*2.2. Culture Extraction and LC-MS Analysis*

MK samples were extracted in 8 mL of 50% methanol in water. Samples were centrifuged (10 min at 16,100 g, 4 ◦C), and the supernatants were collected. These, as well as samples from liquid cultures, were filtered through 0.2 μm polyvinylidene fluoride filters (Chromacol, Welwyn Garden City, UK).

SM profiling was carried out through a 6540 Ultra High Definition (UHD) Accurate Quadrupole Time-of-Flight (Q-TOF) Liquid Chromatography tandem Mass-Spectrometry (LC-MS/MS) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a Dual Electrospray Ionization (ESI) source, coupled to a 1200 series Rapid Resolution High Performance Liquid Chromatography (HPLC) with a Diode Array Detector (DAD) system (all from Agilent Technologies). Samples (7 μL) were injected onto a Poroshell 120EC-C18 1.8 pm, 2.1 × 5 mm reverse phase analytical column (Agilent Technologies) at a constant temperature (35 ◦C). Mobile phases consisted of (A) water (Cromasolv® Plus, LC-MS-Sigma) and (B) acetonitrile (Cromasolv® Plus, LC-MS-Sigma) both acidified with 0.1% LC-MS grade formic acid. The analyses were carried out at a flow rate of 0.6 mL min−<sup>1</sup> with the following gradient: 0 min—5% B; 12 min—100% B; 15 min—100% B; 17 min—95% B; 20 min—95% B; 2 min post-time. The UV spectra were collected by DAD every 0.4 s from 190 to 750 nm with a resolution of 2 nm. The source conditions for electrospray ionization were the following: nitrogen gas temperature was 350 ◦C with a drying gas flow rate of 11 L min−<sup>1</sup> and a nebulizer pressure of 45 psig. The fragmentor voltage was 180 V and skimmer voltage 45 V. The range acquisition of TOF spectra was from 50 to 1600 m/z with an acquisition rate value of 3 spectra s−1. Data were collected in positive ion mode. The real-time lock mass correction was performed by using two reference mass solutions including purine (C5H4N4 at m/z 121.050873, 10 μmol L−1) and hexakis (1H,1H,3H-tetrafluoropentoxy)-phosphazene (C18H18O6N3P3F24 at m/z 922.009798, 2 μmol L<sup>−</sup>1). These solutions were purchased from Agilent Technologies and injected into MS by an isocratic pump at a constant flow rate (0.06 mL min−1). Solvents were LC–MS grade, and all other chemicals were analytical grade. All were from Sigma-Aldrich (Steinheim, Germany) unless otherwise stated.

Mass spectra were analyzed through the MassHunter Qualitative Analysis Software B.06.00 (Agilent Technologies), and then through the MassProfile Professional Software (Agilent Technologies) to compute the annotation and statistical analyses. LC-MS data were compared to known compounds included in an in-house database, as previously described [37,38].

Graphical representations were performed using ClustVis, a web-based multivariate data analysis tool. The principal component analysis (PCA) was performed using the Singular Value Decomposition (SVD) with imputation algorithm in ClustVis online tool. Data on SMs were summarized using the heatmap function in ClustVis tool with row centered and unit variance scaling applied. The hierarchical clustering was obtained using correlation method. Compounds with normalized intensity values >2 were used to analyze common and unique entities in the different treatments by Venn diagrams with the online tool jvenn (http://jvenn.toulouse.inra.fr/app/index.html).
