A qualitative untargeted metabolomics approach was used to identify candidate small molecules responsible for motility inhibition in the “sibling rivalry” inhibition zone between two <i>Marinobacter algicola</i> colonies in collaboration with the Carrano lab within the Chemistry department. Using artificial sea water media plates solidified with 1.5% agar, two sibling colonies were plated 1-3 cm equidistant from a center line and grown in the dark until an inhibition zone formed. Then, the agar in between the two colonies, as well as the agar on the other side of each colony from the inhibition zone, were excised and separately centrifuged to extract all the liquid from the agar. The supernatant was then sterile filtered, pH adjusted to 2.5, then extracted with ethyl acetate. The ethyl acetate layers were then dried. All samples were reconstituted with 1mL of 1:1 acetonitrile:water prior to analysis on the liquid chromatograph-mass spectrometer (LCMS) system. Reverse phase chromatography (RP) in positive mode was used in combination with hydrophilic interaction liquid chromatography (HILIC) in negative mode to analyze the samples. We primarily relied on the bioinformatics platforms XCMS Online for spectral alignment, feature detection and statistical analysis, while the Global Natural Products Social Molecular Networking (GNPS) was employed for metabolite identification. Statistically significant features on XCMS Online were filtered such that only those with a fold change ≥5, p-value ≤0.05, and maximum intensity ≥10000 were used for further compound identification, yielding 296 features upregulated in the inhibition zone to parse. Using MS2 fragmentation against various databases followed by standard confirmation, we verified the following compounds: salicylic acid, 2-isopropylmalic acid, dethiobiotin, guanine, hypoxanthine and D-pantethine. Preliminary motility assays in Carrano’s lab have shown salicylic acid as a promising candidate. Quantitative analysis has confirmed highest concentrations of salicylic acid in the inhibition zone at biologically active levels. We are now working on identifying how salicylic acid is biosynthesized in the bacterium.

LCMS analysis of secondary metabolites involved in formation of “sibling rivalry” zone in <i>Marinobacter algicola</i>

ACS Spring 2022

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LCMS analysis of secondary metabolites involved in formation of “sibling rivalry” zone in Marinobacter algicola