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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 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.
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gks891-F2: 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.

Mentions: The results of whole genome SMRT sequencing analysis are shown in Figure 2 and demonstrate that the putative GATC MTase is expressed, methylates the adenine residues on both strands to form m6A, but actually recognizes the more specific sequence, 5′-RGATCY-3′, although methylation seems not to be complete during normal growth. This MTase is called M.CsaI. The specificity was very strict as the number of hits observed for 5′-NGATCN-3′, but not conforming to 5′-RGATCY-3′, was 0 (Supplementary Figure S3). The Type I system is very well defined and recognizes the usual bipartite sequence pattern recognized by Type I enzymes, but this particular recognition sequence 5′-CCAC(N)6CTC-3′ has not been reported previously (5). As usual for Type I systems, the MTase, M.CsaII, acts on the single adenine residue in each DNA strand forming m6A. The putative prophage-encoded MTase appears not to be expressed. That the 5′-RGm6ATCY-3′ signal is due to expression of Csal_1368 and is not a combination of expression of both Type II ORFs was tested by cloning Csal_1401 separately in the methylation deficient E. coli strain ER2796 (22). The resulting clone showed that the MTase was non-specific and methylated most, but not all, A residues in the plasmid (Supplementary Figure S4). Motif analysis indicated the following specificity rules for this relatively non-specific MTase: 5′-m6AB-3′ and 5′-Sm6AAM-3′ (>96% of all hits with a kinetic score >100 fell into these motifs; B = not A; S = G or C, M = A or C).Figure 2.


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

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

gks891-F2: 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.
Mentions: The results of whole genome SMRT sequencing analysis are shown in Figure 2 and demonstrate that the putative GATC MTase is expressed, methylates the adenine residues on both strands to form m6A, but actually recognizes the more specific sequence, 5′-RGATCY-3′, although methylation seems not to be complete during normal growth. This MTase is called M.CsaI. The specificity was very strict as the number of hits observed for 5′-NGATCN-3′, but not conforming to 5′-RGATCY-3′, was 0 (Supplementary Figure S3). The Type I system is very well defined and recognizes the usual bipartite sequence pattern recognized by Type I enzymes, but this particular recognition sequence 5′-CCAC(N)6CTC-3′ has not been reported previously (5). As usual for Type I systems, the MTase, M.CsaII, acts on the single adenine residue in each DNA strand forming m6A. The putative prophage-encoded MTase appears not to be expressed. That the 5′-RGm6ATCY-3′ signal is due to expression of Csal_1368 and is not a combination of expression of both Type II ORFs was tested by cloning Csal_1401 separately in the methylation deficient E. coli strain ER2796 (22). The resulting clone showed that the MTase was non-specific and methylated most, but not all, A residues in the plasmid (Supplementary Figure S4). Motif analysis indicated the following specificity rules for this relatively non-specific MTase: 5′-m6AB-3′ and 5′-Sm6AAM-3′ (>96% of all hits with a kinetic score >100 fell into these motifs; B = not A; S = G or C, M = A or C).Figure 2.

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