The unusual convergence of steroid catabolic pathways in .

, an opportunistic pathogen responsible for pulmonary infections, contains genes predicted to encode two steroid catabolic pathways: a cholesterol catabolic pathway similar to that of and a 4-androstenedione (4-AD) catabolic pathway. Consistent with this prediction, grew on both steroids. In contrast to , RHA1, and other Actinobacteria, the cholesterol and 4-AD catabolic gene clusters of the complex lack genes encoding HsaD, the -cleavage product (MCP) hydrolase. However, ATCC 19977 harbors two homologs elsewhere in its genome. Only one of the encoded enzymes detectably transformed steroid metabolites. Among tested substrates, HsaD and HsaD of had highest substrate specificities for MCPs with partially degraded side chains thioesterified with coenzyme A (/ = 1.9 × 10 and 5.7 × 10 mMs, respectively). Consistent with a dual role in cholesterol and 4-AD catabolism, HsaD also transformed nonthioesterified substrates efficiently, and a Δ mutant of grew on neither steroid. Interestingly, both steroids prevented growth of the mutant on acetate. The Δ mutant of excreted cholesterol metabolites with a fully degraded side chain, while the corresponding RHA1 mutant excreted metabolites with partially degraded side chains. Finally, the Δ mutant was not viable in macrophages. Overall, our data establish that the cholesterol and 4-AD catabolic pathways of are unique in that they converge upstream of where this occurs in characterized steroid-catabolizing bacteria. The data further indicate that cholesterol is a substrate for intracellular bacteria and that cholesterol-dependent toxicity is not strictly dependent on coenzyme A sequestration.