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

Phylogenetic tree of the four <i>B</i>. <i>trehalosi</i> isolates sequenced in this work.

<p>The tree was built from an amino acid MUSCLE alignment of all the core genes (a total of 1,969,970 amino acid residues per genome). This alignment was used to compute a Kimura distance matrix that was input to PHYLIP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161499#pone.0161499.ref034" target="_blank">34</a>] to create the Neighbor-Joining tree as described at the <a href="https://edgar.computational.bio.uni-giessen.de/cgi-bin/edgar.cgi?action=view&type=phylogeneticTree&project=EDGAR_Bibersteinia" target="_blank">EDGAR website</a>. The genome of a related Pasteurellales species, <i>Mannheimia haemolytica</i> 183 (<a href="http://www.ncbi.nlm.nih.gov/nuccore/472256744" target="_blank">CP004752.1</a>), was used as the outgroup to root the tree. The tree topology showed 100% concordance with 500 bootstrapping iterations.</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

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

Orthologs in the four <i>B</i>. <i>trehalosi</i> isolates.

<p>The pan genome as determined by EDGAR, consisting of 2,650 ORFs, was used to build the Venn diagram. Numbers in intersecting regions correspond to the number of orthologs shared by those strains. Numbers in non-intersecting regions correspond to the number of ORFs without an ortholog in any of the other three strains, and in parentheses are the numbers of these that are singletons (that is, without non-orthologous homologs, and therefore unique to the strain). Numbers in black represent total ORFs, and numbers in red represent MTases (i.e., represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161499#pone.0161499.t002" target="_blank">Table 2</a>).</p

MTase genes in four <i>B</i>. <i>trehalosi</i> strains.^a

<p>MTase genes in four <i>B</i>. <i>trehalosi</i> strains.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161499#t002fn001" target="_blank">^a</a></p

Genome sequence data for four <i>B</i>. <i>trehalosi</i> strains.

<p>Genome sequence data for four <i>B</i>. <i>trehalosi</i> strains.</p

EcoBLMcrX, a classical modification-dependent restriction enzyme in <i>Escherichia coli</i> B: Characterization <i>in vivo</i> and <i>in vitro</i> with a new approach to cleavage site determination

<div><p>Here we characterize the <u>m</u>odification-<u>d</u>ependent restriction <u>e</u>nzyme (MDE) EcoBLMcrX <i>in vivo</i>, <i>in vitro</i> and in its genomic environment. MDE cleavage of modified DNAs protects prokaryote populations from lethal infection by bacteriophage with highly modified DNA, and also stabilizes lineages by reducing gene import when sparse modification occurs in the wrong context. The function and distribution of MDE families are thus important. Here we describe the properties of EcoBLMcrX, an enzyme of the <i>E</i>. <i>coli B</i> lineage, <i>in vivo</i> and <i>in vitro</i>. Restriction <i>in vivo</i> and the genome location of its gene, <i>ecoBLmcrX</i>, were determined during construction and sequencing of a B/K-12 hybrid, ER2566. In classical restriction literature, this B system was named <i>r</i>_<i>6</i> or <i>rglA</i>_<i>B</i>. Like many genome defense functions, <i>ecoBLmcrX</i> is found within a genomic island, where gene content is variable among natural <i>E</i>. <i>coli</i> isolates. <i>In vitro</i>, EcoBLMcrX was compared with two related enzymes, BceYI and NhoI. All three degrade fully cytosine-modified phage DNA, as expected for EcoBLMcrX from classical T4 genetic data. A new method of characterizing MDE specificity was developed to better understand action on fully-modified targets such as the phage that provide major evolutionary pressure for MDE maintenance. These enzymes also cleave plasmids with ^m5C in particular motifs, consistent with a role in lineage-stabilization. The recognition sites were characterized using a site-ranking approach that allows visualization of preferred cleavage sites when fully-modified substrates are digested. A technical constraint on the method is that ligation of one-nucleotide 5' extensions favors G:C over A:T approximately five-fold. Taking this bias into account, we conclude that EcoBLMcrX can cleave 3' to the modified base in the motif R^m5C|. This is compatible with, but less specific than, the site reported by others. Highly-modified site contexts, such as those found in base-substituted virulent phages, are strongly preferred.</p></div

ECD_02034 is EcoBLMcrX.

<p>Products of <i>in vitro</i> translation (IVT) were incubated with three experimental substrates with complete base substitution of C. ^hm5C-T4: T4<i>gt</i>, sensitive to restriction <i>in vivo</i>; WT-T4: wild type T4, glucosylated ^hm5C, resistant <i>in vivo</i>; XP12: ^m5C-substituted phage, as described in Materials and Methods. Triangles signify 3-fold dilutions of IVT products. V: IVT product of empty vector. ECD1: IVT product of ECD_02033, ECD2: IVT product of ECD_02034; ECD1+2: mixture of the separately translated products; ECD1-2: IVT with cotranscribed, cotranslated genes.</p

Illumina data analysis strategy.

<p>Illumina data analysis strategy.</p