Figure 6. Metabolomics analysis shows differences in the utilization of pollen-derived glycosides across the four Lactobacillus species.

(A) Volcano plots displaying ions with significant fold changes (FC) for each of the four species after 16 hr of growth in pollen extract versus glucose. Each dot corresponds to an ion in the untargeted metabolomics dataset. Different colors represent ions that significantly change over time in one, two, three, or four species. Dashed black lines represent the significance thresholds: p-value < 0.01 and log2FC < −1 or > 1. (B) Culture wells of the four species grown in cfMRS + 0.05% rutin after 16 hr of incubation. The yellow precipitate is only visible for Lmel. (C) Rutin and quercetin detection in spent medium of Lmel and Lkul grown in cfMRS + 0.05% rutin after 16 hr of incubation (n=5). (D) Changes in key metabolites during growth measured by GC-MS (n=5). Log2FC relative to time point 0 is plotted. Time is reported in hours. * Indicates metabolites whose identity was confirmed using analytical standards. For m/z values see Supplementary file 10.

Figure 6—figure supplement 1. PCA in vitro metabolomics.

Principal component analysis (PCA) of the metabolome profile of each species based on the log2FC values calculated between the two time-points for each ion. The larger the distance between species on the PCA axes, the more they differ in their metabolome profiles. The arrows, that is the environmental vectors, point in the direction of the maximum correlation with the environmental variable, that is the ions. The ions on the tip of the longest arrows are the ones that explain the most of the distribution of the data within the PCA. Only the top 24 ions explaining the data distribution are displayed.

Figure 6—figure supplement 2. Definition of pollen-derived ions.

Volcano plot displaying R2 values obtained from the pollen dilution series regression lines and the log2FC calculated between undiluted pollen extract and water. The lines represent the thresholds that we set to define an ion as pollen-derived: log2FC > two and R2 > 0.75. Within the light green area are included the ions that we consider pollen-derived (n = 406).

Figure 6—figure supplement 3. Untargeted metabolomics: key metabolites discussed in the main text.

In vitro metabolomics of spent medium of the four species grown in cfMRS + PE for 16 hr. The log2FC was obtained comparing the ion intensities at the end and at the beginning of the experiment.

Figure 6—figure supplement 4. GC-MS detection of key metabolites over time.

Log2FC relative to T0 is plotted. Time is reported in hours. For m/z values see Supplementary file 10.

Figure 6—figure supplement 5. Logistic regression growth curve of the four species.

Growth-curve data were obtained for the four species at the four time-points included in the second metabolomics experiment (growth in presence of pollen extract) by qPCR (copy number) and fitted to a standard form of the logistic equation. Each point represents one replicate (n=5).