Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1.

A 4-nitrophenol (4-NP)-degrading bacterium was isolated from activated sludge and identified as a Rhodococcus sp. This bacterium, designated as strain PN1, could utilize 4-NP as a sole carbon, nitrogen and energy source. Degradation tests of 4-NP using cell suspensions of strain PN1 revealed that the degradation was induced by 4-NP and that 4-nitrocatechol (4-NC) was one of the metabolites. A gene library was constructed from the total DNA of strain PN1 and introduced into Rhodococcus rhodochrous ATCC 12674. Two recombinant strains showed 4-NP hydroxylase activity, and a 9.1-kb DNA fragment encoding the activity was isolated from one of the strains. In addition, a 2.4-kb smaller fragment expressing the activity was subcloned from the 9.1-kb fragment and sequenced. The sequence analysis showed that the fragment encodes a two-component 4-NP hydroxylase, the predicted amino acid sequence of which exhibits significant similarity to those of phenol hydroxylases and 4-hydroxyphenylacetate 3-hydroxylases belonging to the two-component flavin diffusible monooxygenase (TC-FDM) family proposed by Galán et al. (J. Bacteriol., 182, 627-636, 2000).

Mutational analysis of the metabolism of 2,6-naphthalenedisulfonate by Pigmentiphaga sp. NDS-2.

The metabolism of 2,6-naphthalenedisulfonate (26NDS) by Pigmentiphaga sp. NDS-2 was analyzed by isolating a mutant (NDS2-002) which slowly grew on a minimal medium containing 26NDS as the sole source of carbon. Liquid chromatography/mass spectrometry (LC/MS) analysis of metabolic intermediates in this strain revealed that 5-sulfosalicylate (5SSA) is accumulated during the degradation of 26NDS. To analyze the lower metabolic pathway, a mutant strain NDS2-008, which could not grow either on 26NDS, 5SSA or gentisate, but could on succinate as the carbon source, was isolated. When the resting cells of NDS2-008 were incubated with 5SSA or gentisate, a substance deduced to be maleylpyruvate (Zhou et al., J. Bacteriol., 183, 700-708, 2001) was commonly detected upon HPLC analysis. These results suggest that Pigmentiphaga sp. NDS-2 degrades 26NDS via 5SSA, gentisate and maleylpyruvate as intermediates.