benzofurans and 3-chlorodibenzofuran

Rhodococcus sp. strain HA01, isolated through its ability to utilize dibenzofuran (DBF) as the sole carbon and energy source, was also capable, albeit with low activity, of transforming dibenzo-p-dioxin (DD). This strain could also transform 3-chlorodibenzofuran (3CDBF), mainly by angular oxygenation at the ether bond-carrying carbon (the angular position) and an adjacent carbon atom, to 4-chlorosalicylate as the end product. Similarly, 2-chlorodibenzofuran (2CDBF) was transformed to 5-chlorosalicylate. However, lateral oxygenation at the 3,4-positions was also observed and yielded the novel product 2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran. Two gene clusters encoding enzymes for angular oxygenation (dfdA1A2A3A4 and dbfA1A2) were isolated, and expression of both was observed during growth on DBF. Heterologous expression revealed that both oxygenase systems catalyze angular oxygenation of DBF and DD but exhibited complementary substrate specificity with respect to CDBF transformation. While DfdA1A2A3A4 oxygenase, with high similarity to DfdA1A2A3A4 oxygenase from Terrabacter sp. strain YK3, transforms 3CDBF by angular dioxygenation at a rate of 29% +/- 4% that of DBF, 2CDBF was not transformed. In contrast, DbfA1A2 oxygenase, with high similarity to the DbfA1A2 oxygenase from Terrabacter sp. strain DBF63, exhibited complementary activity with angular oxygenase activity against 2CDBF but negligible activity against 3CDBF. Thus, Rhodococcus sp. strain HA01 constitutes the first described example of a bacterial strain where coexpression of two angular dioxygenases was observed. Such complementary activity allows for the efficient transformation of chlorinated DBFs.

Topics: Benzofurans; Dioxins; Dioxygenases; DNA, Bacterial; DNA, Ribosomal; Gene Expression Profiling; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Multigene Family; Oxidation-Reduction; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Rhodococcus; RNA, Ribosomal, 16S; Salicylates; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Substrate Specificity

Dibenzofuran uptake-associated kinetic parameters of suspended and attached Sphingomonas sp. strain HH19k cells were compared. The suspended cells were studied in a batch system, whereas glass beads in percolated columns were used as the solid support for attached cells. The maximum specific activities of cells in the two systems were the same. The apparent half-maximum uptake rate-associated concentrations (Kt') of attached cells, however, were considerably greater than those of suspended cells and depended on cell density and on percolation velocity. A mathematical model was developed to explain the observed differences in terms of substrate transport to the cells. This model was based on the assumptions that the intrinsic half-maximum uptake rate-associated concentration (Kt) was unchanged and that deviations of Kt' from Kt resulted from the stereometry and the hydrodynamics around the cells. Our calculations showed that (i) diffusion to suspended cells and to single attached cells is efficient and therefore only slightly affects Kt'; (ii) diffusion to cells located on crowded surfaces is considerably lower than that to single attached cells and greatly increases Kt', which depends on the cell density; (iii) the convective-diffusive transport to attached cells that occurs in a percolated column is influenced by the liquid flow and results in dependency of Kt' on the flow rate; and (iv) higher specific affinity of cells correlates with higher susceptibility to diffusion limitation. Properties of the experimental system which limited quantitative proof of exclusively transport-controlled variations of Kt' are discussed.

Topics: Bacterial Adhesion; Benzofurans; Biodegradation, Environmental; Diffusion; Gram-Negative Aerobic Bacteria; Kinetics; Osmolar Concentration; Solubility

The dibenzofuran-degrading bacterial strain Pseudomonas sp. HH69 showed high oxidative activity towards 3-chlorodibenzofuran (3CDF). During the co-metabolic turnover of 3CDF large amounts of 4-chlorosalicylate and temporarily small amounts of salicylate were excreted. Simultaneously a yellow colour appeared due to the excretion of two polar products. Conversion of 3CDF by a mutant, derived from Pseudomonas sp. HH69 and defective in 2,3-dihydroxybiphenyl-1,2-dioxygenase led to the formation of equal quantities of 4'-chloro-2,2',3-trihydroxybiphenyl (4'CTHBP) and 4-chloro-2,2',3-trihydroxybiphenyl (4CTHBP). Crude extracts of the wild type transformed 4'CTHBP to 4-chlorosalicylate, whilst 4CTHBP was transformed to salicylate. Hence, we propose a non-selective initial attack on both aromatic rings of 3CDF and a degradative pathway for the resulting chlorotrihydroxybiphenyls.

Topics: Benzofurans; Biotransformation; Pseudomonas

3-Chlorodibenzofuran was the only markedly mutagenic isomer among the four monochlorodibenzofurans. Although it was mutagenic even in the absence of 9,000g supernatant fraction (S9) of rat liver, it was further activated by the addition of S9. Metabolic activation of this compound in mutagenicity was studied using liver S9s and cell fractions which were prepared from rats treated with two inducers. 1,1-Dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) was used as an inducer of phenobarbital inducible cytochrome P-450, and beta-naphthoflavone (beta NF) was used as an inducer of 3-methylcholanthrene inducible cytochrome P-448. S9, microsomal, mitochondrial, and cytosolic fractions were obtained from four groups of rats, i.e., untreated, DDE treated, beta NF treated, and DDE and beta NF treated groups. Mutagenicity was tested using Salmonella typhimurium tester strain TA98, because this strain is more sensitive to 3-chlorodibenzofuran than strain TA100. This experiment showed that 3-chlorodibenzofuran was activated most highly by beta NF-induced microsomes. However, it was also activated by the cytosolic fraction. Moreover, it was highly activated in rat livers which were not treated with inducers. The activity of aryl hydrocarbon hydroxylase (AHH) of each fraction was measured. AHH did not always become an index of the metabolic activation of 3-chlorodibenzofuran. This study showed that 3-chlorodibenzofuran is activated not only by cytochrome P-448, which is induced by 3-methylcholanthrene type inducers, but also by the enzymes existing in normal rat liver. This result suggests a risk of manifestation of its toxicity to normal animals.

Topics: Aminopyrine N-Demethylase; Animals; Aryl Hydrocarbon Hydroxylases; Benzofurans; Biotransformation; Cytochrome P-450 Enzyme System; Cytosol; Enzyme Induction; Liver; Male; Microsomes, Liver; Mitochondria, Liver; Mutagenicity Tests; Mutagens; Rats; Rats, Inbred Strains; Salmonella typhimurium