Chemical quantification of N-acyl alanine methyl ester (NAME) production and impact on temporal gene expression patterns in Roseovarius tolerans EL-164

• Published in: BMC microbiology Vol. 24; no. 1; pp. 489 - 13
• Main Authors: Leinberger, Janina, Koteska, Diana, Boldt, Judith, Petersen, Jörn, Shivaramu, Sahana, Tomasch, Jürgen, Schulz, Stefan, Brinkhoff, Thorsten
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 21.11.2024; BioMed Central; BMC
• Subjects: Acyl-Butyrolactones - metabolism; Alanine; Alanine - analogs & derivatives; Alanine - metabolism; Amino acids; Analysis; Antibiosis; Antibiotics; Antimicrobial activity; Bacteria; Biological activity; Biosynthesis; Cellular stress response; Chloride; Chromatography; Esters; Esters - metabolism; Firing pattern; Gene expression; Gene Expression Regulation, Bacterial - drug effects; Genes; Genetic aspects; Growth phases; Identification and classification; Investigations; Lactones; Metabolites; Names; Properties; Quantification; Rhodobacteraceae - genetics; Rhodobacteraceae - metabolism; Roseobacter; Secondary metabolites; Signaling; Solvents; Stationary phase; Transcriptomes; Germany; United States > US; Time course; Signaling; Secondary metabolites; Quantification; Differential gene expression; Transcriptomics; Growth phases; Antibiosis; Roseobacter
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/s12866-024-03624-7

Previous 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. To 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 (2.0-16.9 µM) and 5.3-86.4 µg L (15.0-244.3 nM), respectively. Co-quantification of the C14:1-AHL showed concentrations ranging between 17.5 and 58.7 mg L (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. Our 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.