The 17β-estradiol (E2)-degrading bacterium Rhodococcus sp.RCBS9 previously showed remarkable resistance to the combined stresses of low temperature and E2. In this study, physiological experiments and transcriptomic analysis were performed to investigate the mechanisms underlying the strain's low-temperature adaptation and briefly analyze how it maintains its ability to degrade E2 at low temperature. The results showed that the strain's signal transduction functions, adaptive changes in cell membrane and cell wall structure, gene repair functions, and synthesis of antioxidants and compatible solutes are key to its ability to adapt to low temperature. In addition, its stress proteins in response to low temperature were not typical cold shock proteins, but rather universal stress proteins (USPs) and heat shock proteins (HSPs), among others. The strain also upregulated biofilm production, transporter proteins for carbon source uptake, and proteins for fatty acid degradation to ensure energy generation. The strain's multiple stress responses work synergistically to resist low-temperature stress, ensuring its adaptability to low-temperature environments and ability to degrade E2. Finally, six genes related to survival at low temperature (identified in the transcriptome analysis) were expressed in E. coli BL21, and they were found to contribute to recombinant E. coli growth at low temperature.
The molecular mechanism by which microorganisms degrade the endocrine-disrupting contaminant 17β-estradiol (E2) at low temperatures remains largely unclear, with only limited studies available. Here, we investigated the adaptive strategy of the cryotolerant bacterium Rhodococcus sp . RCBS9 for sustaining E2 degradation at 10 °C, a temperature at which efficient estrogen degradation has rarely been reported. Using time-resolved multi-omics and phenotypic assays, we characterized a dynamic, staged adaptation process involving changes in oxidative stress defense, membrane composition, transcription factors, signaling factors, and stress proteins. We uncovered three major novelties. First, RCBS9 employs a distinct degradation pathway that first cleaves the saturated sterol rings, in contrast to the canonical 4,5-seco pathway that initiates with aromatic ring A cleavage. Second, we identified and functionally validated a cold-tolerant short-chain dehydrogenase/reductase (SDR) encoded by gene RQCS_RS03565 that converts E2 to estrone (E1), as confirmed by heterologous expression and site-directed mutagenesis. Third, subsequent sterol ring cleavage proceeds via a suite of oxidoreductases and hydrolases. The resulting aromatic intermediates are then mineralized through the β-ketoadipate pathway, as suggested by the coordinated upregulation of intradiol dioxygenase and carboxymuconolactone decarboxylase. Additionally, trace iron (50 μM) promoted E2 degradation and reduced E1 accumulation, partly attributable to enhanced metabolic activity and upregulation of iron-requiring enzyme genes. Overall, this work provides fundamental insights into a novel estrogen biodegradation logic under cold stress and proposes a practical iron-mediated strategy for low-temperature bioremediation.
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