Quantification of N-acyl alanine methyl ester (NAME) production by Roseovarius tolerans EL-164 enables dose-dependant activity testing

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. Three setups were compared: spike-in of NAME, spike-in of AHL, spike-in of both metabolites. For each setup, biological triplicates were sampled at three different time points (tS1=26 h (NAME spike), tS2=49 h, tS3=53 h (AHL spike)). All cultures were sampled at all time points, irrespective whether or not they did receive the respective metabolite at that time point. However, not all sequencing was successful, resulting in a reduced number of replicates for some time points and conditions.