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The methylomes of six bacteria.

Murray IA, Clark TA, Morgan RD, Boitano M, Anton BP, Luong K, Fomenkov A, Turner SW, Korlach J, Roberts RJ - Nucleic Acids Res. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, 240 County Road, Ipswich, MA 01938, USA.

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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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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gks891-F6: 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.

Mentions: Our main goal was to characterize the Type I system and also ascertain the sites of methylation by the MTases not addressed in the previous study. The Type I system, now called BceSVI, was clearly active and recognized the sequence 5′-TAm6AGN7TGG-3′, where again the underlined T indicates m6A on the complementary strand (Figure 6; Supplementary Figures S1f and S2f). This system is a little unusual in that, it contains two M subunits. Because we did not clone the individual components of this system, we cannot say whether one or both M subunits are active. The sites of modification of the three other Type II MTases are indicated in Table 1, while the Type III MTase, which had been identified earlier by cloning, is shown to be completely active in the genome.Figure 6.


The methylomes of six bacteria.

Murray IA, Clark TA, Morgan RD, Boitano M, Anton BP, Luong K, Fomenkov A, Turner SW, Korlach J, Roberts RJ - Nucleic Acids Res. (2012)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526280&req=5

gks891-F6: 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.
Mentions: Our main goal was to characterize the Type I system and also ascertain the sites of methylation by the MTases not addressed in the previous study. The Type I system, now called BceSVI, was clearly active and recognized the sequence 5′-TAm6AGN7TGG-3′, where again the underlined T indicates m6A on the complementary strand (Figure 6; Supplementary Figures S1f and S2f). This system is a little unusual in that, it contains two M subunits. Because we did not clone the individual components of this system, we cannot say whether one or both M subunits are active. The sites of modification of the three other Type II MTases are indicated in Table 1, while the Type III MTase, which had been identified earlier by cloning, is shown to be completely active in the genome.Figure 6.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, 240 County Road, Ipswich, MA 01938, USA.

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.

Show MeSH