Dibenzofuran (DF) is one of the dioxin carbon skeletal compounds used as a model to study the microbial degradation of dioxins. This study analyzed the transcriptional regulation of the DF dioxygenase genes dfdA1 to dfdA4 in the DF-utilizing actinomycetes Rhodococcus sp. strain YK2 and Terrabacter sp. strain YK3. An open reading frame designated dfdR was detected downstream of the dfdC genes. The C-terminal part of the DfdR amino acid sequence has high levels of similarity to several LuxR-type DNA binding helix-turn-helix domains, and a GAF domain sequence in the central part was detected by a domain search analysis. A derivative of YK2 with dfdR disrupted was not able to utilize DF and did not exhibit DF-dependent dfdA1 transcriptional induction ability, and these dysfunctions were compensated for by introduction of dfdR. Promoter analysis of dfdA1 in Rhodococcus strains indicated that activation of the dfdA1 promoter (P dfdA1 ) was dependent on dfdR and DF and not on a metabolite of the DF pathway. The cell extract of a Rhodococcus strain that heterologously expressed DfdR showed electrophoretic mobility shift (EMS) activity for the P dfdA1 DNA fragment in a DF-dependent manner. In addition, P dfdA1 activation and EMS activity were observed with hydrophobic aromatic compounds comprising two or more aromatic rings, suggesting that DfdR has broad effector molecule specificity for several hydrophobic aromatic compounds.Concern about environmental pollution caused by chlorinated dioxins is increasing as these compounds are unintentionally produced as by-products of several industrial processes. We studied the microbial degradation of dioxins by using dibenzofuran (DF) and dibenzo-p-dioxin as model compounds. To date, several DF-utilizing bacterial strains have been isolated, and the routes of DF and dibenzo-pdioxin catabolism have been analyzed (35,55). The gramnegative DF-utilizing bacterium Sphingomonas wittichii RW1 has been isolated (56) and analyzed in detail. High-GϩC-content gram-positive bacterial strains capable of utilizing DF as a carbon and energy source, such as Terrabacter sp. strain DBF63 (34), Terrabacter sp. strain YK3, and Rhodococcus sp. strain YK2 (21), have also been isolated. In addition, we recently reported isolation of a low-GϩC-content spore-forming DF-utilizing bacterium, Paenibacillus sp. strain YK5 (22).The initial pathway for bacterial degradation of DF and the enzymes catalyzing the reactions in DF-utilizing actinomycetes are shown in Fig. 1A. The first step of DF degradation is catalyzed by the Rieske nonheme iron oxygenases DfdA1 to DfdA4; these enzymes have regiospecificities for the oxygenation of DF at position 4,4a and are thus termed angular dioxygenases. The 4,4a-dihydroxylated reaction product (compound II) is unstable and spontaneously converted to 2,2Ј,3-trihydroxybiphenyl (compound III). Subsequent reactions are catalyzed by extradiol dioxygenase (DfdB) and hydrolase (DfdC) and produce salicylate (compound V) and 2-hydroxypenta-2,4-dienoate (compound VI). Previously, we r...