Departamento de Biología Molecular (Área de Bioquímica y Biología Molecular), Facultad de Veterinaria, Universidad de León, León, Spain.
Methods Mol Biol. 2023;2704:51-83. doi: 10.1007/978-1-0716-3385-4_4.
The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and/or other steroids (e.g., testosterone and 4-androsten-3,17-dione) under aerobic conditions through the 9,10-seco pathway. From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemically defined media containing some cyclopentane-perhydrophenanthrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the route involved in bile acids and androgens, the 9,10-seco pathway in this bacterium. In this manuscript, we describe the most relevant methods used in our lab for the identification of the chromosomal location and nucleotide sequence of the catabolic genes (or gene clusters) encoding the enzymes of this pathway, and the tools useful to establish the role of some of the enzymes that participate in this route.
从不同生境中分离得到的微生物种的研究，使得人们能够鉴定和描述出能够在有氧条件下通过 9,10-裂环途径同化胆汁酸和/或其他甾体（如睾酮和 4-雄烯-3,17-二酮）的细菌。从土壤样品中，我们分离到了几株属于假单胞菌属的菌株，它们能够在含有某些环戊烷-全氢菲衍生物作为碳源的化学定义培养基中高效生长。对其中一种细菌（P. putida DOC21）进行的遗传和生化研究，使得能够鉴定出与胆汁酸和雄激素途径相关的基因和酶，即该细菌中的 9,10-裂环途径。在本文中，我们描述了我们实验室中用于鉴定编码该途径中酶的代谢基因（或基因簇）的染色体位置和核苷酸序列的最相关方法，以及用于确定参与该途径的一些酶的作用的工具。
