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. 2012 Dec;40(22):11450-62.
doi: 10.1093/nar/gks891. Epub 2012 Oct 2.

The methylomes of six bacteria

Iain A Murray  1 , Tyson A Clark, Richard D Morgan, Matthew Boitano, Brian P Anton, Khai Luong, Alexey Fomenkov, Stephen W Turner, Jonas Korlach, Richard J Roberts
Affiliations

The methylomes of six bacteria

Iain A Murray et al. Nucleic Acids Res. 2012 Dec.

Erratum in

  • Nucleic Acids Res. 2014 Apr;42(6):4140

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 N(6)-methyladenine ((m6)A) and N(4)-methylcytosine ((m4)C) 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 ((m5)C) recognition motifs from the SMRT® sequencing data because this modification produces weaker signals using current methods. However, all predicted (m6)A and (m4)C 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.

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Figures

Figure 1.
Figure 1.
Methylome determination of G. metallireducens GS-15. (a) Example trace of kinetic variation, showing three instances of methylated sequence regions. (b) Scatter plot of sequencing coverage and kinetic score for all genomic positions. The colors indicate the bases as shown in the upper left of the panel. The cutoff for detected genomic positions is indicated by the dashed line. (c) MTase specificities determined from the genomic positions detected as methylated. They are highlighted as gray boxes in the example trace (a). (d) Summary of detected methylated positions across the genome.
Figure 2.
Figure 2.
Methylome determination of C. salexigens. (a and b) Example traces of kinetic variation, showing two instances of methylated positions. (c) MTase specificities determined from the genomic positions detected as methylated. (d) Summary of detected methylated positions across the genome.
Figure 3.
Figure 3.
Methylome determination of V. breoganii 1C-10. (a–c) Example traces of kinetic variation, showing instances of the detected methylated motifs. (d) MTase specificities determined from the genomic positions detected as methylated. (e) Summary of detected methylated positions across the genome.
Figure 4.
Figure 4.
Methylome determination of C. jejuni 81-176. (a–e) Example traces of kinetic variation, showing instances of the detected methylated motifs. (f) MTase specificities determined from the genomic positions detected as methylated. (g) Summary of detected methylated positions across the genome.
Figure 5.
Figure 5.
Methylome determination of C. jejuni NCTC 11168. (a–d) Example traces of kinetic variation, showing instances of the detected methylated motifs. (e) MTase specificities determined from the genomic positions detected as methylated. (f) Summary of detected methylated positions across the genome.
Figure 6.
Figure 6.
Methylome determination of B. cereus ATCC 10987. (a–c) Example traces of kinetic variation, showing instances of the detected methylated motifs. (d) MTase specificities determined from the genomic positions detected as methylated. (e) Summary of detected methylated positions across the genome.
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