Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360

Abstract

The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K. Due to the environmental significance of its chlorinated derivatives, the metabolism of dibenzofuran has been studied intensively in the last few years (10, 14, 16-18, 48, 58, 59, 65). Accordingly, bacterial mineralization of dibenzofuran is initiated by dibenzofuran 4,4a-dioxygenase, catalyzing the angular dioxygenation of dibenzofuran. This gives rise to the chemically labile hemiacetal 4,4a-dihydro-4,4a-dihydroxydibenzofuran, which, after spontaneous rearomatization, yields 2,2Ј, 3-trihydroxybiphenyl (16, 59). A multicomponent dioxygenase of this angular dioxygenase type was purified from Sphingomonas sp. strain RW1 (9) as a four-component class IIA dioxygenase system. Ring cleavage of 2,2Ј,3-trihydroxybiphenyl was shown to be catalyzed by extradiol dioxygenases yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid (16, 59), which is subsequently hydrolyzed to 2-oxopent-4-enoate and salicylate (8,16,59). From Sphingomonas sp. strain RW1, a 2,2Ј,3-trihydroxybiphenyl-cleaving extradiol dioxygenase which was suggested to be involved in dibenzofuran mineralization was cloned and characterized (24). In this paper, three distinct extradiol dioxygenases from Terrabacter sp. strain DPO360 are shown to be involved in total mineralization of dibenzofuran and characterized according to their function in the degradative pathway.- MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. Terrabacter sp. strain DPO360 was isolated from tar-contaminated soil in Germany (57), and Terrabacter sp. strain DPO1361 was isolated from Rhine water (58). Both grampositive isolates were catalase positive and cytochrome oxidase negative. Biochemical characterization of these strains using the API-CH50 and API-20NE test kits (Biomerieux) gave identical results, apart from positive reactions for esculin and 5-ketogluconate with Terrabacter sp. strain DPO1361, reactions which were negative for Terrabacter sp. strain DPO360. Both strains were cultivated as described earlier (58) with dibenzofuran as a source of carbon. Yeast extract (0.005% [wt/vol]) was added to the minimal medium to supply vitamins. L broth (42) was used as a complex medium for Terrabacter sp. and Escherichia coli strains.[47] was used as a recipient for library construction and screening, and E. coli JM109 [recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ⌬(lac-proAB) FЈ(traD36 proAB ϩ lacI q lacZ⌬M15) [69] was used in all other cloning experiments. The cloning vectors pUC20 and pUC21 (identical with pUC20, except for an inverted polylinker) were obtained from Boehringer Mannheim Biochemicals. Plasmids constructed in the present study are shown in Fig. 3.Chemicals. Chemicals were of the highest purity commercially available (Merck, Darmstadt, Germany; Sigma-Aldrich, Steinheim, Germany). 3-Phenylcatechol was obtained from Wako Chemicals (Neuss, Germany). 3-C...

Biodegradation of Dibenzofuran by Janibacter terrae Strain XJ-1

“…However, strain XJ-1 highly accumulates gentisic acid instead of catechol in the final products. Compared with YY-1 and those DF-degrading Janibacter and Terrabacter strains isolated from Europe [20,26], XJ-1 is more similar to YY-1 in the DF-metabolizing pathway and to European strains in the 16S rDNA sequence. It has been found that DF-degrading genes are dispersed on both chromosomal and extrachromosomal DNA.…”

“…When grown on DF, Terrabacter sp. strains DBF63, DPO 360, and DPO1361-the latter a heterotypic synonym to Janibacter terrae-produced intermediates 2,2¢,3-trihydroxybiphenyl and salicylic acid by way of initial dioxygenation by angular dioxygenase [15,16,20,26]. Salicylic acid can be converted to catechol and gentisic acid [16].…”

Bacterial degradation of aromatic compounds via angular dioxygenation.

“…2,2Ј,3-Trihydroxybiphenyl is a compound analogous to 2,3-dihydroxybiphenyl, which is a metabolic intermediate of biphenyl, and is converted to benzoic acid by FN Engesser et al, 1989;Strubel et al, 1989 Terrabacter sp. strain DPO1361 DF 4,4a-dioxygenase DF, FN Engesser et al, 1989;Schmid et al, 1997;Strubel et al, 1989Strubel et al, , 1991Trenz et al, 1994 Terrabacter sp. strain DPO360 DF 4,4a-dioxygenase DF Schmid et al, 1997 Sphingomonas sp.…”

“…strain DPO1361 DF 4,4a-dioxygenase DF, FN Engesser et al, 1989;Schmid et al, 1997;Strubel et al, 1989Strubel et al, , 1991Trenz et al, 1994 Terrabacter sp. strain DPO360 DF 4,4a-dioxygenase DF Schmid et al, 1997 Sphingomonas sp. strain HH69 DF 4,4a-dioxygenase DF, DD Fortnagel et al, 1990;Harms et al, 1990Harms et al, , 1991Harms et al, , 1995Schreiner et al, 1997 Sphingomonas wittichii strain RW1 DF 4,4a-dioxygenase DF, DD, 9-Fluorenone Timmis, 1997, 1998;, (Dioxin dioxygenase) 1999, 2000Bertini et al, 1995;Halden et al, 1999;Happe et al, 1993;Keim et al, 1999;Megharaj et al, 1997;Schläfli et al, 1995;Wilkes et al, 1996;Wittich et al, 1992;Yabuuchi et al, 2001 Terrabacter sp.…”

“…Based on the specific constants k cat /K m of these meta cleavage enzymes, both BphC1 and BphC2 were shown to be responsible for the meta cleavage of 2,2Ј,3-trihydroxybiphenyl. The DNA region containing the bphC1 gene was cloned and sequenced (Schmid et al, 1997). In this region, meta cleavage compound hydrolase (bphD) is located directly downstream of bphC1 gene, although the nucleotide sequence of 3Ј-terminal region has not been determined completely.…”