Cloning of a genetically unstable cytochrome P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl ether by Rhodococcus ruber.
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.
Chauvaux
S
S
Unité Microbiologie et Environnement and URA 2172, France. chauvaux@pasteur.fr
Chevalier
F
F
Le Dantec
C
C
Fayolle
F
F
Miras
I
I
Kunst
F
F
Beguin
P
P
D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D005019:Ethyl Ethers / Q000378:metabolism*; D005288:Ferredoxins / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D010088:Oxidoreductases / Q000235:genetics; D012240:Rhodococcus / Q000235:genetics* / Q000378:metabolism
D005019:Ethyl Ethers; D005288:Ferredoxins; C098546:ethyl tert-butyl ether; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases