Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase

Abstract

Nitrilotriacetate (NTA), a synthetic chelating agent, has been used for various radionuclide processing and decontamination procedures. NTA has been codisposed with radionuclides and heavy metals to soils and subsurface sediments (4, 27, 32), where it can greatly increase the mobility of these metals in the environment by forming soluble complexes with them. Microbial degradation of NTA may ultimately aid in immobilizing these radionuclides. Several NTA-degrading bacteria have been isolated (2,7,13,18,23,42). An NTA monooxygenase (NTA-Mo) of Chelatobacter heintzii ATCC 29600 has been identified (16) (47). NTA oxidation is proposed to be catalyzed by a heterodimer of components A (cA) and B (cB), but it is unclear how these two components interact with each other or what the function of each component is (44). In order to provide additional information on the function of cA and cB and to facilitate understanding natural attenuation or engineered bioremediation of NTA in the environment through the use of specific PCR primers or gene probes, we cloned, sequenced, and analyzed a gene cluster involved in NTA degradation. On the basis of DNA sequence and activity analysis, the two components were shown to be two separate enzymes, an monooxygenase that oxidized NTA at the expense of reduced flavin mononucleotide (FMNH 2 ) and O 2 and an NADH:flavin mononucleotide (FMN) oxidoreductase that uses NADH to reduce FMN to FMNH 2 .(A preliminary account of this work was presented at the 1995 American Society for Microbiology General Meeting [46], and the DNA sequence and gene organization have been published in a master's degree thesis [46]). MATERIALS AND METHODSBacterial strains and plasmids. The plasmids used or constructed in this study are listed in Table 1. C. heintzii ATCC 29600 was obtained from the American Type Culture Collection (Rockville, Md.) and was cultured with using a mineral salt medium with NTA as the sole carbon source (42). Escherichia coli HB101 was used as the host for pRK311, strain DH5␣ was used as the host for pBS, and strain JM105 was used as the host for pTrc99A. E. coli strains were routinely grown at 37ЊC in Luria-Bertani (LB) medium or on LB agar (37). Tetracycline and ampicillin (Sigma, St. Louis, Mo.) were used at 25 and 100 g/ml, respectively.Gene cloning. The two components of NTA-Mo were purified according to the method of Uetz et al. (44), subjected to discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (24), and then electroblotted onto a polyvinylidene difluoride membrane (28, 31) for N-terminal amino acid sequence analysis on an ABI 470 protein sequencer at the Department of Biochemistry and Biophysics, Washington State University.Oligonucleotides were 5Ј end labeled with 32 P by polynucleotide kinase (37). C. heintzii ATCC 29600 genomic DNA was isolated by a combination of largescale CsCl gradient preparation and hexadecyltrimethyl ammonium bromide and phenol extraction (3, 37). The PolarPlex Chemiluminescent Kit of Millipore (Bedford, Mass.) was used to ...

The Pix pilus adhesin of the uropathogenic Escherichia coli strain X2194 (O2 : K− : H6) is related to Pap pili but exhibits a truncated regulatory region

“…This PAI spans a 58 kb region, and among the 44 ORFs identified it contains several virulence genes, including a complete pap gene cluster. Additionally, the authors described the R6 gene as showing a highly significant homology to a gene inside a gene cluster encoding nitrilotriacetate monooxygenase and NADH : flavin mononucleotide oxidoreductase in C. heintzii ATCC 29600 (Xu et al, 1997). By sequence alignment an ORF of 1467 bp encoding a protein of 488 aa was suggested.…”

“…The R6 gene of E. coli CFT073 has recently been reported to express significant homology to a transposase gene of Chelatobacter heintzii (Xu et al, 1997). It was interesting to see whether the R6 gene fragment represented just the remnant of a transposable element or if this sequence might be involved in the regulation of Pix pilus expression.…”

Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD

“…Thermostability-Studies on the thermostability of PheA1 and PheA2 were performed by incubating the enzymes (46 M PheA1 or 48 M PheA2) in closed vials at different temperatures (50,60,70, and 85°C) in 25 mM phosphate (pH 7.0) for 2 h. At timed intervals, aliquots were withdrawn from the incubation mixtures and assayed for residual phenol hydroxylase (PheA1) and NADH:flavin reductase (PheA2) activities.…”

Reduced Flavins Promote Oxidative DNA Damage in Non-respiringEscherichia coli by Delivering Electrons to Intracellular Free Iron

“…Whereas FADH 2 most commonly serves as a redox cofactor when it is tightly bound to enzymes, free FADH 2 is used as a co-substrate for monooxygenase reactions (42)(43)(44)(45)(46), including several from E. coli strains (28,47,48). Indirect evidence has suggested that FADH 2 may also assist in the activation of ribonucleotide reductase (33).…”