Finding novel oxidases specific to hydrocortisone in bacterial genomes
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Abstract
Steroids play various roles in the body, such as regulating metabolism, immune response, and maintaining homeostasis. Cortisol, a hormone in the glucocorticoid subclass of steroids, is crucial for stress response and cognitive functions, with its levels indicating adrenal health. Monitoring cortisol levels within the human body is beneficial to health management. Current urine and saliva assays for cortisol measurement are not real-time and require strict testing conditions. More innovative and practical methods that simplify the process of monitoring cortisol levels, making it accessible and manageable for individuals, whether in a clinical setting or at home, are in need to be developed.
Some bacteria have evolved enzymes that exhibit high specificity and selectivity for particular molecules. For instance, Pseudomonas putida contains the nicA2 gene, which encodes a nicotine oxidase that specifically reacts to nicotine. This suggests that bacteria could serve as valuable resources in the search for novel oxidases to cortisol and other hormones of related structure playing relevant roles in humans. Accordingly, we have observed that the bacterium Stenotrophomonas acidaminiphila isolated from a wastewater can use hydrocortisone (HCRT) as a single carbon source, indicating that S. acidaminiphila likely codes for an enzyme that binds HCRT specifically for its utilization.
In our lab, we built and utilized a reporter system that generates fluorescence via the hydrogen peroxide produced by enzymes with oxidase activity upon the addition of substrates of interest. This system, combined with a high-throughput screening of fluorescent cells was successfully deployed to identify the nicotine oxidase nicA2 gene from a genomic DNA library of P. putida. Therefore, it is plausible to follow this approach to identify putative hydrocortisone specific genes in S. acidaminiphila or other hydrocortisone utilizing bacteria.
In the present work I describe the optimization of the functional assay to identify genes coding for hydrocortisone oxidases, from validation of gene enrichment of well-known nicotine oxidase through several rounds of genomic screening, to the test of different HCRT and fluorogenic substrate amplex ultra red (AUR) concentrations, as well as incubation time to identify the assay conditions that maximize the fluorescence of a positive control towards HCRT. Assay conditions found allow between 101 and 102 fold more fluorescence of the positive control than a pooled DNA library of four bacterial genomes. This is the highest fluorescence ever recorded for a positive control to HCRT ever recorded with our assay, increasing chances of finding novel genes responsive to HCRT.