The spectral width in the carbon dimension was 170 p.p.m. and 180 p.p.m., respectively. All spectra were processed and analyzed using Bruker’s topspin

(v3.0) software. Usually, zero-filling was applied to double the number of real points in each dimension. Chemical shifts were referenced to the HDO resonance at 4.7 p.p.m. Chemical shift assignments for 13C were determined indirectly from HSQC and HMBC spectra. Pseudomonas sp. strain Chol1 was subjected to random transposon mutagenesis Y-27632 by insertion of the transposon mini-Tn5 Km1 and screened for transposon mutants showing altered growth with cholate as described previously (Birkenmaier et al., 2007). One mutant, strain G12, was analyzed further. Strain G12 could not grow with cholate as the sole substrate, but it could grow with succinate in the presence of cholate. HPLC analysis of supernatants from these cultures revealed that strain G12 did not transform cholate at all. We then checked this website whether strain G12 could grow with intermediates of cholate degradation. With supernatants containing DHADD (VIII), strain G12 could grow after a long lag phase. Notably, cells of strain G12 induced for growth with DHADD were also induced for cholate transformation during growth with succinate in

the presence of cholate. HPLC analysis revealed that cholate was transformed into several compounds with an absorption maximum at 244 nm, which is indicative of steroids with a 3-keto-1,4-diene structure of the A-ring (Philipp et al., 2006). In the next step, we identified the gene in strain G12, in which the mini-Tn5 Km1 had been inserted. The transposon Cyclooxygenase (COX) was inserted into an

ORF of 1212 bp at bp 333. The predicted protein had 403 amino acids and showed high identity to nonspecific lipid transfer proteins from various bacteria. Among these were two bacteria, for which growth with cholate had been demonstrated, namely Pseudoalteromonas haloplanktis strain TAC125 (Birkenmaier et al., 2007) and Comamonas testosteroni strain KF-1 (Rösch et al., 2008). The nonspecific lipid transfer proteins from strains TAC125 and KF-1 showed 80% and 68% identity, respectively, to the gene product from strain Chol1 (Fig. 2). This gene was named skt (for steroid β-ketothiolase) for reasons that will be described below. To investigate the function of skt for cholate degradation further, we decided to construct a defined mutant of this gene by subjecting strain Chol1 to insertional mutagenesis with the suicide vector pKnockoutG. The resulting strain Chol1-KO[skt] could not grow with cholate; growth with cholate was restored when an intact copy of skt was provided in trans on the vector pBBR1MCS-5 (Fig. 3a). This complementation clearly showed that the phenotype of this mutant was caused by the inactivation of skt. Strain Chol1-KO[skt] could grow with succinate in the presence of cholate (Fig. 3b).