Functional classification of genes identified in members of the <i>Methylocystaceae</i>.

<p>The gene content of strain SC2 (red), strain Rockwell (blue) and <i>Ms. trichosporium</i> OB3b (green) and that of the core genome shared by them (grey) was subjected to functional classification by the RAST server. CDS were classified into 27 functional categories using the SEED subsystem. Numbers in parentheses next to the strain names indicate the number of CDS assigned to the SEED subsystem out of the total number of CDS present in the particular genome. The proportion of CDS (x-axis) assigned to a particular subsystem was calculated by dividing the number of CDS assigned to this category by the total number of CDS assigned to the SEED subsystem database. The functional categories were arranged according to the number of CDS assigned for strain SC2 to each category. The number of CDS classified for the individual strains and their core genome into each SEED subsystem was subjected to statistical analysis using STAMP. A <i>p</i>-value cutoff of 0.05 was used to determine significant differences. Subsystems showing significant differences are marked by an asterisk.</p

Denitrification-mediated N₂ production by strain SC2.

<p>³⁰N₂ production was measured after fifteen days for cells incubated in NMS containing either K¹⁵NO₃ (blue) or KNO₃ (orange). The assays were performed under both anaerobic and aerobic conditions. Data points are means ±SD of three separate experiments.</p

Prediction of the <i>oriC</i> region by Ori-Finder.

<p>(A) 1,063-bp sequence (2,008,193 bp to 2,009,255 bp) of the predicted <i>oriC</i> site. Three <i>dnaA</i> box motifs identified using the <i>Escherichia coli</i> specific <i>dnaA</i> boxes are bold-faced and highlighted. Palindromic repeats identified in this region are marked by arrows at the top. (B, C) The Z-curves measuring the disparity between the percent content of AT (red lines), GC (green lines), RY (blue lines) and MK (yellow lines) for the original sequence (B) and the rotated sequence (C). It should be noted that the coordinate origin of the rotated sequence begins and ends in the maximum of the GC disparity curve. Short vertical red lines at the top show the locations of indicator genes, such as <i>dnaA</i>, <i>dnaN</i>, <i>gidA</i>, and <i>hemE</i>. The upward black arrow indicates the position of the predicted <i>oriC</i>. Purple peaks with diamonds indicate DnaA box clusters. (D) Pairwise alignment between the <i>dif</i> sites located in the genomes of <i>E. coli</i> and strain SC2. In strain SC2, the <i>dif</i>-like sequence is located from nucleotide position 276,895 to 276,922 (almost halfway of the deduced <i>oriC</i>) and matches at 20 nucleotide positions with the 28-bp <i>dif</i> sequence of <i>E. coli</i>.</p

N₂O production by strain SC2.

<p>Cells were incubated in NMS, either in the presence (filled symbol) or absence (open symbol) of 10% acetylene. Assays were performed both under anaerobic (orange) and aerobic (green) conditions. Data points are means ±SD of three separate experiments. The inset shows the same graph with a y-axis zoomed in for the range 0 to 0.8.</p

Venn diagram showing the number of CDS unique to and shared by the <i>Methylocystaceae</i> members.

<p>Data analysis was performed using the genomes of strain SC2 (red), strain Rockwell (blue) and <i>Ms. trichosporium</i> OB3b (green). Numbers in circles indicate the number of unique CDS, while those in intersections represent the number of orthologous CDS common to two or more strains. Orthologs were detected by reciprocal best BLASTP matches with the EDGAR software.</p

N₂ fixation by strain SC2.

<p>(A) Growth dynamics (OD₆₀₀) of strain SC2 in batch cultures on N-free medium (with atmospheric N₂ as sole nitrogen source). Oxygen concentrations of 1% (blue), 5% (green), 10% (red), 15% (brown) and 20% (black) were used to test their effect on N₂ fixation-mediated growth. Note that the x-axis is not in scale. (B) Effect of oxygen on the nitrogenase activity (acetylene reduction assay) in strain SC2. Ethylene production was measured after 24 hours of incubation under different concentrations of oxygen in the headspace. Data points are means ±SD of three separate experiments.</p

Neighbor-joining tree constructed for the methanotrophic core genome.

<p>The tree is based on the alignment of 154 CDS that are common to all eight methanotroph genomes used for comparative analysis. Non-matching parts of the alignments were eliminated prior to tree construction. The individual gene alignments were combined into one concatenated alignment. The neighbor-joining tree was constructed using EDGAR. All branches of the phylogenetic tree showed 100% bootstrap support based on 500 replications. See ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074767#s3" target="_blank">Materials and Methods</a>’ for further details.</p

Phylogenetic tree of nine <i>C. perfringens</i> conjugative plasmids.

<p>The phylogenetic tree was inferred using the Neighbor-joining algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049753#pone.0049753-Saitou1" target="_blank">[35]</a>. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All positions containing gaps were eliminated from the dataset (Pairwise deletion option). Phylogenetic analyses were conducted in MEGA5.</p

PFGE Southern blot of plasmids from NE <i>C. perfringens</i> poultry strains.

<p>Southern blotting of PFGE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049753#pone-0049753-g001" target="_blank">Figure 1</a>) was performed with DIG-labelled probes for <i>netB</i> and <i>hdhA</i>. Results from both <i>netB</i> and <i>hdhA</i> probes are shown overlayed. In all lanes with two bands, the upper band represents <i>netB</i> and the lower band <i>hdhA</i>. M: Mid-Range II PFG molecular DNA ladder (Kb).</p

PFGE analyses of plasmids from NE <i>C. perfringens</i> poultry strains.

<p>Agarose plugs containing DNA from each specified isolate were digested with <i>Not</i>I and subjected to PFGE and staining with ethidium bromide. Line numbers indicate isolate numbers M: Mid-Range II PFG molecular DNA ladder (Kb).</p