Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches.
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.
Ono
Akira
A
Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan.
Miyazaki
Ryo
R
Sota
Masahiro
M
Ohtsubo
Yoshiyuki
Y
Nagata
Yuji
Y
Tsuda
Masataka
M
D001709:Biotechnology / Q000379:methods; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D049308:Dioxygenases; D005648:Fuel Oils; D015723:Gene Library; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000378:metabolism*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005648:Fuel Oils; D009097:Multienzyme Complexes; D009281:Naphthalenes; D012989:Soil Pollutants; C031721:naphthalene; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase