Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation From the Bacterium BNC1

Appl Environ Microbiol. 2001 Feb;67(2):688-95. doi: 10.1128/AEM.67.2.688-695.2001.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins*
  • Biodegradation, Environmental
  • Cloning, Molecular
  • Edetic Acid / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Genes, Bacterial*
  • Gram-Negative Bacteria / enzymology*
  • Gram-Negative Bacteria / genetics
  • Molecular Sequence Data
  • Multigene Family
  • NADH, NADPH Oxidoreductases / genetics*
  • NADH, NADPH Oxidoreductases / metabolism
  • Oxidoreductases Acting on CH-NH Group Donors / genetics*
  • Oxidoreductases Acting on CH-NH Group Donors / metabolism
  • Oxygenases / genetics*
  • Oxygenases / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA

Substances

  • Bacterial Proteins
  • Edetic Acid
  • Oxygenases
  • EmoA protein, bacterium BNC1
  • Oxidoreductases Acting on CH-NH Group Donors
  • NADH, NADPH Oxidoreductases
  • EmoB protein, bacterium BNC1
  • EDTA monooxygenase