DNA Methylation Impacts Gene Expression and Ensures Hypoxic Survival of Mycobacterium tuberculosis

Scarlet S Shell,Erin G Prestwich,Sarah M Fortune,Seung-Hun Baek,Christopher M Sassetti,Rupal R Shah,Peter C Dedon,William R Bishai

doi:10.1371/journal.ppat.1003419

Abstract

DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N6-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces the expression of a number of genes. Each has a MamA site located at a conserved position relative to the sigma factor −10 binding site and transcriptional start site, suggesting that MamA modulates their expression through a shared, not locus-specific, mechanism. While strains lacking MamA grow normally in vitro, they are attenuated in hypoxic conditions, suggesting that methylation promotes survival in discrete host microenvironments. Interestingly, we demonstrate strikingly different patterns of DNA methyltransferase activity in different lineages of M. tuberculosis, which have been associated with preferences for distinct host environments and different disease courses in humans. Thus, MamA is the major functional adenine methyltransferase in M. tuberculosis strains of the Euro-American lineage while strains of the Beijing lineage harbor a point mutation that largely inactivates MamA but possess a second functional DNA methyltransferase. Our results indicate that MamA influences gene expression in M. tuberculosis and plays an important but strain-specific role in fitness during hypoxia.

Highlights

Mycobacterium tuberculosis is a pathogen of tremendous global significance, causing 9 million cases of tuberculosis annually and latently infecting up to a third of the world’s population [1]
In order to investigate the determinants of DNA methylation in the M. tuberculosis genome, we began by examining a site in the lppC gene that had been previously reported to be protected from restriction enzyme cleavage [33]
Consistent with the published data, we confirmed that this site was largely protected from cleavage by PvuII in M. tuberculosis strains from the Euro-American lineage and the vaccine strain M. bovis BCG (Figure 1, A and B), but was fully susceptible to PvuII in strain HN878, a member of the Beijing lineage of M. tuberculosis (Figure 1, B and C)

Summary

Introduction

Mycobacterium tuberculosis is a pathogen of tremendous global significance, causing 9 million cases of tuberculosis annually and latently infecting up to a third of the world’s population [1]. M. tuberculosis can persist for decades in the infected host. Over such timescales, the bacterium must tune gene expression patterns to match conditions in the host environment, including hypoxia, nutrient deprivation, and low pH, and maintain these adaptations over long periods of time. While eukaryotes use a variety of mechanisms to heritably ensure expression states, DNA methylation is the only known mechanism by which prokaryotes might achieve epigenetic inheritance. Both adenine and cytosine can be methylated in DNA, resulting in N6-methyladenine, N4-methylcytosine, and 5methylcytosine (accurately termed N6-methyl-29deoxyadenosine, N4-methyl-29deoxycytidine, and 5-methyl-29deoxycytidine, and abbreviated here as N6-MdA, N4-MdC, and 5-MdC, respectively). In prokaryotes N6-MdA is the best-characterized epigenetic regulator of gene expression [4,5,6,7,8,9]

Methods

Results

Conclusion