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Genes involved in the methyl tert-butyl ether (MTBE) metabolic pathway of Mycobacterium austroafricanum IFP 2012

¹ Institut Français du Pétrole, 1–4, avenue de Bois-Préau, F-92852 Rueil-Malmaison, France
² Biotechnology Research Institute, NRCC, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P2R2

Correspondence
Françoise Fayolle-Guichard
francoise.fayolle{at}ifp.fr

Methyl tert-butyl ether (MTBE) is a persistent pollutant of surface and groundwater, and the reasons for its low biodegradability are poorly documented. Using one of the rare bacterial strains able to grow in the presence of MTBE, Mycobacterium austroafricanum IFP 2012, the protein profiles of crude extracts after growth in the presence of MTBE and glucose were compared by SDS-PAGE. Ten proteins with molecular masses of 67, 64, 63, 55, 50, 27, 24, 17, 14 and 11 kDa were induced after growth in the presence of MTBE. Partial amino acid sequences of N-terminal and internal peptide fragments of the 64 kDa protein were used to design degenerate oligonucleotide primers to amplify total DNA by PCR, yielding a DNA fragment that was used as a probe for cloning. A two-step cloning procedure was performed to obtain a 10 327 bp genomic DNA fragment containing seven ORFs, including a putative regulator, mpdR, and four genes, mpdC, orf1, mpdB and orf2, in the same cluster. The MpdB protein (64 kDa) was related to a flavoprotein of the glucose–methanol–choline oxidoreductase family, and the MpdC protein (55 kDa) showed a high similarity with NAD(P) aldehyde dehydrogenases. Heterologous expression of these gene products was performed in Mycobacterium smegmatis mc2 155. The recombinant strain was able to degrade an intermediate of MTBE biodegradation, 2-methyl 1,2-propanediol, to hydroxyisobutyric acid. This is believed to be the first report of the cloning and characterization of a cluster of genes specifically involved in the MTBE biodegradation pathway of M. austroafricanum IFP 2012.

The GenBank/EMBL/DDBJ accession number for the genomic DNA sequence reported in this paper is DQ147773.

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