Isolation and characterization of a new Mycobacterium austroafricanum strain, IFP 2015, growing on MTBE

Degradative strains of fast-growing Mycobacterium spp. are commonly isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soils. Little is known, however, about the ecology and diversity of indigenous populations of these fast-growing mycobacteria in contaminated environments. In the present study 16S rRNA genes were PCR amplified using Mycobacterium-specific primers and separated by temperature gradient gel electrophoresis (TGGE), and prominent bands were sequenced to compare the indigenous Mycobacterium community structures in four pairs of soil samples taken from heavily contaminated and less contaminated areas at four different sites. Overall, TGGE profiles obtained from heavily contaminated soils were less diverse than those from less contaminated soils. This decrease in diversity may be due to toxicity, since significantly fewer Mycobacterium phylotypes were detected in soils determined to be toxic by the Microtox assay than in nontoxic soils. Sequencing and phylogenetic analysis of prominent TGGE bands indicated that novel strains dominated the soil Mycobacterium community. Mineralization studies using [(14)C]pyrene added to four petroleum-contaminated soils, with and without the addition of the known pyrene degrader Mycobacterium sp. strain RJGII-135, indicated that inoculation increased the level of degradation in three of the four soils. Mineralization results obtained from a sterilized soil inoculated with strain RJGII-135 suggested that competition with indigenous microorganisms may be a significant factor affecting biodegradation of PAHs. Pyrene-amended soils, with and without inoculation with strain RJGII-135, experienced both increases and decreases in the population sizes of the inoculated strain and indigenous Mycobacterium populations during incubation.

Metabolism of the environmental pollutant benzo[ a ]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[ a ]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[ a ]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis -4,5-dihydro-4,5-dihydroxybenzo[ a ]pyrene (benzo[ a ]pyrene cis -4,5-dihydrodiol), cis -11,12-dihydro-11,12-dihydroxybenzo[ a ]pyrene (benzo[ a ]pyrene cis -11,12-dihydrodiol), trans -11,12-dihydro-11,12-dihydroxybenzo[ a ]pyrene (benzo[ a ]pyrene trans -11,12-dihydrodiol), 10-oxabenzo[ def ]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[ a ]pyrene. The ortho -ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[ a ]pyrene cis -4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[ a ]pyrene cis -4,5-dihydrodiol had 30% 4 S ,5 R and 70% 4 R ,5 S absolute stereochemistry. Benzo[ a ]pyrene cis -11,12-dihydrodiol adopted an 11 S ,12 R conformation with 100% optical purity. The enantiomeric composition of benzo[ a ]pyrene trans -11,12-dihydrodiol was an equal mixture of 11 S ,12 S and 11 R ,12 R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[ a ]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[ a ]pyrene in M. vanbaalenii PYR-1.

A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012, was also able to degrade tert-amyl methyl ether and tert-amyl alcohol. Ethyl tert-butyl ether was weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co2+ during growth of M. austroafricanum IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE- or TBA- grown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE- or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE- and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.