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. 2024 Mar 9;15:2161. doi: 10.1038/s41467-024-46449-8

Fig. 5. Inactivation of the methionine synthase MetH causes B12 toxicity in M. tuberculosis resulting in alleviating compensatory mutations in the metE B12-Riboswitch.

Fig. 5

a Expected phenotypes of the M. tuberculosis H37Rv ΔmetH knockout compared to the wild type strain in presence and absence of B12. b and c in vitro growth in liquid and in solid media of the M. tuberculosis H37Rv wild type strain (b) and its ΔmetH mutant (c) in absence or presence of exogenous B12 and/or L-methionine. d and f Survival rates from groups of SCID mice fed with normal diet (d) and B12-deficient diet (f), and inoculated by the intranasal route with M. tuberculosis H37Rv and the ΔmetH mutant. Each curve represents the pool from 2 independent experiments (n = 12). Statistical analysis was performed using Log-rank (Mantel-Cox) test. e and g Bacterial loads in the lungs of control (e) and B12 anemic (g) C57BL/6 mice after 24 h, or 4 weeks infected with M. tuberculosis H37Rv and its ΔmetH mutant. Data are mean ± SD of n ≥ 6 biological replicates per mice group. Statistical analysis was performed using unpaired t-test. p-values are as follows: ***0.001 > p > 0.0001; ns not significant, p ≥ 0.05. Results show that inactivation of the MetH isoform attenuates the mutant exclusively in animals with normal serum B12-levels (h) 5’ UTR sequences of the metE B12-Riboswitch. Negative numbers indicate nucleotides immediately upstream of the metE initiation codon. Bases showing mutations in M. tuberculosis H37Rv ΔmetH colonies grown with B12 are highlighted in red in the Sanger chromatograms. Aditional mutations are shown in Fig. S14. i Secondary structure prediction of the metE B12-riboswitch. Location of the identified mutations in the B12-resistant M. tuberculosis H37Rv ΔmetH colonies are indicated by red arrows. Invariant residues across ~200 B12-riboswitches are indicated in capital letters. Lowercase letters indicate M. tuberculosis specific residues. The conserved B12-box is highlighted in yellow. The metE 5’UTR region is shown in blue letters with the possible RBS underlined. The start of the metE CDS is shown with a purple arrow and the initiation codon is highlighted in red.