Transposon sequencing reveals metabolic pathways essential for Mycobacterium tuberculosis infection
Fig 4
M. tuberculosis requires Phe synthesis for survival in vitro in the absence of amino acid supplementation and in mice.
(A) Phenylalanine biosynthesis in M. tuberculosis. Single arrows represent one-way reactions. Double arrows represent reversible reactions. Proteins predicted to catalyze each step are indicated. (B-C) Strains grown in 7H9 supplemented with 0.5% casamino acids were washed in PBS-T and diluted to OD600 = 0.01 in 7H9 or 7H9 supplemented with 0.5% casamino acids. Bacterial growth and survival were measured by (B) optical density at 600 nm and (C) serial dilutions and plating to recover viable CFU. (D-E) C57BL/6J mice were infected by aerosol with the indicated strains. Mice (n = 6) were euthanized at days 1, 7, 21, and 42 post-infection. Lung (D) and spleen (E) homogenates were serially diluted and plated to recover viable CFU. In (C-E), WT and pheA::Tn pMV-pheA were plated on 7H10 agar; pheA::Tn was plated on 7H10 agar with 0.5% casamino acids. Data represent the mean ± standard error of three biological replicates (B-C) or six animals (D-E). Asterisks indicate P-value < 0.05; n.d. indicates not detected (detection limit = 1 CFU).