Characterization of Transgenic Tobacco Plants Containing Bacterial bphc Gene and Study of Their Phytoremediation Ability

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene – enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified after purification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected.Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with non-transgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.     

Cloning and sequence analyses of a 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (<Emphasis Type="Italic">bphC</Emphasis>) from<Emphasis Type="Italic"> Comamonas</Emphasis> sp. SMN4 for phylogenetic and structural analysis

A genomic library of biphenyl-degrading Comamonas sp. SMN4 for isolating fragments containing the 2,3-dihydroxybiphenyl 1,2-dioxygenase (23DBDO) gene was constructed. The smallest subclone (pNPX9) encoding 23DBDO activity was sequenced and analyzed. The C-terminal domain of 23DBDO from Comamonas sp. SMN4 had five catalytically essential residues and was more highly conserved than the N-terminal domain. Phylogenetic and structural relationships of 23DBDO from Comamonas sp. SMN4 were analyzed. Electronic Publication     

The bphC gene-encoded 2,3-dihydroxybiphenyl-1,2-dioxygenase is involved in complete degradation of dibenzofuran by the biphenyl-degrading bacterium Ralstonia sp. SBUG 290

AIMS: Biphenyl-degrading bacteria are able to metabolize dibenzofuran via lateral dioxygenation and meta-cleavage of the dihydroxylated dibenzofuran produced. This degradation was considered to be incomplete because accumulation of a yellow-orange ring-cleavage product was observed. In this study, we want to characterize the 1,2-dihydroxydibenzofuran cleaving enzyme which is involved in dibenzofuran degradation in the bacterium Ralstonia sp. SBUG 290. METHODS AND RESULTS: In this strain, complete degradation of dibenzofuran was observed after cultivation on biphenyl. The enzyme shows a wide substrate utilization spectrum, including 1,2-dihydroxydibenzofuran, 2,3-dihydroxybiphenyl, 1,2-dihydroxynaphthalene, 3- and 4-methylcatechol and catechol. MALDI-TOF analysis of the protein revealed a strong homology to the bphC gene products. We therefore cloned a 3.2 kb DNA fragment containing the bphC gene of Ralstonia sp. SBUG 290. The deduced amino acid sequence of bphC is identical to that of the corresponding gene in Pseudomonas sp. KKS102. The bphC gene was expressed in Escherichia coli and the meta-fission activity was detected using either 2,3-dihydroxybiphenyl or 1,2-dihydroxydibenzofuran as substrate. CONCLUSIONS: These results demonstrate that complete degradation of dibenzofuran by biphenyl degraders can occur after initial oxidation steps catalysed by gene products encoded by the bph-operon. The ring fission of 1,2-dihydroxydibenzofuran is catalysed by BphC. Differences found in the metabolism of the ring fission product of dibenzofuran among biphenyl degrading bacteria are assumed to be caused by different substrate specificities of BphD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time that the gene products of the bph-operon are involved in the mineralization of dibenzofuran in biphenyl degrading bacteria.