Two Angular Dioxygenases Contribute to the Metabolic Versatility of Dibenzofuran-Degrading Rhodococcus sp. Strain HA01

Abstract

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.Biarylethers, comprising dibenzo-p-dioxin (DD), dibenzofuran (DBF), diphenyl ether, and their halogenated derivatives, are widespread environmental pollutants. Polychlorinated DDs and DBFs, the contaminating by-products formed during the manufacture of pesticides or the incineration of industrial and domestic wastes, can cause a wide range of serious health effects (1,4,13).Microorganisms play important roles in the degradation and mineralization of xenobiotic and aromatic compounds in natural environments. Their aerobic degradation is frequently initiated by Rieske nonheme iron oxygenases which catalyze the incorporation of two oxygen atoms into the aromatic ring to form arene cis-diols (15). This is then followed by a dehydrogenation reaction catalyzed by a cis-dihydrodiol dehydrogenase to give catechol or substituted catechols, which serve as substrates for oxygenolytic aromatic ring cleavage. Rieske nonheme iron oxygenases are typically composed of a terminal oxygenase (iron-sulfur protein) and different electron transport proteins (8). The catalytic iron-sulfur proteins are homoor heteromultimers with the ␣-subunit containing a Riesketype [2Fe-2S] cluster, a mononuclear nonheme iron oxygen activation center, and a substrate-binding site (8) that is responsible for substrate specificity (15). Comparison of the amino acid sequences of the ␣-subunits ...

Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter

“…DF412 (BAG06223)1 dxnA1 Miyauchi et al, 2008d2 Rhodococcus sp. HA01 (ACC85677)1Aly et al, 2008d2 Terrabacter sp. YK1 (BAG80728)1Iida et al, 2002ad2 Rhodococcus sp.…”

The GAF-Like-Domain-Containing Transcriptional Regulator DfdR Is a Sensor Protein for Dibenzofuran and Several Hydrophobic Aromatic Compounds

“…strain DF412 (33) and Rhodococcus sp. strain HA01 (3). These results suggest that DF-utilizing actinomycetes recently acquired dfd gene clusters by lateral gene transfer.…”

“…The dfd catabolic pathway genes have been identified in at least four genera of actinomycetes (3,20,21,33,44), and the levels of nucleotide sequence conservation are high. By using Southern hybridization analysis, we determined that the transcription regulator gene dfdR was highly conserved in all six strains of DF-utilizing actinomycetes that we isolated and analyzed (data not shown), suggesting that dfdR was laterally transferred as a part of the dfd gene cluster in several DFutilizing actinomycetes.…”

Expression inEscherichia coliof Biphenyl 2,3-Dioxygenase Genes from a Gram-Positive Polychlorinated Biphenyl Degrader,Rhodococcus jostiiRHA1

“…HA01 expressed no enzyme activity in E. coli. 14,19,21) The biphenyl dioxygenase genes bpdC1C2BA of Rhodococcus sp. strain M5 expressed slight activity in E. coli, 42) but no reports have described the details of the reason they were scarcely expressed.…”

“…13 ing difficulty in expressing enzyme activity of actinomycete enzyme genes other than BphA in E. coli. [16][17][18][19][20][21] In this study, expression of RHA1 BphA genes, bphAaAbAcAd was examined in detail to achieve functional expression of it in E. coli. We found that the lack of bphAaAbAcAd-coding enzyme activity in E. coli originates from an inability of RHA1 bphAc to express its catalytic activity in E. coli.…”