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Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches

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Abstract

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.

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Acknowledgements

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Agriculture, Forestry, and Fisheries (HC-06-2323), Japan. R. M. was supported by the Japan Society for the Promotion of Science Research Fellowships for Young Scientists.

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Correspondence to Masataka Tsuda.

Additional information

A.O. and R.M. contributed equally to this work.

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Ono, A., Miyazaki, R., Sota, M. et al. Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches. Appl Microbiol Biotechnol 74, 501–510 (2007). https://doi.org/10.1007/s00253-006-0671-4

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Keywords

  • PAH
  • Metagenome
  • Mobile genetic element
  • NDO