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:BackgroundPrevious studies have identified structurally diverse N-acyl amino acid methyl esters (NAMEs) in culture extracts of Roseovarius tolerans EL-164 (Roseobacteraceae). NAMEs are structural analogues of the common signaling compounds N-acyl homoserine lactones (AHLs), but do not participate in AHL-mediated signaling. NAMEs show minor antialgal and antimicrobial activity, but whether this activity serves as the primary ecological role remains unclear.ResultsTo enable dose-dependent bioactivity-testing, we have established a chromatographic method for quantification of NAMEs in bacterial culture extracts. The concentrations determined for the two major NAMEs produced by EL-164, C16:1-NAME and C17:1-NAME, ranged between 0.685 and 5.731 mg L- 1 (2.0-16.9 µM) and 5.3-86.4 µg L- 1 (15.0-244.3 nM), respectively. Co-quantification of the C14:1-AHL showed concentrations ranging between 17.5 and 58.7 mg L- 1 (56.6-189.7 µM). We observed distinct production patterns for NAMEs and AHLs, with a continuous NAME production during the entire incubation period. We conducted a spike-in experiment, using the determined metabolite concentrations. By comparing the transcriptomes of pre- and post-metabolite-spikes, we identified three clusters of differentially expressed genes with distinct temporal expression patterns. Expression levels of stress response genes differed between NAME- and AHL-spiked EL-164 cultures in the stationary phase.ConclusionsOur findings support previous studies suggesting an ecological role for C16:1-NAME as antibiotic, by proving that NAME concentrations in batch cultures were higher than the minimal inhibitory concentrations against Maribacter sp. 62 - 1 (Flavobacteriia) and Skeletonema costatum CCMP 1332 (Coscinodiscophyceae) reported in the literature. Our study further exemplified the broad application range of dose-dependent testing and highlighted the different biological activities of NAMEs and AHLs.
