METABOLISM OF NAPHTHALENE, FLUORENE, AND PHENANTHRENE - PRELIMINARY CHARACTERIZATION OF A CLONED GENE-CLUSTER FROM PSEUDOMONAS-PUTIDA NCIB-9816

A modified cloning procedure was used to obtain large DNA insertions ( 20 to 30 kb) from Pseudomonas putida NCIB 9816 that expressed polycycl ic aromatic hydrocarbon (PAH) transformation activity in Escherichia c oli HB101. Four subclones (16 [in both orientations], 12, and 8.5 kb i n size) were constructed from the initial clones. Naphthalene, fluoren e, and phenanthrene transformations were investigated in these eight N CIB 9816 clones by a simple agar plate assay method, which was develop ed to detect and identify potential PAH metabolites Results indicated that the necessary genes encoding the initial ring fission of the thre e PAHs in E. coli cells are located in an 8.5-kb EcoRI-XhoI portion, b ut the lower-pathway genes are not present in a 38-kb neighborhood reg ion. These NCIB 9816 clones could transform naphthalene and phenanthre ne to salicylic acid and 1-hydroxy-2-naphthoic acid, respectively. Wit h the same clones, fluorene was degraded to 9-hydroxyfluorene, 9-fluor enone, and two unidentified compounds. Genetic similarity between the NAH7 upper-pathway genes and the cloned NCIB 9816 genes was confirmed by Southern blot DNA-DNA hybridization. In spite of this genetic simil arity, the abilities of the two clusters to transform multiple PAHs we re different. Under our experimental conditions, only the metabolites from naphthalene transformation by the NAH7 clone (pE317) were detecte d, whereas the NCIB 9816 clones produced metabolites from all three PA Hs.