Mechanism of 4-nitrophenol oxidation in Rhodococcus sp. Strain PN1: characterization of the two-component 4-nitrophenol hydroxylase and regulation of its expression

Abstract

4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil. Rhodococcus sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PN1 contained a gene cluster (nphRA1A2) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XylS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a Rhodococcus strain revealed that the transcription of nphA1 is induced by only 4-NP (of several phenolic compounds tested) in the presence of nphR, which is constitutively expressed. Disruption of nphR abolished transcriptional activity, suggesting that nphR encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in Escherichia coli and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide (FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC.

FIG. 1.

Physical map of pKPN3 containing the 4-NP hydroxylase gene cluster of Rhodococcus sp. strain PN1 and its derivatives. A catechol 2,3-dioxygenase gene (atdB) from Acinetobacter sp. strain YAA (42, 45) was inserted into the StuI site and the EcoRV site of pKPN3 as a reporter gene to construct pKPN32 and pKPN35, respectively. For the disruption of nphR, a tetracycline resistance gene (tet) from pBR322 (4) was introduced into the EcoRV site of pKPN32 to construct pKPN33. These derivatives were used to investigate the transcriptional activity of nphA1 and nphR in R. rhodochrous ATCC 12674 in the presence or absence of various inducers.

FIG. 2.

Enzymatic conversion of 4-NP (a) and other phenolic compounds (b and c) using NphA1 and His-NphA2. Enzymatic conversion was carried out in the standard reaction mixture, except for the buffer pH, which was 8.0. In 4-NP conversion (a), 4-NC was monitored as the product. Open circles, 4-NP; closed circles, 4-NC; squares, 3-NP; diamonds, phenol; triangles, 4-chlorophenol.

FIG. 3.

Genetic organization of the NP degradation gene clusters identified so far (a) and proposed NP degradation pathways (b). The genetic organization and the proposed pathways were constructed based on the following sequences and references: the nph gene cluster, AB081773 and this study; the npc gene cluster, AB154422 and reference ; and the npd gene cluster, EF052871 and reference .