Role of gene mpeB0561 in the methyl tert-butyl ether biodegradation pathway in Methylibium petroleiphilum PM1

A number of environment-isolated bacteria have been demonstrated to utilize MTBE as its sole carbon and energy source, therefore, characterization of the enzymes involved in MTBE degradation is crucial to better understand the limitations of its metabolism. The gene mpeB0561 proposed to be involved in HIBA degradation in Methylibium petroleiphilum PM1, the best characterized MTBE degrader, was selected as target for in-vitro mutagenesis with the EZ-Tn5 in-vitro mutagenesis system from Epicentre. A PM1 mutant strain was obtained with a disrupted copy of gene mpeB0561 and growth and degradation capabilities where tested in the laboratory. In order to determine the effect of the mutation in HIBA degradation by PM1, a Reverse Phase HPLC method was developed. The method was able to consistently resolve HIBA peak and the limit of quantitation (LOQ) obtained was 0.84 ppm.;MdpH appeared to be involved in the degradation of 2-methyl-2-hydroxy-isopropanol: The mutation affected the ability of mutant PM1 to grow on MTBE, TBA and 2-methy1-2-hydroxy-isopropanol, but not on HIBA; and degradation rates for MTBE and tert-butyl alcohol (TBA) where similar between wild type and mutant PM1.