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Journal of Bacteriology, November 2001, p. 6551-6557, Vol. 183, No. 22
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.22.6551-6557.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Cloning of a Genetically Unstable Cytochrome P-450 Gene Cluster Involved in Degradation of the Pollutant Ethyl tert-Butyl Ether by Rhodococcus ruber

Sylvie Chauvaux,1,* Fabien Chevalier,2 Corinne Le Dantec,3 Françoise Fayolle,4 Isabelle Miras,1 Frank Kunst,2 and Pierre Beguin1

Unité Microbiologie et Environnement and URA 2172,1 Laboratoire de Génomique des Microorganismes Pathogènes,2 and Laboratoire de Référence des Mycobactéries,3 Institut Pasteur, 75724 Paris Cedex 15, and Département de Microbiologie, Institut Français du Pétrole, 92852 Rueil-Malmaison Cedex,4 France

Received 9 July 2001/Accepted 29 August 2001

Rhodococcus ruber (formerly Gordonia terrae) IFP 2001 is one of a few bacterial strains able to degrade ethyl tert-butyl ether (ETBE), which is a major pollutant from gasoline. This strain was found to undergo a spontaneous 14.3-kbp chromosomal deletion, which results in the loss of the ability to degrade ETBE. Sequence analysis of the region corresponding to the deletion revealed the presence of a gene cluster, ethABCD, encoding a ferredoxin reductase, a cytochrome P-450, a ferredoxin, and a 10-kDa protein of unknown function, respectively. The EthB and EthD proteins could be easily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were induced by ETBE in the wild-type strain. Upstream of ethABCD lies ethR, which codes for a putative positive transcriptional regulator of the AraC/XylS family. Transformation of the ETBE-negative mutant by a plasmid carrying the ethRABCD genes restored the ability to degrade ETBE. Complementation was abolished if the plasmid carried ethRABC only. The eth genes are located in a DNA fragment flanked by two identical direct repeats of 5.6 kbp. The ETBE-negative mutants carry a single copy of this 5.6-kbp repeat, suggesting that the 14.3-kbp chromosomal deletion resulted from a recombination between the two identical sequences. The 5.6-kbp repeat is a class II transposon carrying a TnpA transposase, a truncated form of the recombinase TnpR, and a terminal inverted repeat of 38 bp. The truncated TnpR is encoded by an IS3-interrupted tnpR gene.

* Corresponding author. Mailing address: Unité Microbiologie et Environnement, Département des Biotechnologies, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33 1 40 61 37 04. Fax: 33 1 45 68 87 90. E-mail: chauvaux{at}pasteur.fr.

Journal of Bacteriology, November 2001, p. 6551-6557, Vol. 183, No. 22
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.22.6551-6557.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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