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 bacte rial strains able to degrade ethyl tert-butyl ether (ETBE), which is a majo r pollutant from gasoline. This strain was found to undergo a spontaneous 1 4.3-kbp chromosomal deletion, which results in the loss of the ability to d egrade ETBE. Sequence analysis of the region corresponding to the deletion revealed the presence of a gene cluster, ethABCD, encoding a ferredoxin red uctase, a cytochrome P-450, a ferredoxin, and a 10-kDa protein of unknown f unction, respectively. The EthB and EthD proteins could be easily detected by sodium dodecyl sulfate-polyacrylamide get electrophoresis and were induc ed by ETBE in the wild-type strain. Upstream of ethABCD lies ethR, which co des for a putative positive transcriptional regulator of the AraC/XylS fami ly. Transformation of the ETBE-negative mutant by a plasmid carrying the et hRABCD genes restored the ability to degrade ETBE. Complementation was abol ished if the plasmid carried ethRABC only. The eth genes are located in a D NA fragment flanked by two identical direct repeats of 5.6 kbp. The ETBE-ne gative 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 carry ing a TnpA transposase, a truncated form of the recombinase TnpR, and a ter minal inverted repeat of 38 bp. The truncated TnpR is encoded by an IS3-int errupted tnpR gene.