The bacterial uptake of steroids and their metabolites remains poorly understood. We investigated two transporters associated with cholate catabolism in Rhodococcus jostii RHA1. Reverse transcriptase quantitative-PCR indicated that an ATP-binding cassette (ABC) transporter and a major facilitator superfamily (MFS) transporter were upregulated 16.7-and 174-fold, respectively, during the exponential phase of growth on cholate compared to growth on pyruvate. Gene knockout analysis established that these transporters are required for the reassimilation of distinct metabolites that accumulate during growth on cholate. The ABC transporter, encoded by camABCD, was essential for uptake of 1␤(2=-propanoate)-3a␣-H-4␣(3؆(R)-hydroxy-3؆-propanoate)-7a␤-methylhexahydro-5-indanone and a desaturated analog. The MFS transporter, encoded by camM, was essential for uptake of 3,7(R),12(S)-trihydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oate. These metabolites differ from cholate metabolites reported to be excreted by proteobacteria in that they retain an isopropanoyl side chain at C-17. The uptake of these metabolites was necessary for maximal growth on cholate: a ⌬camB mutant lacking the permease component of the ABC transporter and a ⌬camM mutant lacking the MFS transporter grew to 74% and 77%, respectively, of the yield of the wild type. This study demonstrates for the first time the requirement for specific transporters for uptake of cholate metabolites and highlights the importance and complexity of transport processes associated with bacterial steroid catabolism.

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CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding interpretable loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, but no genome-scale guide libraries have been reported. We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

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