Figure 3.

MetM is essential for methionine-mediated PAS antagonism and uptake of methionine in M. bovis BCG. (A) Growth kinetics of M. bovis BCG metM::himar1 and M. bovis BCG wild type during PAS exposure when antagonistic metabolites are added. Growth was assessed by OD₆₀₀ readings every 2–3 days. (B) M. bovis BCG strains were grown to an OD₆₀₀ of ~0.5, washed three times to remove residual sulfate with sulfate-free Sautons medium, and resuspended in sulfate-free Sautons medium to a starting OD₆₀₀ of 0.01 and cells were starved for sulfur for 5 days. Following the exhaust period, sulfur-sources were added, and cells were incubated for 7 days to resume growth. The fold-change in growth was assessed as a ratio of the final OD₆₀₀ over the starting OD₆₀₀ following the exhaust period (final OD₆₀₀/starting OD₆₀₀). (C) Uptake of L-methionine-(methyl-d₃) was measured using LC-MS/MS. Samples that fell outside the quantifiable lower limit of the standard curve for L-methionine-(methyl-d₃) (25.00–0.39 μM) were labeled #, not quantifiable. (B,C) p-values of pairwise comparisons (denoted by bracketed lines) were calculated using the Student t-test. ^*p < 0.05, ^**p < 0.005, ns indicates no significant difference (p > 0.05). Error bars denote standard deviation and are representative of 3 separate experiments.