Two N. meningitidis serogroup A strains Z2491 and NM1264 display divergent DNA modifications.

<p>DNA modification scores are plotted against the coverage in SMRT sequencing of Tet1 converted samples. Each dot represents a position on either strand with a modification score larger than 20, the color specifying the nucleotide base, on which the modification was detected. Modified adenosines (red dots) are predominantly detected in strain NM1264. The horizontal line indicates the threshold score 50 applied for subsequent motif finding.</p

Methylation-sensitive restriction assays to validate DNA methylation derived from SMRT sequencing (A) DNA samples separated via gel electrophoresis, lane labels indicating (M) 1 kb Plus DNA Ladder (Invitrogen), (gDNA) whole genomic DNA preparations, and (NlaIV) restriction digest products targeting sites 'GGNNCC' in a methylation-sensitive manner. Samples from 2 different strains (Z2491 and NM1264, genome size: 2.18 Mb) display differential resistance consistent with DNA methylation profiles.(B) single position resolution of modification signal averaged over ~1800 'GGNNCC' sites in the respective genome assemblies. The fractions of sites exhibiting a modification score above 50 are displayed for each position and strand. (C) For comparison, adenosine methylation featuring enhanced sensitivity and positional resolution averaged over ~4000 'ACACC' sites.

<p>Methylation-sensitive restriction assays to validate DNA methylation derived from SMRT sequencing (A) DNA samples separated via gel electrophoresis, lane labels indicating (M) 1 kb Plus DNA Ladder (Invitrogen), (gDNA) whole genomic DNA preparations, and (NlaIV) restriction digest products targeting sites 'GGNNCC' in a methylation-sensitive manner. Samples from 2 different strains (Z2491 and NM1264, genome size: 2.18 Mb) display differential resistance consistent with DNA methylation profiles.(B) single position resolution of modification signal averaged over ~1800 'GGNNCC' sites in the respective genome assemblies. The fractions of sites exhibiting a modification score above 50 are displayed for each position and strand. (C) For comparison, adenosine methylation featuring enhanced sensitivity and positional resolution averaged over ~4000 'ACACC' sites.</p

Phase variability at modA12 locus: Alignment of representative read sequences derived from genome sequencing (strains NM1264 or Z2491).

<p>The number of repeat units is indicated in the second column, with blue cell background indicating a resulting ON status of the ORF, and yellow for an OFF status. The third column indicates the total number of reads matching the corresponding repeat configuration.</p

Variability at DNA methyltransferase loci.

<p>101 N. meningitidis isolates clustered according to SNP distance, yielding in two sequence type (ST) groups. Each column represents an isolate and rows specify the ORF status of 13 DNA methyltransferases (Rebase geneIDs of Z2491 reference strain). Bars in grey at the bottom represent the number of repeat units determining ON/OFF status of the phase-variable modA12 (M.NmeAORF1589P)</p

Methylated nucleotides display higher mutation rates than non-methylated positions.

<p>Positional co-occurrences of (in total 6031) SNPs at positions within (methylation) target motifs. Methylated positions highlighted in red within five target motifs (bold), as detected in the present study, with, each compared to two similar control sequences. Black bars in histogram represent nucleotides in methylated motifs, gray shades represent sequences not known as DNA-methylation targets. For each motif, counts of overlapping SNPs (for 5m-cytosine motifs: C/G in reference →N; or for 6m-adenosine: A/T→N) at each position are normalized by the genome-wide motif occurrences (numbers for methylated motifs in inset box). The dashed lines indicate the corresponding number of SNPs expected from random occurrence (G/C or A/T) across the genome and over-representation was tested with the χ2 statistics (*p value < 10–5).</p

Depletion of cytosine methylated motifs in the immediate upstream region of ORFs.

<p>Occurrence counts of five (most frequent) methylation target motifs are plotted against their position (in bp) relative to 1997 oriented ORFs (genome annotation N. meningitidis strain Z2491). Motif counts are presented as sum over all ORFs within 50 bp windows, centered at position zero. Red lines in each panel compare to the GC content percentage (y-axis label to the right), averaged over all ORF regions. Dashed horizontal lines represent the averaged motif occurrences corresponding to statistically significant (p value 0.05) depletion of the corresponding motif, derived from a comparison to equally sized sets of random loci. The lower right panel represents occurrence counts of a set of six non-methylated control motifs with similar base composition (identical positions in bold), and similar occurrence frequencies as the non-palindromic target motifs T5mCTGG and AC6mACC. None of the control motifs display significant depletions at ORFs comparable to that of methylated motifs.</p

Histogram of distances of methylation motifs to TSS in the promoter regions.

<p>(<i>A</i>) Distances for the 5′-CT<i>A</i>T-3′ motif to TSS. (<i>B</i>) Distances for the 5′-G<i>A</i>N₇TAY-3′/3′-CTN₇<i>A</i>TR-5′ motif to TSS.</p

Genome-wide distribution of 5′-CT

<p><b><i>A</i></b><b>T-3′</b><b> motif.</b> (<i>A</i>) Hot spots of methylation for the 5′-CT<i>A</i>T-3′ motif. The graph represents the number of motifs per 1 kb window. The red line indicates the threshold (5). Regions with more than five 5′-CT<i>A</i>T-3′ motifs are considered “hot spots of methylation”. Red arrows indicate the most enriched regions. (<i>B</i>) Circular representation of the <i>M. pneumoniae</i> genome. Red marks indicate genome locations of the three main enriched regions of methylation. (<i>C</i>) Methylation in the putative origin of replication of <i>M. pneumoniae</i>. Blue boxes indicate putative DnaA boxes. Red arrows and lines indicate methylation sites. Black arrows indicated a common distance (24 bp) from methylation sites to the TSSs of the three MPNs. (<i>D</i>) Motif sequences of two putative cell division “check points” (L, left and R, right). Noteworthy only in L motif contains the recognition motif 5′-CT<i>A</i>T-3′ (showed in red letters) on the complementary strand. In contrast, the R motif contains 5′-TTAT-3′ (showed in blue letters) on the complementary strand instead. The three large grey arrows are the three non-coding RNAs (MPNs200, MPNs201 and MPNs381).</p

Levels of RNA and protein for different proteins of M-R systems of <i>M. pneumoniae</i>.

<p>Data for microarrays and tiling was taken from Guell et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003191#pgen.1003191-Guell1" target="_blank">[43]</a>. Data regarding protein copy number was taken from Maier et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003191#pgen.1003191-Maier1" target="_blank">[44]</a> and from unpublished MS analysis done by M. Lluch-Senar. ORFs labeled with an asterisk indicate proteins found to bind to DNA by doing affinity chromatography with a DNA column followed by salt elution and MS (manuscript in preparation). HsdM (Methyltransferase), HdsS (DNA specificity recognition protein), HdsR (Restriction enzyme), M.MpnI (name assigned to the methyltransferase that recognizes 5′-CT<i>A</i>T-3′), M.MpnII (name assigned to the putative methyltransferase of the Type I motif) and S.MpnII (HsdS subunit associated to Type I restriction modification system).</p>δ<p>Essential genome of <i>M. pneumoniae</i> has been determined by using a library of minitransposon mutants (manuscript in preparation Lluch-Senar, M. et al.). NE, non essential gene; E, essential gene.</p

Unmethylated sites.

<p>A) IPD ratio plot of a 5′-CT<i>A</i>T-3′ site not detected as methylated (shadowed in yellow). B) IPD ratio plot of an unmethylated 5′-G<i>A</i>N₇TAY-3′/3′-CTN₇<i>A</i>TR-5′ motif (shadowed in yellow). For comparison of signal intensity, a methylated 5′-CT<i>A</i>T-3′ is also shown in the bottom plot (shadowed in red).</p