Figure 5

qRT-PCR validation of the expression of the selected genes in various growth conditions. Selected genes represent the main metabolic pathways discussed in this study. (A) Differential expression of the representative cholesterol breakdown (kstD, fadE30) and import (mce4, supA) genes. (B) Differential expression of the representative genes of Mtb central metabolism (prpR, icl1) and TAG synthesis gene tgs-1. (C) Differential expression of the representative genes of Mtb virulence lipid synthesis (fadD26, papA5) and the methylmalonyl pathway (MMP) (mutA, mutB). (D) Change in expression of PrpR operon genes (represented here by prpD) in response to vitamin B₁₂ in Mtb wild-type growing in standard 7H9/10% OADC medium. The expression of metE was used as an example of confirmed B₁₂-dependent regulation of gene expression. qRT-PCR analysis of espA (representative of the EspR regulon) expression in medium with or without B₁₂ supplementation confirms the discussed influence of vit. B₁₂ on the expression of ESX-1 associated genes. GLC Mtb growing on glycerol, CHO Mtb growing on cholesterol, CHO+B12 Mtb growing on cholesterol and vitamin B₁₂, ∆prpR CHO ∆prpR mutant growing on cholesterol, ∆prpR CHO+B12 ∆prpR mutant growing on cholesterol and vitamin B₁₂. Statistical significance: one, two and three stars denote p values ≤0.05, ≤0.01 and ≤0.001, respectively (Student’s t-test).