The extradiol dioxygenase diversity of a site highly contaminated with aliphatic and aromatic hydrocarbons under air-sparging treatment was assessed by functional screening of a fosmid library in Escherichia coli with catechol as substrate. The 235 positive clones from inserts of DNA extracted from contaminated soil were equivalent to one extradiol dioxygenase-encoding gene per 3.6 Mb of DNA screened, indicating a strong selection for genes encoding this function. Three subfamilies were identified as being predominant, with 72, 55 and 43 fosmid inserts carrying genes, related to those encoding TbuE of Ralstonia pickettii PK01 (EXDO-D), IpbC of Pseudomonas sp. JR1 (EXDO-K2) or DbtC of Burkholderia sp. DBT1 (EXDO-Dbt), respectively, whereas genes encoding enzymes related to XylE of Pseudomonas putida mt-2 were not observed. Genes encoding oxygenases related to isopropylbenzene dioxygenases were usually colocalized with genes encoding EXDO-K2 dioxygenases. Functional analysis of representative proteins indicated a subcluster of EXDO-D proteins to show exceptional high affinity towards different catecholic substrates. Based on Vmax/Km specificity constants, a task-sharing between different extradiol dioxygenases in the community of the contaminated site can be supposed, attaining a complementary and community-balanced catalytic power against diverse catecholic derivatives, as necessary for effective degradation of mixtures of aromatics.
Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), which are provided by two enzymic systems (dioxygenases and monooxygenases), have been identified. The monooxygenase attacks methyl or ethyl substituents of the aromatic ring, which are subsequently transformed by several oxidations to corresponding substituted pyrocatechols or phenylglyoxal, respectively. Alternatively, one oxygen atom may be first incorporated into aromatic ring while the second atom of the oxygen molecule is used for oxidation of either aromatic ring or a methyl group to corresponding pyrocatechols or protocatechuic acid, respectively. The dioxygenase attacks aromatic ring with the formation of 2-hydroxy-substituted compounds. Intermediates of the "upper" pathway are then mineralized by either ortho- or meta-ring cleavage ("lower" pathway). BTEX are relatively water-soluble and therefore they are often mineralized by indigenous microflora. Therefore, natural attenuation may be considered as a suitable way for the clean-up of BTEX contaminants from gasoline-contaminated soil and groundwater.
Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4'-dichlorobiphenyl (3,4'-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Krickel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CCI and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4'-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 ,ug ml-' and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction ofP. acidovorans M3GY, with the novel capability to utilize 3,4'-DCBP, thus involves the complete use ofmeta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.
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