Metagenomics reveals diversity and abundance of meta‐cleavage pathways in microbial communities from soil highly contaminated with jet fuel under air‐sparging bioremediation

Abstract

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.

From Rare to Dominant: a Fine-Tuned Soil Bacterial Bloom during Petroleum Hydrocarbon Bioremediation

“…However, in this study, the molar C:N:P ratio was 100:4:0.8 in the original soil, 100:2.5:0.4 after the first diesel amendment, and ϳ100:1.8:0.3 after the second amendment (see Table S3 in the supplemental material). Despite the high diesel concentration that emulated highly polluted soil (61,62), the small number of bacterial inoculations (2 times), nutrient limitation, and nonregulated temperature, degradation was significantly higher in both bioaugmented microcosms than in the noninoculated C1 soil after 8 weeks. Future studies should focus on additional field conditions.…”

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway

“…This type of toluene degradation mechanism, twosuccessive monooxygenation of toluene, has also been proposed for the recently sequenced Alycycliphilus denitrificans (34). The presence of a complete monooxygenation pathway for the degradation of toluene is also supported by the presence of genes encoding extradiol dioxygenases (catechol 2,3-dioxygenases; (BAH90326 and AAS75778), which commonly cluster together with phenol hydroxylase encoding genes in toluene-degrading strains (27). Two other possible degradation pathways for toluene degradation were suggested by the microarray results, although each of them was found only in one of the reactors.…”

“…The 16S rRNA gene of each strain was amplified by PCR using universal primers (25), the purified PCR products were sequenced, and the sequences were phylogenetically assigned using the BLASTn tool (http://www.ncbi.nlm.nih.gov/BLAST/). The axenic cultures were examined for the presence of catabolic genes involved in toluene degradation using several previously described degenerate primers (26)(27)(28)(29) and the newly designed primers PHE-F (5=-GAYCCBTTYCGYHTRACCAT GGA) and PHE-R (5=-GGCARCATGTARTCCWKCATCAT) for amplifying a 700-bp gene fragment of the ␣-subunit of phenol-methylphenol monooxygenases of the soluble diiron monooxygenases (19,30). The PHE-F and PHE-R primers were used at a final concentration of 0.5 M according to the following PCR program: 95°C for 15 min; 35 cycles of 95°C for 45 s, 52°C for 45 s, and 72°C for 1 min; and a final elongation at 72°C for 8 min.…”

Construction and validation of metagenomic DNA libraries from landfarm soil microorganisms

“…Estradiol dioxygenases are key enzymes in degrading aromatic compounds and many of these proteins and their coding sequences have been described, purified, and characterized in recent decades (Brennerova et al, 2009). Catechol 2,3-dioxygenases are a group that display significant activity against catechol (Eltis and Bolin, 1996).…”