Comamonas testosteroni ⌬Comamonas (formerly Pseudomonas) testosteroni is a soil microorganism able to grow at the expense of testosterone, progesterone, and various bile acids as unique sources of carbon and energy. Complete assimilation of these substrates is achieved through a complex metabolic pathway involving many enzymatic steps of oxidation responsible for the breakdown of the steroid nucleus (9,25,29). The introduction of double bonds into ring A at the early stage of this catabolic process is critical since it initiates the molecule cleavage. Upon induction with testosterone, C. testosteroni synthesizes three different dehydrogenases able to promote the desaturation of ring A of 3-ketosteroids: one (Fig. 1). All three enzymes have properties specific to flavoproteins, but only ⌬ 4 (5ß)DH was purified and characterized as being a flavin adenine nucleotide (FAD)-dependent enzyme (10,30).In a previous work, we isolated and characterized the gene encoding ⌬ 1 DH from C. testosteroni ATCC 17410 (37). A Tn5-insertion mutant (named 06) defective in testosterone utilization was found to express very low levels of both ⌬ 1 -and ⌬ 4 (5␣)DH activities while producing other quantifiable steroid-modifying enzymes at parental levels. These results strongly suggested that the genes coding for ⌬ 1 DH and ⌬ 4 (5␣)DH belong to the same transcription unit. In the present study, we demonstrate that the two genes are adjacent on the chromosome of C. testosteroni. The putative ⌬ 4 (5␣)DH peptide deduced from the gene sequence showed weak homology with the ⌬ 1 DH protein except in the N terminus, where a highly conserved FAD-binding site is present. These findings indicate that both dehydrogenases, though having closely related enzymatic activities, are probably not derived from a common ancestor. MATERIALS AND METHODSBacterial strains, phages, and plasmids. Bacterial strains, phages, and plasmids used in this study are listed in Table 1.Media and growth conditions. Escherichia coli and Pseudomonas putida were grown routinely in LB broth (38) or on Mueller-Hinton agar plates (Pasteur Diagnostics, France). When necessary, the following antibiotics were added to growth media: ampicillin (50 g/ml), chloramphenicol (20 g/ml), and tetracycline (12.5 g/ml) for E. coli, and chloramphenicol (300 g/ml) for P. putida. C. testosteroni was grown in YE broth as described previously (37). E. coli XL-1 Blue, used for single-stranded DNA production, was cultured in nutrient broth composed of (per liter) Bacto Tryptone (35 g), Bacto Yeast Extract (20 g; Oxoid Ltd., Basingtoke, England), and NaCl (5 g) at pH 7.5. All bacterial cultures were incubated at 30ЊC with shaking.Biotransformation of steroids. Recombinant bacteria harboring DNA fragments from C. testosteroni were assayed for the expression of various enzyme activities of the steroid catabolic pathway (see below). Enzyme-catalyzed conversion of steroids into specific metabolites was demonstrated by thin-layer