Metabolism of 2-hydroxy-1-naphthoic acid and naphthalene via gentisic acid by distinctly different sets of enzymes in Burkholderia sp. strain BC1

Abstract

Burkholderia sp. strain BC1, a soil bacterium, isolated from a naphthalene balls manufacturing waste disposal site, is capable of utilizing 2-hydroxy-1-naphthoic acid (2H1NA) and naphthalene individually as the sole source of carbon and energy. To deduce the pathway for degradation of 2H1NA, metabolites isolated from resting cell culture were identified by a combination of chromatographic and spectrometric analyses. Characterization of metabolic intermediates, oxygen uptake studies and enzyme activities revealed that strain BC1 degrades 2H1NA via 2-naphthol, 1,2,6-trihydroxy-1,2-dihydronaphthalene and gentisic acid. In addition, naphthalene was found to be degraded via 1,2-dihydroxy-1,2-dihydronaphthalene, salicylic acid and gentisic acid, with the putative involvement of the classical nag pathway. Unlike most other Gram-negative bacteria, metabolism of salicylic acid in strain BC1 involves a dual pathway, via gentisic acid and catechol, with the latter being metabolized by catechol 1,2-dioxygenase. Involvement of a nonoxidative decarboxylase in the enzymic transformation of 2H1NA to 2-naphthol indicates an alternative catabolic pathway for the bacterial degradation of hydroxynaphthoic acid. Furthermore, the biochemical observations on the metabolism of structurally similar compounds, naphthalene and 2-naphthol, by similar but different sets of enzymes in strain BC1 were validated by real-time PCR analyses.

Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1

“…strain BC1 describing 2-naphthol, gentisaldehyde, and gentisic acid as pathway intermediates (27). Moreover, the presence of a strictly inducible nonoxidative decarboxylase was also observed in the cell extract of 2H1NA-grown culture catalyzing the enzymatic transformation of 2H1NA to 2-naphthol.…”

“…2H1NA decarboxylase activity was previously reported to be strictly inducible in the presence of 2H1NA and a differentially expressing ϳ32-kDa protein band was observed only in the cell extract of 2H1NA-grown cells compared to that of 2-naphtholgrown cells (27). For detailed characterization, 2H1NA decarboxylase was partially purified from crude cell extract of strain BC1 grown on 2H1NA using differential protein precipitation steps and hydrophobic interaction chromatography ( Table 1).…”

“…A final extension was performed at 72°C for 7 min. The resulting PCR product was sequenced as reported previously (27).…”

“…Nonoxidative decarboxylase activity was qualitatively determined by UV-visible light spectral analysis as described previously (27) while the activity was quantitatively determined based on the formation of 2-naphthol, analyzed by high-pressure liquid chromatography (HPLC) using a methanol-water (50:50 vol/vol) isocratic solvent system with a flow rate of 1 ml/min (27). A standard curve of 2-naphthol created by HPLC under identical analytical conditions was used for quantitative estimation.…”

“…Native 2H1NA decarboxylase was purified from crude cell extract of BC1 cells grown for 16 h at 28°C in 4 liters of mineral salt medium (MSM) (20) containing 0.5 g/liter 2H1NA. Crude cell extract was prepared as described previously (27) and was then fractionated by sequential protein precipitation using ammonium sulfate. The 30 to 50% ammonium sulfate saturated fraction was centrifuged at 12,000 ϫ g for 30 min, and the resulting pellet was dissolved in buffer A (50 mM K 2 HPO 4 -KH 2 PO 4 buffer [pH 7.0]) and dialyzed against buffer B [50 mM K 2 HPO 4 -KH 2 PO 4 buffer (pH 7.0) containing 0.8 M (NH 4 ) 2 SO 4 ].…”