Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1

Abstract

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.

FIG. 1

The restriction sites and gene organization of the 15,603-bp DNA fragment. BamHI (B), HindIII (H), and EcoRI (E) target sites are indicated. Orientation of the arrows indicates the direction of gene transcription. The genes represented by black arrows may directly participate in EDTA degradation.

FIG. 2

SDS-PAGE of the purified EmoA and EmoB proteins. Lane 1 contains 0.6 μg of nonfusion EmoA, lane 2 contains molecular weight markers (low-range SDS-PAGE markers; Bio-Rad, Hercules, Calif.), and lane 3 contains 2 μg of EmoB (C-terminal fusion). The gel was stained with Gel Code Blue.

FIG. 3

The time course of EDTA degradation by EmoA/EmoB. Symbols: ⧫, EDDA accumulation; ■, EDTA degradation. The arrows indicate the subsequent addition of NADH. Error bars indicate the standard deviations for triplicate samples.

FIG. 4

RT-PCR of emo genes. Molecular markers are in lane 1 (1 kb DNA ladder; Lifetech, Rockville, Md.). Lane 2, emoB-induced cells; lane 3, emoAB-induced cells; lane 4, emoB-uninduced cells; lane 5, emoAB-uninduced cells; lane 6, emoB amplification by normal PCR (no reverse transcriptase)-induced cells; and lane 7, emoB amplification by normal PCR-uninduced cells.

FIG. 5

The EDTA degradation by EmoA in the bacterium BNC1.