Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution Maria Lluch-Senar 1,2. *, Khai Luong 3. , Vero ´ nica Llore ´ ns-Rico 1,2 , Javier Delgado 1,2 , Gang Fang 4 , Kristi Spittle 3 , Tyson A. Clark 3 , Eric Schadt 4 , Stephen W. Turner 3 , Jonas Korlach 3 , Luis Serrano 1,2,5 1 EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Barcelona, Spain, 2 Universitat Pompeu Fabra (UPF), Barcelona, Spain, 3 Pacific Biosciences, Menlo Park, California, United States of America, 4 Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, United States of America, 5 Institucio ´ Catalana de Recerca i Estudis Avanc ¸ats (ICREA), Barcelona, Spain 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 N 6 -methyladenine (6 mA) and N 4 -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 (59-CTAT-39), as well as a more complex Type I m6A sequence motif in M. pneumoniae (59-GAN 7 TAY-39/39- CTN 7 ATR-59). 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. Citation: Lluch-Senar M, Luong K, Llore ´ns-Rico V, Delgado J, Fang G, et al. (2013) Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution. PLoS Genet 9(1): e1003191. doi:10.1371/journal.pgen.1003191 Editor: Paul M. Richardson, Progentech, United States of America Received September 12, 2012; Accepted November 8, 2012; Published January 3, 2013 Copyright: ß 2013 Lluch-Senar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Institutes of Health grants 1RC2HG005618-01 (NHGRI) and 1RC2GM092602-01 (NIGMS). The LS group was supported by the European Research Council (ERC) advanced grant, the Fundacio ´ n Marcelino Botin, and the Spanish Ministry of Research and Innovation to the ICREA researcher LS. VL-R was funded by the Fundacio ´ n La Caixa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: KL, TAC, KS, SWT, and JK are full-time employees at PacificBiosciences, a company commercializing single-molecule, real-time nucleic acid sequencing technologies. * E-mail: maria.lluch@crg.es . These authors contributed equally to this work. 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]. For instance, in many eukaryotes, methylated cytosine residues at 59-CG-39 (CpG) sequences are recognized by methyl-CpG binding proteins that usually repress the transcription of local DNA regions [2–5]. In the bacterial world, methylation is most commonly associated with restriction-modification (R-M) systems that provide a defense mechanism against invading foreign genomes [6]. In addition, it is known that a variety of enzymes capable of methylating DNA at adenine [7] and cytosine [8,9] play functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, transposition and conjugal transfer of plasmids, and regulation of gene expression [7,10–16]. Phenomena involving inheritance of DNA methylation patterns are also 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]. Notably, most adhesion genes in Escherichia coli are regulated by DNA methylation patterns [19,20]. Little is known about how widespread heritable epigenetic control is in the bacterial world or the roles that epigenetic regulatory systems play in bacterial biology, including pathogenesis. For instance, it has been shown that DNA methylation in Streptococcus mutans up-regulates the expression of virulence factors like gbpC and bacteriocins [21]. It has also 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]. In some gram-positive and gram-negative species that have been studied, adenine methylation plays a critical role in regulating chromosome replication. Adenine is generally methyl- ated by members of the Dam family of methyltransferases, such as Dam in E. coli and DpnII in Streptococcus pneumoniae, that recognize the sequence motif 59-GATC-39 [23]. In these bacteria, the PLOS Genetics | www.plosgenetics.org 1 January 2013 | Volume 9 | Issue 1 | e1003191