The methylomes of six bacteria

Iain A Murray,Brian P Anton,Alexey Fomenkov,Stephen W Turner,Khai Luong,Richard J Roberts,Richard D Morgan,Jonas Korlach,Tyson A Clark,Matthew Boitano

doi:10.1093/nar/gks891

Iain A Murray, Brian P Anton + Show 8 more

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https://doi.org/10.1093/nar/gks891

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Abstract

Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and C. jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases, it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTase genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. Two of these proved active. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT® sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active but also revealing their recognition sequences.

Highlights

We are becoming accustomed to the ever-increasing speed and reduced cost with which DNA can be sequenced
We have known for a long time about the epigenetic role of 5-methylcytosine (m5C), sometimes called the fifth base, and more recently it has been found that 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are present [1,2,3,4]
G. metallireducens GS-15, C. salexigens and V. breoganii 1C-10 had never been tested for active MTases previously, while three other strains, B. cereus ATCC 10987, C. jejuni subsp. jejuni 81-176 and C. jejuni NCTC 11168 were all known to contain several active MTases [25,26,27]

Summary

Introduction

We are becoming accustomed to the ever-increasing speed and reduced cost with which DNA can be sequenced. What is often lost in this frenzy of sequencing is the fact that DNA consists of more than just four bases. We have known for a long time about the epigenetic role of 5-methylcytosine (m5C), sometimes called the fifth base, and more recently it has been found that 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are present [1,2,3,4]. Two more modified bases, N6-methyladenine (m6A) and N4-methylcytosine (m4C), are common in bacterial genomes, where they function as components of restriction–modification (RM) systems [5]. Until recently, these have usually been ignored because of the lack of simple methods to determine their locations. With the advent of single-molecule, real-time (SMRT) sequencing [6,7,8], it has suddenly become possible to detect these modified bases as a part of the routine sequencing procedure

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