Figure 3. In vitro evolution of 18:2 resistance in lactobacilli.

(A) Five cultures of L. reuteri strain LR0 and five cultures of L. johnsonii strain LJ0 were passaged twice daily via a 100x dilution in liquid culture supplemented with 18:2. The 18:2 concentration was increased each week by 1 mg/ml from 5 to 10 mg/ml over a total of 6 weeks. (B) Disc diffusion (as in Figure 1) of L. reuteri and L. johnsonii starting strains LR0 and LJ0 and evolved populations LR2 and LJ4. Tested compounds: A. SBO, B. Saline, C. DMSO, D. 16:0, E. 18:0, F. 18:1, G. 18:2, H. 18:3. Growth curve of C) L. reuteri starting strain LR0, evolved isolate LR2-1 (from population LR2), (D) L. johnsonii starting strain LJ0, and evolved isolate LJ41072 (from population LJ4) in liquid medium with and without 18:2. Each point represents triplicate cultures and standard deviations are shown. See also Figure 3—figure supplements 1 and 2 and Supplementary file 2 to 6.

Figure 3—figure supplement 1. Lactobacillus strains, populations, and isolates involved in the in vitro evolution experiment.

Mouse derived strains LR0 (L. reuteri) and LJ0 (L. johnsonii) were inoculated into five cultures each. These cultures were passaged twice daily in media containing 18:2 for six weeks. The resulting populations for LR0 were LR1 to LR5 and for LJ0, LJ1 to LJ5. From population LR2, isolate LR2-1 was further analyzed as was isolate LJ41072 from LJ4.

Figure 3—figure supplement 2. Lactobacillus populations passaged in 18:2 have increased resistance to 18:2.

A) Disc diffusions for all L. reuteri and L. johnsonii populations and their ancestor strains with 18:2 and 18:3. (B) Individual L. reuteri fatty acid mutations in FabT and the hydrolase gene created in the fatty acid sensitive strain PTA 6475.