UBC Theses and Dissertations
Two novel transporters essential for the reassimilation of cholic acid metabolites excreted by Rhodococcus jostii RHA1 Swain, Kendra Lynn
In this study, I investigated two novel transporters associated with cholic acid catabolism in Rhodococcus jostii RHA1. Reverse-transcriptase quantitative-PCR indicated that an ABC transporter was upregulated 16.7-fold and an MFS transporter was upregulated 174-fold during the exponential phase of growth on cholic acid compared to pyruvate. With gene knockout analysis, I discovered that these transporters are required for the reassimilation of distinct cholic acid metabolites. The ABC transporter, encoded by the camABCD genes, was essential for uptake of 12-hydroxy-9-oxo-1,2,3,4,10,19,23,24-octanorcholan-5,22-dioic acid and 12-hydroxy-9-oxo-1,2,3,4,10,19,23,24-octanorchol-6-en-5,22-dioic acid. The MFS transporter, encoded by the camM gene, was essential for uptake of 3,7,12-trihydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oic acid. The uptake of these metabolites is necessary for maximal growth on cholic acid: the ΔcamB mutant, lacking the permease component of the ABC transporter, and the ΔcamM mutant, lacking the MFS transporter, only achieved 74% and 77%, respectively, of the final wild type protein yield. These metabolites differ from previously reported cholic acid metabolites from Proteobacteria in that they retain an isopropionyl side chain at the C17 position. This study is the first to demonstrate the function of putative cholic acid genes through targeted mutagenesis, as well as the first to provide evidence for the requirement for transporters involved in cholic acid metabolite uptake. This work highlights the importance and complexity of transport processes associated with bacterial catabolism and may contribute to industrial applications involving bacterial steroid transformation.
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