Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches
Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches
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DOI：
10.1007/s00253-006-0671-4
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发表时间：
2007-02-01
影响因子：
5
通讯作者：
Tsuda, Masataka
中科院分区：
文献类型：
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作者：
Ono, Akira;Miyazaki, Ryo;Tsuda, Masataka
在日本，采用了两种不依赖培养的不同方法从受石油污染的土壤中分离萘双加氧酶（NDO）基因。一种方法是构建基于广宿主范围粘粒的宏基因组DNA文库，另一种是所谓的外源质粒分离技术。我们在这两种方法中对NDO基因的筛选是基于恶臭假单胞菌（Pseudomonas putida）菌株的功能互补，这些菌株含有Tn4655K，这是一种携带整套萘降解（nah）基因但缺乏编码NDO基因的转座子。在前一种方法中，我们获得了一个粘粒克隆（pSLX928 - 6），它携带一个用于将萘转化为水杨酸的nah上游途径操纵子，并且这个操纵子与IncP - 9萘降解质粒pDTG1上相应的操纵子具有非常高的相似性。在后一种方法中，将来自土壤的微生物组分与一株无质粒且含有Tn4655K染色体拷贝的恶臭假单胞菌菌株进行接合，获得了转接合子，它们接收了一个200kb或80kb的质粒，该质粒包含用于萘完全降解的所有nah基因。随后的分析表明：（1）这两个质粒都属于IncP - 9不相容群；（2）它们的nah上游途径操纵子与pDTG1和pSLX928 - 6的操纵子显著相似，但不完全相同；（3）这些质粒携带通过间位裂解途径进行水杨酸代谢的基因。
Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway.