Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing - PubMed (original) (raw)

. 2012 Dec;30(12):1232-9.

doi: 10.1038/nbt.2432. Epub 2012 Nov 8.

Diana Munera, David I Friedman, Anjali Mandlik, Michael C Chao, Onureena Banerjee, Zhixing Feng, Bojan Losic, Milind C Mahajan, Omar J Jabado, Gintaras Deikus, Tyson A Clark, Khai Luong, Iain A Murray, Brigid M Davis, Alona Keren-Paz, Andrew Chess, Richard J Roberts, Jonas Korlach, Steve W Turner, Vipin Kumar, Matthew K Waldor, Eric E Schadt

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Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

Gang Fang et al. Nat Biotechnol. 2012 Dec.

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Abstract

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

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Figures

Figure 1

Figure 1

Extensive kinetic variation detected in C227-11. (a) Representative KV events detected at two loci in the C227-11 genome. For each position the loglikelihood ratio (LLR) statistic is plotted and represents the likelihood that the enzyme kinetics at the indicated position in the native C227-11 DNA is significantly (P < 2.58 × 10–8; FDR < 1%) different than the kinetics at the corresponding position in whole genome amplified C227-11 DNA. The higher the LLR, the more support exists for KV events. The only KV signals in this region are at the indicated positions and correspond to CTGCAG and GATC motifs on both the plus and minus strands. (**b**) Scatter plot and histogram of the extensive KV events detected at adenines detected across the C227-11 genome (see Supplementary Table 2 for summaries of the detections at G, C and T bases). The _x_ axis represents the amount of sequence coverage supporting the tested position. At a 1% FDR (corresponding to an LLR of 15.5; dotted horizontal line) 49,311 adenines were detected as KV events (1.9% of all adenines). (**c**) Genome-wide detections of KV events at adenines identified in **b** are represented around the C227-11 chromosome as a circos plot. The LLRs of adenine sites on the positive and negative strands are plotted as the red curve on the outer and inner loops of the Circos plot, respectively. We subsampled 50,000 sites on each strand for illustration purposes. The black circles represent the 1% FDR line (LLR > 15.5). Blue, green, orange and black hash marks correspond to the locations of GATC, CTGCAG, ACCACC and CCACN8TGAY/RTCAN8GTGG motifs, respectively.

Figure 2

Figure 2

Identification and annotation of the MTases targeting the different sequence motifs in the C227-11 genome. (a,b) Locations of the nine predicted MTases (Table 1) are shown around the C227-11 chromosome (a) and ESBL plasmid (b). The triangle expansions at each MTase site are color-coded according to type, and the genes represented in each MTase region are also color-coded by gene type. Each MTase gene was cloned into plasmid pRRS and expressed in a methylase-free strain of E. coli. The plasmid constructs for each of the MTases are depicted as a circos plots at each of the MTase locations, with the inside of the annulus representing the coordinates of the plasmid, the blue hash marks representing the locations of adenines contained in the corresponding motif targeted by the MTase, and the two red curves around the plasmid representing the –log10(P value) for the LLR model for the two DNA strands. Associated with each circos plot is a Weblogo plot based on the top adenine sites detected in each case (FDR <0.1%). The adenines in all of the contexts indicated were detected as modified at an FDR <0.1%.

Figure 3

Figure 3

ϕ104 encodes a functional restriction-modification system. Ten microliters of serial 100-fold dilutions of the indicated phage were spotted (left to right) on top agar-seeded lawns of the E. coli strain K12/K37 and lysogenic derivatives thereof. λ** has the immunity of lambdoid phage 434 (ref. ), but is primarily l. The modified lysate, HK022(mod), was obtained by growing lambdoid phage HK022 (ref. ) in the lysogen of K12/K37 that carries a ϕ104 prophage with a deletion substitution of the Pst1 endonuclease gene (K37ϕ104Δ_endpst_).

Figure 4

Figure 4

The RM system associated with M.EcoGIII regulates the expression of many genes and pathways. (a) Several genes are identified as differentially expressed at the 1% FDR level in C227-11 relative to C227ΔRM and in C227-11 relative to 55989. Significant overlap between the signatures is observed, likely reflecting common effects of the absence of this RM system. These signatures are also enriched for common pathways (Supplementary Table 5). (b) Heat map of 109 genes that are differentially expressed in three replicates of C227-11 relative to three replicates of C227ΔRM (Supplementary Table 8), and that are in the pathways indicated in Supplementary Table 5 (cation transport, cell motility, cell projection and/or flagellum). Genes that are downregulated (upregulated) in C227-11 relative to C227ΔRM are colored different shades of blue (yellow) depending on the degree of downregulation (upregulation). The Z score reflects the degree of down (Z score < 0) or up (_Z_ score > 0) regulations, computed by subtracting the mean of the log transformed expression values and dividing by the s.d. for each gene over all samples scored.

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References

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