A GENE-CLUSTER ENCODING STEPS IN CONVERSION OF NAPHTHALENE TO GENTISATE IN PSEUDOMONAS SP. STRAIN U2

Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in V enezuela by selective enrichment on naphthalene as the sole carbon sou rce. The genes for naphthalene dioxygenase were cloned from the plasmi d DNA of strain U2 on an 8.3-kb BamHI fragment. The genes for the naph thalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb (for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations an d by nucleotide sequencing. The genes for nagB (for naphthalene cis-di hydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAb were two open reading kames, homologs of which have al so been identified in similar locations in two nitrotoluene using stra ins (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 18 1:57-61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926-4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet, Eng. 19:257-269, 1997), Recombinant Esch erichia coli strains with plasmids carrying this region were able to c onvert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance . The first open reading frame, designated nagG, encodes a protein wit h characteristics of a Rieske-type iron-sulfur center homologous to th e large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated nagH, encodes a protein with limited homolog y to the small subunits of the same dioxygenases. Cloned together in E . coli, nagG, nagH, and nagAb, were able to convert salicylate (2-hydr oxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore enco de a salicylate 5-hydroxylase activity. Single-gene knockouts of nagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunit s of salicylate 5-hydroxylase linked to an electron transport chain co nsisting of NagAb and NagAa, although E. coli appears to be able to pa rtially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization, However growth on both nap hthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2, Sequenc e comparisons suggest that the novel gene order nagAa-nagG-nagH-nagAb- naGAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.