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Description
Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen known for its repeated emergence of drug-resistance despite low mutation and replication rates. This contradiction begs the question: How does an organism considered especially static, genetically speaking, exhibit such rapid adaptation to drug pressure? One possibility is epigenetic variability: heritable changes independent from chromosomal alterations. In particular, the DNA base modification N6-methyl-adenine (DNAm) occurs heterogeneously between cells of other prokaryotes and drives phenotypic variability in genetically clonal populations. This work implemented single-molecule, real time (SMRT) sequencing to characterize the methylome of extensively drug-resistant M. tuberculosis and its degree of conservation across lineage and individual isolates. From these characterizations several hypotheses for how DNAm affects phenotype were put forth. Eighty-three mostly extensively drug resistant clinical isolates from five (four high-burden) countries were sequenced using Pacific Biosciences SMRT technology, de novo assembled, circularized, and their methylomes determined. A custom annotation of reference strain H37Rv with experimentally determined transcription start sites (TSSs) was transferred to the de novo assemblies. Analysis of DNAm in relation to TSSs and sigma factor consensus binding motifs (SFBMs) revealed 47 genes a common TSS-SFBM-DNAm configuration coincident with the -10 binding site of the putative promoter, a location likely to affect transcription. Oxidative stress response, Iron and Sulfur homeostasis, host lipid catabolism, and cell wall anabolism were starkly overrepresented in this gene set, suggesting a potential DNAm- mediated transcriptional program that affects these processes. Remarkably, primary reference strain H37Rv's methylome is dissimilar to most clinical strains'. In fact, bases different due to DNAm exceeds SNPs in most isolates but are largely unacknowledged in the literature. This prompts re-evaluation of how broadly applicable transcription initiation and DNA-binding in H37Rv studies are and compels greater focus on lab work with clinical strains, investigation into how environment affects DNAm, and how these DNAm changes influence phenotype.
