The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3-and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the "classical" naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.The classical pathway for naphthalene catabolism in bacteria such as Pseudomonas putida strains NCIMB9816 and PpG7 (7, 52, 53) is via dihydroxylation and cleavage of the first ring and removal of the resulting aliphatic side chain to produce salicylate (2-hydroxybenzoate). This is then converted by the action of salicylate 1-hydroxylase to catechol (1,2-dihydroxybenzene), which undergoes extradiol cleavage via the same route used for a wide range of other aromatic compounds such as toluene and the xylenes (16) and phenol (38). The nah genes are located on two separate operons: the upper pathway operon encoding the conversion of naphthalene to salicylate and the lower (or meta) pathway operon encoding the conversion of salicylate to acetyl coenzyme A and pyruvate (5, 52). The induction of the two operons is linked by a common regulator protein, NahR (34).Although a route for naphthalene catabolism involving the alternative conversion of salicylate to gentisate (2,5-dihydroxybenzoate) has been known for some time (14,25,30,39,41,53), genetic analysis of such a pathway, in Ralstonia (formerly Pseudomonas) sp. strain U2, has only recently been reported (12, 54). There is similarity between the nag genes of strain U2 and the classical nah genes, but only in the conversion of naphthalene to salicylate, for which the genes are homologous and in the same ord...

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During growth of Pseudomonas putida strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a max of 446 nm. The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC). This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water. It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3. Using the cloned pnb structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC. This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site.During the metabolism of 4-nitrotoluene (4NT) as the sole carbon and nitrogen source by Pseudomonas putida strain TW3, the nitro group is retained during the initial sequential oxidations of the methyl group to form 4-nitrobenzoate (4NB) (9,10,15). This is then further metabolized to 3,4-dihydroxybenzoate (protocatechuate [PCA]) with release of the nitro group as NH 4 ϩ (15) through the sequential action of 4NB reductase (PnbA) and 4-hydroxylaminobenzoate lyase (PnbB), the genes for which have been cloned and characterized ( Fig. 1) (8). During growth of strain TW3 on either 4NT or 4NB, the growth medium progressively becomes yellow to orange as the degradation proceeds. In this paper we demonstrate that this is due to accumulation of the novel phenoxazinone 2-aminophenoxazin-3-one-7-carboxylate (APOC). The results suggest that APOC is formed as a by-product during the conversion of 4-hydroxylaminobenzoate (4HAB) to PCA by PnbB. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1 and shown in Fig. 1B.Chemicals and growth media. Aromatic and aliphatic substrates were obtained from Aldrich Chemical Co. P. putida TW3 was grown on minimal salts medium supplemented with either solid 4NT (0.5 g/liter), the sodium salt of 4-nitrobenzoic acid (5 mM), or sodium succinate (10 mM). Escherichia coli strains were routinely grown on Luria-Bertani (LB) medium. Where appropriate, ampicillin was added at 100 g/ml, streptom...

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