Effect of myricetin treatment on Roseovarius tolerans EL-164 during exponential and early stationary phase growth.

Discovery of cryptic natural products can be expedited by the use of high throughput elicitor screening (HiTES). In this study, we discover that catechol-containing flavonoids elicit the production of a new group of ceramide lipids in the marine bacterium Roseovarius tolerans EL-164. To investigate the mechanism of elicitation of myricetin, a model catechol flavonoid, we used RNA-seq to determine the molecular response. The transcriptomic dataset illustrated that iron-starvation genes, as well as the import and catabolism of carbon sources, specifically branched chain amino acids, were upregulated upon myricetin treatment. Simultaneously, complex IV of the electron transport chain was universally downregulated as were oxidative stress responses. Together, along with further follow up studies, we were able to support the model that myricetin reduces the bioavailability of iron and reduced oxidative phosphorylation and the proton motive force. Our data suggests that the hampering of the proton motive force is what initiates the upregulation of the ceramide lipids. Overall design: R. tolerans starter cultures were used to inoculate 25 mL of ½ YTSS liquid media in 125 mL flasks at a starting OD600 of 0.01 containing either 12.5 or 0 µM myricetin (1% DMSO for all) and grown at 30 ºC and 40 RPM. Growth curves were monitored by OD600 and 3 mL samples were collected in Rnase-free centrifuge tubes when cultures reached an OD600 of ~0.3 and ~0.75. This was completed in biological triplicate. The samples were centrifuged, and the pellets were resuspended in 1 mL of RNAlaterTM stabilization solution (Thermo) and stored at 4 ºC for 24 hours. To isolate total RNA for RNA-seq, the samples were centrifuged, the RNAlaterTM solution was discarded, and total RNA was isolated using the Rneasy kit (QIAGEN). Any contaminating genomic DNA was removed by treating the total RNA with Dnase (TURBO Dnase Kit), per manufacturer's instructions. RNA integrity was confirmed through gel electrophoresis