Project description: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.
Project description:N-acyl amino acid methyl esters (NAMEs), which are structurally similar to the signalling compounds N-acyl homoserine lactones (AHLs), have been identified in culture extracts of Roseovarius tolerans EL-164. However, previous studies have shown that NAMEs do not participate in AHL-mediated signalling and thus their ecological role remains unclear. To enable dose-dependent bioactivity-testing of NAMEs, we have established a quantification method for NAMEs. The concentrations determined for the major NAMEs produced by EL 164, C16:1- and C17:1-NAME, ranged between 0.7-5.7 mg L-1 and 5.3-86.4 µg L-1, respectively. We observed opposing production patterns for NAMEs and AHLs, with a continuously increasing NAME production during exponential growth and an accumulation in the stationary phase. We further conducted a spike-in experiment, using the previously determined metabolite concentrations. By comparing the transcriptomes of pre- and post-NAME or AHL spikes, we identified distinct impacts on gene expression patterns. Different genetic neighbourhoods were significantly up- or downregulated in NAME and AHL-spiked cultures. Yet no synergetic effect was observed. These findings exemplify the broad application range of dose-dependent testing and highlight the different biological activities of NAMEs and AHLs.
Project description:Transcriptional profiling of an El Tor biotype crp mutant The virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991) and an isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein were grown in LB medium to optical density at 600 nm of 1.5. The cultures were chilled in ice, cells quickly collected by centrifugation and total RNA imediately extracted. RNAwas extracted and purified using the Trizol plus RNA purification system (Invitrogen) followed RNEasy miniElute cleanup (Qiagen). RNA samples were conserved at - 80 C and used within a week. Keywords: Genetic modification
