabolizes PCB. By utilizing a surfactant not readily available to indigenous populations and a constitutive promoter, selective growth and PCB-degradative gene expression are decoupled from biphenyl. Since PCB degradation via the biphenyl degradation pathway is nonadaptive in the absence of biphenyl, there is no selective pressure for PCB gene maintenance. The recombinant strains exhibited degradative activity against 25 of 33 PCB congeners in Aroclor 1248 in the absence of biphenyl. Whole-cell enzyme assays indicated that PCB-degradative activity of a recombinant strain carrying the PCB genes on a plasmid was approximately twice that of the same strain carrying the PCB genes on a transposon. Plasmid loss rates in the absence of antibiotic selection averaged 7.4% per cell division and were highly variable between experiments. Surfactant-amended slurries of PCB-contaminated electric power plant substation soil were inoculated with approximately 10(5) recombinant cells per ml. The populations of the added strains increased to greater than 10(9) cells per ml in 2 days, and cell growth coincided with PCB degradation. By 15 days, 50 to 60% of the indicator congener 2,3,2',5'-tetrachlorobiphenyl was degraded. The effectiveness of PCB degradation by the plasmid-containing strain depended on plasmid stability. The transposon-encoded PCB genes were much more stable, and in surfactant-amended soil slurries, PCB degradation was more consistent between experiments.
多氯联苯（PCB）降解基因在组成型启动子的控制下，被克隆到一个广宿主范围的质粒和一个转座子中。这些构建体被插入到一株利用表面活性剂的菌株——恶臭假单胞菌IPL5中，以创建一个现场应用载体（FAV），在该载体中，一种降解表面活性剂的生物体可共代谢多氯联苯。通过利用本地菌群不易获得的一种表面活性剂和一个组成型启动子，选择性生长和多氯联苯降解基因的表达与联苯解耦。由于在没有联苯的情况下，通过联苯降解途径进行的多氯联苯降解是非适应性的，因此不存在多氯联苯基因维持的选择压力。在没有联苯的情况下，重组菌株对Aroclor 1248中33种多氯联苯同系物中的25种表现出降解活性。全细胞酶分析表明，携带多氯联苯基因的重组菌株在质粒上的多氯联苯降解活性大约是携带转座子上多氯联苯基因的同一菌株的两倍。在没有抗生素选择的情况下，质粒丢失率平均每细胞分裂为7.4%，并且在实验之间变化很大。用每毫升约10⁵个重组细胞接种受多氯联苯污染的发电厂变电站土壤的表面活性剂改良泥浆。添加菌株的数量在2天内增加到每毫升大于10⁹个细胞，并且细胞生长与多氯联苯降解同时发生。到第15天，指示同系物2,3,2',5'-四氯联苯有50%至60%被降解。含质粒菌株对多氯联苯的降解效果取决于质粒的稳定性。转座子编码的多氯联苯基因更稳定，并且在表面活性剂改良的土壤泥浆中，多氯联苯降解在实验之间更一致。
