Molecular characterization of a novel ortho-nitrophenol catabolic gene cluster in Alcaligenes sp. strain NyZ215

Abstract

Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cis-muconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723.

FIG. 1.

(A) Proposed initial reactions of ONP catabolism catalyzed by the onpABC gene products in vivo. (B) Organization of the ONP gene cluster involved in the initial reactions of ONP degradation in Alcaligenes sp. strain NyZ215. The arrows indicate the sizes and directions of transcription of each ORF or gene. The arrow above onpC indicates the region where the genome walking started in both directions. Plasmid pZWXW1 was constructed by cloning the 5-kb fragment from the first cycle of genome walking into T-vector. E. coli containing this plasmid exhibited ONP 2-monooxygenase activity. The locations of primer sets RTA, RTB, RTC, RTAB, and RTBC and the amplified DNA fragments for RT-PCR are indicated. (C) Analysis of onpABC transcription by RT-PCR. Total RNAs of ONP-grown strain NyZ215 were prepared for RT-PCR, and reactions performed without RT were used as negative controls. Lanes M, molecular size markers; lanes 2, 4, 6, 8, and 10, products amplified using the RTA, RTB, RTC, RTAB and RTBC primer sets, respectively, with products of RT; lanes 1, 3, 5, 7, and 9, corresponding negative controls.

FIG. 2.

Spectral changes associated with the transformation of ONP by OnpA. Sample and reference cuvettes contained 0.1 mM NADPH, 4 mM Mg²⁺, and cell extracts containing OnpA (about 10 μg/ml) in 500 μl of 20 mM phosphate buffer (pH 7.5). Reactions were started by addition of ONP (at a final concentration of 0.02 mM) to the sample cuvettes, and spectra were recorded every 3 min.

FIG. 3.

Phylogenetic relationships of ONP 2-monooxygenase and related proteins having known functions based on the results of a BLASTP search of the GenBank database. Construction of the phylogenetic tree (using the neighbor-joining method) and multiple-sequence alignment were performed with the Mega 3.1 software. Gap and gap length penalties were set at 10. The bootstrap confidence limits are indicated at the nodes, and the scale at the bottom indicates sequence divergence.