The complex methylome of the human gastric pathogen Helicobacter pylori

Juliane Krebes 1 2 Richard D. Morgan 0 Boyke Bunk 1 4 Cathrin Spro er 1 4 Khai Luong 3 Raphael Parusel 2 Brian P. Anton 0 Christoph Ko nig 3 Christine Josenhans 1 2 Jo rg Overmann 1 4 Richard J. Roberts 0 Jonas Korlach 3 Sebastian Suerbaum 1 2 0 New England Biolabs, 240 County Road, Ipswich, MA 01938, USA 1 German Center for Infection Research , Hannover-Braunschweig Site, Carl- Neuberg-Strae 1, 30625 Hannover, Germany 2 Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School , Carl-Neuberg-Strae 1, 30625 Hannover, Germany 3 Pacific Biosciences , 1380 Willow Road, Menlo Park, CA 94025, USA 4 Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures , Inhoffenstrae 7B, 38124 Braunschweig, Germany - The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT ) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction. INTRODUCTION The Gram-negative human pathogen, Helicobacter pylori, chronically infects more than half of the world population. Helicobacter pylori infection induces inflammation of the gastric mucosa, which can give rise to sequelae, such as peptic ulcer disease and gastric cancer (1). Helicobacter pylori is the bacterial pathogen with the highest genetic diversity and variability (24), which is believed to contribute to lifelong persistence by enabling adaptation to its host (2,3). In addition to a high mutation rate (5), recombination between different H. pylori strains during mixed infections with multiple strains within one stomach is the major driving force of allelic diversification (68). The naturally competent H. pylori differs from other bacteria by integrating unusually short fragments of DNA into its chromosome after natural transformation (9). The reasons for the small sizes of imports are largely unknown, but differences of the genomic content of active restriction-modification (R-M) systems have been suggested to limit recombination between H. pylori strains (1012). R-M systems are widely distributed among bacteria and are found in >90% of the analyzed genomes (13). Bacterial R-M systems were initially described as a defence mechanism against bacteriophage infection (14,15). They comprise two enzymatic activities: (i) a methyltransferase (MTase) activity that catalyzes the addition of a methyl group from the donor S-adenosyl methionine (SAM) to adenine or cytosine, and (ii) a restriction endonuclease (REase) activity that cleaves internal phosphodiester bonds of the DNA backbone. Both enzyme activities of the same system (cognate enzymes) recognize the same specific nucleotide sequence (recognition site), and methylation of the recognition site prevents restriction. The three major groups of R-M systems are classified as Type I, II and III, according to their subunit composition, cofactor requirements, structure of their recognition sequence and mode of action [for detailed reviews see (16,17)]. Type I systems are the most complex and form a heteropentamer (HsdR2M2S) that exerts three functions: restriction (HsdR), modification (HsdM) and specificity (HsdS). This complex works both as REase and MTase, but HsdM2S alone is sufficient for methylation. Sequence specificity of HsdR and HsdM is achieved by HsdS, which is typically composed of two target recognition domains (TRDs) mediating sequence recognition on both DNA strands. The simplest systems are the Type IIP R-M systems, which consist of two separate polypeptides (REase, MTase) that act independently of each other. Type III systems are also encoded by two genes (mod and res). While the Mod subunit alone achieves DNA modification, both subunits are required for restriction. In contrast to typical Type II MTases, which usually methylate 48 bp palindromic sites on both DNA strands, Mod catalyzes hemi-methylation of the DNA at 46 bp asymmetric recognition sites. More recently, a fourth class of R-M systems has been added. Type IV systems are encoded by one or two genes that represent methyl-dependent REases (18). Adenine and cytosine are the only bases known to be enzymatically methylated. In bacteria, three types of methylation, N6-methyladenine (m6A), N4-methylcytosine (m4C) and 5-methylcytosine (m5C) have been detected. While Type I and Type III R-M systems only methylate adenine, all three types of methylation have been reported to be catalyzed by Type II MTases (17). Helicobacter pylori genomes encode an unusually high number of R-M systems (13,1921). The two first H. pylori strains whose genomes were sequenced are 26695 (19) and J99 (21), and their strongly different complements of R-M systems have been analyzed in some detail. The two strains have been proposed to encode members of all four types of R-M systems. While several studies have addressed the activity of Type II MTases (2224), only one Type III MTase of H. pylori 26695 has been functionally characterized so far (25). Apart from that, Type I and Type III R-M systems of H. pylori were mostly uncharacterized and their specificity unknown. An overview about known and predicted R-M genes for many H. pylori strains can be found in the REBASE database [http://rebase.neb.com/rebase/rebase.html, (13)]. It has recently been reported that DNA methylation can be reliably detected at single-base resolution by Single Molecule Real-Time (SMRT ) sequencing technology, which enables the genome-wide detection of m6A, m4C and m5C methylation (26,27). In this next-generation sequencing technology, genome sequencing is achieved by monitoring the action of an engineered phi29-based DNA polymerase, which catalyzes the incorporation of fluorescently labeled nucleotides. Besides the primary sequence, (...truncated)