Bacterial metabolism of steroids, from enzymes to ecosystems

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The current proposal is to continue a productive Discovery Grant research program. The long-term goal of the program remains the same, to discover and understand novel aspects of microbial metabolism, physiology and ecology, with a focus on steroid catabolism. I seek a broad understanding encompassing biochemical, genetic, evolutionary and ecological perspectives. The proposed research builds upon important advances supported by my previous Discovery Grant. In the past five years, we have elucidated a novel pathway for catabolism of cholate by Rhodococcus spp. We have analyzed over 8000 genomes, mapping the occurrence of various steroid catabolism pathways among microbial taxa. And, we have analyzed over 300 shotgun metagenomes, revealing the importance of bacterial steroid catabolism in many natural, engineered and host environments as well as identifying the capacity for steroid catabolism in a number of uncultured taxa. The metagenome analysis revealed the presence of bacteria in marine sponges with very divergent steroid degradation enzymes. We will further investigate these organisms using both culture-dependent and –independent approaches, focusing on novel enzymatic transformations of the diverse steroid structures found in sponges. We have also recently found that Rhodococcus jostii can grow on phosphatidylcholine-cholesterol vesicles, which model the natural state of these lipids in eukaryotic cell membranes. Interestingly, phosphatidylcholine is catabolized only when in this state. Therefore we will investigate the regulatory mechanisms and proteins involved in growth on the vesicles and uptake of the lipids. We have also recently found that R. jostii can both catabolize cholesterol esterified to fatty acids, which occur in eukaryotic cells, as well as produce and store cholesteryl esters within their own cells. We will investigate the enzymes involved in sterol ester catabolism and synthesis, the regulation of these processes and their ecological relevance to R. jostii and other bacteria. These investigations will employ diverse approaches, including metagenomics, genomics, transcriptomics, microbial isolation and culturing, molecular genetics, protein expression and bioinformatics. Applied goals of the research include the discovery of novel biocatalysts that can be employed for the synthesis of chemical products, environmental protection or therapeutics.