Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution

Maria Lluch-Senar,Jonas Korlach,Tyson A Clark,Kristi Spittle,Khai Luong,Verónica Lloréns-Rico,Eric Schadt,Stephen W Turner,Gang Fang,Luis Serrano,Javier Delgado

doi:10.1371/journal.pgen.1003191

Abstract

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N6-methyladenine (6 mA) and N4-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN7TAY-3′/3′-CTN7 ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.

Highlights

Among a few documented mechanisms, methylation of specific DNA sequences by DNA methyltransferases provides one way by which epigenetic inheritance can be orchestrated [1]
We analyzed the genomes of M. pneumoniae and M. genitalium for all the putative methyltransferase genes using comparative sequence analysis and our previous functional assignment [38]
M. pneumoniae Type I system includes a methyltransferase, a DNA specific recognition protein that brings the methyltransferase to the target DNA (HdsS, mpn343), and a restriction enzyme that cleaves unmethylated DNA (HdsR, mpn345)

Summary

Introduction

Among a few documented mechanisms, methylation of specific DNA sequences by DNA methyltransferases provides one way by which epigenetic inheritance can be orchestrated [1]. Phenomena involving inheritance of DNA methylation patterns are known in bacteria. These systems use DNA methylation patterns to pass on information regarding the phenotypic expression state of the mother cell to the daughter cells. Methylation can alter the DNA structure and affect the binding of regulatory protein(s) to its DNA target site, thereby controlling gene expression [17,18]. It has been shown that in E. coli, the expression of the Type IV secretion gene cluster is regulated by a non-stochastic epigenetic switch that depends on methylation of the Fur binding box [22]

Methods

Results

Discussion

Conclusion