Fig. 3. The core B12-dependent transcriptome identifies a gene cluster regulated by B12 in the MTBC and establishes a link with methionine metabolism.

a Experimental procedure for RNA-seq analysis of differentially expressed (DE) genes in response to B12 supplementation of MTBC cultures. Venn diagrams show the resulting DE genes from two independent replicates of M. tuberculosis H37Rv (L4), M. tuberculosis GC1237 (L2), and the animal-adapted M. bovis AF2122. Those DE genes common to the three strains are indicated as the “core B12-regulon”, and consist on 7 downregulated genes in the presence of B12. b RNA-seq profiles showing downregulated gene expression of the gene cluster Rv1129c (prpR)-prpD-prpC-Rv1132-metE in M. tuberculosis H37Rv in response to exogenous B12 supplementation (light blue) relative to the experimental control without B12 (dark blue). Note that in order to visualize expression differences, each genetic section has been represented with its corresponding scale. Expression profiles of the remaining B12-dependent genes are indicated in Fig. S9. c qRT-PCR measures of Rv1129c (prpR), prpD and metE in M. tuberculosis H37Rv cultures grown with or without B12. Relative quantity refers to the differential expression of the selected genes in the presence of B12 in comparison with its expression in the absence of B12. Each gene was normalized against sigA expression in each sample. Graphs represents mean ± SD from three biological replicates. Expression of additional B12-dependent genes is shown in Fig S9. d Quantification of PrpD, PrpC and MetE protein levels of M. tuberculosis H37Rv both in presence and absence of B12 by MRM-MS. Bars represent the area under the curve for every transition from a specific peptide of each protein in both experimental conditions. Equivalent results were obtained for the rest of the analyzed peptides (Fig. S10) in two biological replicates of H37Rv and GC1237 strains of M. tuberculosis.