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

EDTA is a chelating agent, widely used in many industries. Because of its a bility to mobilize heavy metals and radionuclides, it can be an environment al pollutant, The EDTA monooxygenases that initiate EDTA degradation have b een purified and characterized in bacterial strains BNCl and DSM 9103. Howe ver, the genes encoding the enzymes have not been reported. The EDTA monoox ygenase gene was cloned by probing a genomic library of strain BNCl with a probe generated from the N-terminal amino acid sequence of the monooxygenas e, Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichi a coli, and the gene products, EmoA and EmoB, were purified and characteriz ed, Both experimental data and sequence analysis showed that EmoA is a redu ced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH: flavin mononucleotide oxidoreductase, The two-enzyme system oxidized EDTA t o ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiace tate (IDA) with the production of glyoxylate, The emoA and emoB genes were cotranscribed when BNCl cells were grown on EDTA, Other genes in the cluste r encoded a hypothetical transport system, a putative regulatory protein, a nd IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene clus ter is responsible for the initial steps of EDTA and NTA degradation.