The distribution in REBASE of the components of the four Types of restriction systems is shown

<p><b>Copyright information:</b></p><p>Taken from "REBASE—enzymes and genes for DNA restriction and modification"</p><p>Nucleic Acids Research 2007;35(Database issue):D269-D270.</p><p>Published online Jan 2007</p><p>PMCID:PMC1899104.</p><p>© 2006 The Author(s)</p> Red, restriction enzyme genes; blue, methyltransferase genes; and yellow, specificity subunits. Full colors indicate genes whose products have been biochemically characterized, whereas shaded areas represent inferred function based on bioinformatic analysis of DNA sequences. The pop-out slices (with numbers in parentheses) indicate those genes where sequence is available for biochemically characterized enzymes. The adjacent numbers represent those for which only biochemical evidence is available

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by <i>Prevotella intermedia</i> - Fig 8

<p><b>CRISPR-Cas systems of A) <i>P</i>. <i>intermedia</i> ATCC-25611F and B) <i>P</i>. <i>intermedia</i> 17F. CRISPR loci and <i>cas</i> gene organization within <i>P</i>. <i>intermedia</i> genomes.</b> Arrows indicate open reading frames, with gene names indicated above/below each. Predicted <i>cas</i> genes associated with each CRISPR locus are shaded in green while ORFs not identified as part of the CRISPR system or those with currently unknown function are shown in grey. Anti-DR repeats loci are indicated in red with arrows indicating putative tracrRNA and their orientation. CRISPR arrays are represented by hatched rectangles with each square representing a single spacer sequence which we have numbered according to its position relative to the predicted leader sequence (L). Squares shaded yellow indicate that the spacer showed similarity (>87% homology) to phage or plasmids during CRISPRtarget analysis. The consensus DR sequence is indicted below each array (non-conserved nucleotides between systems are underlined). Chr. I and Chr. II indicate the location of the respective CRISPR loci on chromosome I or chromosome II of each strain, while exact genome coordinates of each component are detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185234#pone.0185234.s012" target="_blank">S4</a></b>and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185234#pone.0185234.s013" target="_blank">S5</a> Tables</b>.</p

Comparative Methylome Analysis of the Occasional Ruminant Respiratory Pathogen <i>Bibersteinia trehalosi</i>

<div><p>We examined and compared both the methylomes and the modification-related gene content of four sequenced strains of <i>Bibersteinia trehalosi</i> isolated from the nasopharyngeal tracts of Nebraska cattle with symptoms of bovine respiratory disease complex. The methylation patterns and the encoded DNA methyltransferase (MTase) gene sets were different between each strain, with the only common pattern being that of Dam (GATC). Among the observed patterns were three novel motifs attributable to Type I restriction-modification systems. In some cases the differences in methylation patterns corresponded to the gain or loss of MTase genes, or to recombination at target recognition domains that resulted in changes of enzyme specificity. However, in other cases the differences could be attributed to differential expression of the same MTase gene across strains. The most obvious regulatory mechanism responsible for these differences was slipped strand mispairing within short sequence repeat regions. The combined action of these evolutionary forces allows for alteration of different parts of the methylome at different time scales. We hypothesize that pleiotropic transcriptional modulation resulting from the observed methylomic changes may be involved with the switch between the commensal and pathogenic states of this common member of ruminant microflora.</p></div

Site directed bisulfite sequencing of <i>P</i>. <i>intermedia</i> ATCC-25611F DNA identifies 5-methylcytosine (m5C) modifications of ^5’-GCWGC^-3’ motifs (where W = A or T).

<p>Sequence comparison and alignment of ATCC25611F genomic regions before and after bisulfite conversion. <b>A)</b> Example of region containing GCTGC motifs and <b>B)</b> example of region containing GCAGC motifs. Unmethylated cytosine residues converted to thymine during bisulfite treatment are indicated by white arrows, m5C methylated cytosines protected from deamination are indicated by black arrows (present within GCWGC motifs) while partially converted cytosine residues (shown by dual red and blue peaks in chromatogram) are indicated by grey arrow. DNA was amplified and sequenced using primer set GCWGCregion1_BS and GCWGCregion2_BS, detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185234#pone.0185234.s009" target="_blank">S1 Table</a></b> and full sequence comparison of these regions are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185234#pone.0185234.s003" target="_blank">S3 Fig</a></b>.</p

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by <i>Prevotella intermedia</i>

<div><p><i>Prevotella intermedia</i>, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using <u>S</u>ingle <u>M</u>olecule, <u>R</u>eal-<u>T</u>ime (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, <i>P</i>. <i>intermedia</i> ATCC 25611 and <i>P</i>. <i>intermedia</i> Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the <i>P</i>. <i>intermedia</i> transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for <i>P</i>. <i>intermedia</i>, which will be required for fundamental investigation of this organism’s physiology, metabolism, and pathogenesis in human disease.</p></div

Summary of restriction-modification and orphan methyltransferase systems of <i>Prevotella intermedia</i> ATCC-25611F.

<p>Summary of restriction-modification and orphan methyltransferase systems of <i>Prevotella intermedia</i> ATCC-25611F.</p

Methylated motifs in four <i>B</i>. <i>trehalosi</i> strains.^a

<p>Methylated motifs in four <i>B</i>. <i>trehalosi</i> strains.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161499#t003fn001" target="_blank">^a</a></p

Gene comparison of <i>btr188II</i> R-M system orthologs.

<p>Top row, gene order of orthologous systems from <i>B</i>. <i>trehalosi</i> strains 188, 189 and 192. Bottom row, gene order of orthologous but functionally distinct system from strain 190, <i>btr190I</i>. R-M system genes are shown in solid color, and apparent non-R-M related genes are cross-hatched. Genes from the <i>btr190I</i> system similar to those in the other three strains are shown in blue, and genes unrelated to those in the other three strains are shown in red; the region of apparent replacement is shown by the dotted lines. Note <i>btr190IS</i> is fused to the upstream gene, a homolog of <i>dinD</i>. The TRDs of the two <i>S</i> genes are indicated by lower-case letters, showing that the <i>S</i> gene in the top line comprises two imperfect copies of the same TRD, resulting in a palindromic recognition site.</p

Methyltransferase activities targeting the palindromic GATC motif in <i>P</i>. <i>intermedia</i> strains.

<p><b>A)</b> The methyltransferase associated with the Pin25611FII R-M system (M.Pin25611FII) is responsible for the G^mATC motif modification in <i>Prevotella intermedia</i>. The methyltransferase gene was cloned to plasmid pRRS and expressed in <i>E</i>. <i>coli</i> ER2796, deficient in methyltransferase activity. Plasmid DNA (1 μg), isolated from two separate clones (C1 and C2) of the recombinant construct, was restricted with 1U of either Sau3AI (inhibited by GAT^mC, unaffected by G^mATC) or DpnII (inhibited by G^mATC, unaffected by GAT^mC). Lane M, marker DNA (10kb ladder); lane 1, undigested control plasmid DNA; lane 2 and 3, Sau3AI digested plasmid DNA; lane 4 and 5, DpnII digested plasmid DNA from separate clones (C1 and C2). <b>B)</b> Restriction enzyme digestion of genomic DNA following isolation from <i>P</i>. <i>intermedia</i> 17F, <i>P</i>. <i>intermedia</i> ATCC25611F and the control <i>Fusobacterium nucleatum</i> ATCC25586. Genomic DNA (1 μg) was restricted with 1U of restriction enzymes each recognizing GATC, but differing in their methylation sensitivity, for 1 hour and resolved on a 1% agarose gel. In each gel image: Lane M, marker DNA (10kb ladder); lane 1, undigested control DNA; lane 2, Sau3AI (inhibited by GAT^mC); lane 3, DpnI (methyl-directed endonuclease, requires G^mATC but inhibited by GAT^mC); lane 4, DpnII (inhibited by G^mATC, unaffected by GAT^mC); lane 5, MboI (inhibited by G^mATC and GAT^mC) and lane 6, ApoI treatment (control enzyme, recognizes sequence RAATTY, where R = A or G, and = C or T). <i>P</i>. <i>intermedia</i> DNA appears to be methylated at the adenine and cytosine residues of the GATC motif, while <i>F</i>. <i>nucleatum</i> DNA is unmethylated at both residues. <b>C)</b> The absence of GAT^m5C modification in <i>P</i>. <i>intermedia</i> DNA. Comparison and alignment of untreated ATCC25611F genomic DNA region with the same region after bisulfite conversion indicates the absence of m5C modification within ^5’-GATC^-3’motifs. During bisulfite treatment, unmethylated cytosine or N4-methylcytosine (m4C) is converted to uracil which is read as thymine during sequencing, while 5-methylcytosine (m5C) is not subject to deamination and remains as cytosine. White arrows indicate cytosine residues of the GATC motif converted to thymine. DNA was amplified and sequenced using primer set GATCregion1_BS and GATCregion2_BS, detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185234#pone.0185234.s009" target="_blank">S1 Table</a></b> (full sequence comparison of this and a second GATC region are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185234#pone.0185234.s002" target="_blank">S2 Fig</a></b>).</p

Complete genome map of <i>Prevotella intermedia</i> strains ATCC-25611F and 17F, represented by CGView.

<p>Circular maps of the two chromosomes (I and II) present within <b>A)</b> <i>Prevotella intermedia</i> ATCC-25611F (GenBank: CP019300 and CP019301) and <b>B)</b> <i>Prevotella intermedia</i> strain 17-F (GenBank: CP019302 and CP019303). The outermost two circles indicate coding DNA sequences (CDSs) on the plus and minus strands, respectively. The GC content and GC skew (Green GC+ and purple GC-) are shown in the third and fourth circles (moving towards the center), respectively.</p