Extended Data Fig. 5: The amoebae-specific growth defect of the Δbio mutant strain is not medium-dependent.
To rule out prior culturing of A. castellanii in PYG medium and H. vermiformis and N. gruberi in PYNFH medium as a source of the amoebae-specific requirement for the biotin biosynthetic genes, growth of the Δbio mutant in A. castellanii pre-cultured in PYNFH was compared to A. castellanii pre-cultured in PYG. Under both conditions, the Δbio mutant show a growth defect. While this does not rule out trace amounts of biotin in PYNFH being sufficient for L. pneumophila growth in H. vermiformis and N. gruberi, this result demonstrates that in the same medium, the intracellular levels of biotin in A. castellanii are sufficiently lower than in N. gruberi or H. vermiformis, defining distinct differences in the availability of biotin between these amoebal hosts. Data are the mean ± standard deviation of 2 biological replicates, each generated from 3 technical replicates; *P<0.01, 1-way ANOVA, relative to the WT strain (Source Data for Extended Data Fig. 5).
