TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Vector: (III) is a plasmid, cosmid, phage or a virus.Preferred Bacterium: (IX) is able to cleave an ether fuel additive, and can use ETBE as sole source of carbon and is also capable of degrading tert-butyl alcohol (TBA). The bacterium is also able to completely mineralize an ether fuel additive. Preferably, the recombinant bacterium is able to completely mineralize ETBE.Isolation: (I) is isolated by standard cell culture techniques. EXAMPLE - Rhodococcus ruber (Rr) I-1889, or I-2194, was grown at 30degreesC in LB medium. Rr was plated on MM1 agar with ethyl tert-butyl ether (ETBE) vapor as the carbon source, and independent clones were transferred to liquid LB medium. Cultures were then plated on LB plates, and individual colonies were patched on LB plates and ETBE-containing MM1 plates, including wild type controls. Crude extracts of ETBE-induced Rr were subjected to sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Two polypeptides of 43 and 10 kDa were clearly induced in the wild type upon growth on ETBE. They were also present, although less abundant, in cells of a previously isolated constitutive mutant I-2194(18). Peptide microsequencing yielded the partial sequences HALGDWQTFSSAQGI, FDSVAQWFTR, and SVSNTEMIALWTELG for the 43-kDa protein and GQPTDEAFDTYYS for the 10-kDa protein. The first sequence HALGDWQTFSSAQGI, was 66% identical to a putative cytochrome P-450 from Mycobacterium tuberculosis H37Rv (Genpept Z1771375), suggesting that the 43-kDa polypeptide may be the inducible cytochrome P-450 observed in ETBE-grown Gordonia terrae cells. The GQPTDTEAFDTYYS sequence was 47% identical to Orf4 from Rhodococcus erythropolis (Genpept U171304). The R.erythropolis open reading frame (Orf) 4 gene is part of a cytochrome P-450 gene cluster suggesting that the inducible 10-kDa polypeptide was related to cytochrome P-450 system. To verify the stability of the ETBE-positive phenotype, five independent clones of R.ruber were cultivated in LB broth for 60 generations. Then, cultures were screened for the presence of mutants unable to grow in the presence of ETBE as the sole source of energy and carbon. Twenty to 100% of the clones tested were found unable to degrade ETBE. Five independent mutants, derived from the five original wild type clones, were further characterized. When grown to saturation in minimal medium containing 0.5% glucose and 18 mM ETBE, none of the mutants converted more than 0.3 mM ETBE into tert-butyl ether (TBA), whereas under the same conditions, 10.6 mM TBA was produced by the wild type. The reversion to the ETBE+ phenotype was not detectable suggesting the occurrence of an irreversible genetic rearrangement. Wild type and mutants strains were compared after growth in the presence of 0.5% glucose + 18 mM ETBE. Analysis of crude extracts showed that, in contrast to the wild type, none of the mutants produced the induced 43 and 10 kDa proteins. Mutant resting cells lost the ability to degrade methyl tert-butyl ether (MTBE) and tert-amyl methyl ether (TAME). XbaI-genomic digests of wild type and mutant strains were analyzed by pulsed-field gel electrophoresis. A 125-kbp fragment was present in the wild type strain and was absent in the ETBE-negative mutants. In addition, a 110-kbp fragment was observed in the ETBE-negative mutants only. Southern blot hybridization revealed that the wild-type 125-kbp fragment used as a probe hybridized with the mutant 110-kbp fragment, showing that the 110-kbp fragment was a deleted form of the 125-kbp fragment. This result indicated that ETBE-negative mutants resulted from a 15-kbp chromosomal deletion. The wild-type XbaI-fragment of 125 kbp was purified from a pulsed-field gel electrophoresis gel and was used as probe in Southern-blot analysis. Hybridization of the 125-kb XbaI probe with BamHI-genomic digests showed that a 7.4-kbp band and a 16.3-kbp band, present in the wild-type strain, disappeared in the ETBE-negative mutant. Conversely, a new 9.3-kbp band, which was absent in the wild type strain, was detected in the ETBE-negative mutant. This demonstrated that the 15-kbp deletion identified by pulsed-field gel electrophoresis involved the two BamHI-fragments of 7.4 and 16.3 kbp which were reshuffled into a new BamHI-fragment of 9.3 kbp. In order to determine the sequence of the region corresponding to the deletion, the two wild-type BamHI-fragments of 7.4 kbp and 16.3 kbp were cloned. The 7.4-kbp BamHI fragment was selected by colony hybridization using the 125-kbp XbaI fragment as a probe. The cloned 7.4-kbp fragment was then used as a probe in a Southern blot hybridization. In addition to self hybridization, the 7.4-kb BamHI probe also hybridized with the 16.3-kbp BamHI fragment of the wild type and with the 9.3-kbp BamHI fragment of the ETBE-negative mutant. This indicated that the wild-type 7.4 fragment carried a sequence that was also present in these two fragments. Thus, the wild type 16.3-kbp BamHI fragment and the mutant 9.3-kbp BamHI fragment were cloned by colony hybridization using the 7.4-kb BamHI fragment as a probe. A cluster of four open reading frames with the same orientation and named ethA, B, C, D were identified. Based on protein alignments, ethA (412 amino acids), B (400 amino acids) and C (106 amino acids) could be assigned to individual components of a P-450 system containing monooxygenase.