Relative abundance of Top 100 genera assigned by RDP Classifier at 50% bootstrap cutoffs and percentage of ten divided subsets for all ranked genera.

<p>The genera were ranked based on the average relative abundance for each pyrotag. All rankings in each subfigure were sorted from high to low level and displayed in the right column from bottom to top accordingly. For instance, Ranking 1, 2, 3, …, and 100 represented genera of <i>Zoogloea</i>, <i>Dechloromonas</i>, <i>Flavobacteria</i>, …, and <i>Pseudorhodobacter</i>, respectively. Greek letters of <i>α</i>, <i>β</i>, <i>γ</i>, <i>δ</i>, or <i>ε</i> modified in the terminus of genus name represented classes of <i>α-</i>, <i>β-</i>, <i>γ-</i>, <i>δ-</i>, or <i>ε- Proteobacteria</i>, respectively. Similarly, <i>A</i>, <i>Acido</i>, <i>B</i>, <i>Chla</i>, <i>Chlo</i>, <i>F</i>, <i>N</i>, <i>O</i>, <i>P</i>, <i>S</i>, <i>T</i>, and <i>V</i>, indicated phyla of <i>Actinobacteria</i>, <i>Acidobacteria</i>, <i>Bacteroidetes</i>, <i>Chlamydiae</i>, <i>Chloroflexi</i>, <i>Firmicutes</i>, <i>Nitrospira</i>, <i>OD1</i>, <i>Planctomycetes</i>, <i>Spirochaetes</i>, <i>TM7</i>, and <i>Verrucomicrobia</i>, respectively. The total number of these Top 100 genera assigned into the phyla/class was summarized as: <i>α</i> (13), <i>β</i> (27), <i>γ</i> (9), <i>δ</i> (5), <i>ε</i> (1), <i>A</i> (9), <i>Acido</i> (3), <i>B</i> (13), <i>Chla</i> (1), <i>Chlo</i> (1), <i>F</i> (9), <i>N</i> (1), <i>O</i> (1), <i>P</i> (2), <i>S</i> (1), <i>T</i> (1), and <i>V</i> (3).</p

Cluster analysis of all pyrotags based on relative abundance of Top 100 genera.

<p>‘Average’ represented the average relative abundance. The Past statistical software was used to calculate the distance using Bray-Curtis similarity measure.</p

Schematic representation of experimental design employed in this study.

<p>The full length of <i>E. coli</i> 16S rRNA gene was used as the reference scale. Fourteen blue arrow lines indicated all pyrotags analyzed in this study. S (L)-V1V2 represented the pyrotag contained 16S V1V2 region derived from a Short (Long) amplicon. Four small PCR fragments covered 16S V1V2, V3V4, V5V6, and V7V8V9 regions were defined as Short amplicons. The remainder three large PCR fragments targeted 16S regions of V1–V4, V3–V6, and V5–V9 were defined as Long amplicons.</p

Overview of processed pyrotags.

<p>The raw pyrotags were the original reads generated by the 454 run. After removal of low quality reads, short reads, chimera, noise, and archaea reads, the clean pyrotags were finally obtained and used for downstream bioinformatic analysis.</p

Primers used in this study.

<p>The positions of all primers are referred to the 16S rRNA gene of <i>E. coli</i> str. K12 substr. DH10B. The bold italic nucleotide ‘<i>G</i>’ fused into the 5′ terminus of each barcode is to offer an unbiased ligation between amplicons and 454 adaptors.</p

S-type and L-type pyrotags classified into six different taxonomic units assigned by RDP Classifier at 50% bootstrap cutoffs.

<p>The equal sequencing depths of 23609 reads and 3276 reads were subsampled to make fair comparison for S-type and L-type pyrotags, respectively.</p

Relative abundance at phylum or class (only for <i>Proteobacteria</i>) level assigned by RDP Classifier at 50% confidence thresholds.

<p>The extremely low percentage phyla of ‘<i>Aquificae</i>, <i>BRC1</i>, <i>Caldiserica</i>, <i>Chlorobi</i>, <i>Cyanobacteria</i>, <i>Deferribacteres</i>, <i>Deinococcus-Thermus</i>, <i>Fibrobacteres</i>, <i>Fusobacteria</i>, <i>Gemmatimonadetes</i>, <i>Lentisphaerae</i>, <i>OD1</i>, <i>OP10</i>, <i>OP11</i>, <i>Spirochaetes</i>, <i>SR1</i>, <i>Synergistetes</i>, <i>Tenericutes</i>, <i>Thermotogae</i>, and <i>WS3</i>’ were not displayed in detail and summarized as rare phyla.</p

Statistical data of Top 500 (a) and Top 100 (b) genera not detected for each S-type pyrotag.

<p>Every subset of 100 genera was divided and calculated independently (a). Genera detected by all S-type pyrotags simultaneously were not listed and the not detected genera were all marked with crosses (b).</p

Suppression of <i>dna2</i> by ExoIp Overexpression

<p>Strain W303 <i>dna2–1</i> was transformed with the plasmids pRS424G and pRS424G-EXO1, an empty plasmid vector and a plasmid expressing ExoIp from the <i>GAL110</i> promoter, respectively. W303RAD5 strains <i>dna2–1 7A(pRS424G)</i> and <i>dna2–1(pRS424G-EXO1)</i> were grown to mid log phase and serially diluted on yeast-dextrose galactose- and raffinose-containing plates and incubated at 30 °C for 5 d.</p

Separation-of-Function Checkpoint Mutants <i>mrc1AQ</i> and <i>rad9</i>Δ Are Not Synthetically Lethal with <i>dna2</i> or <i>rrm3</i>Δ Mutants

<div><p><i>mrc1</i>Δ experiments were carried out in the isogenic 4741 strain and are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010061#pgen-0010061-st004" target="_blank">Table S4</a>. <i>mrc1AQ</i> experiments were carried out in strains isogenic with W303 <i>RAD5</i>⁺.</p><p>Panel 1 (top): segregants from a <i>DNA2/dna2–2 MRC1/mrc1</i>Δ diploid. Segregants are isogenic with <i>4741</i>. <i>d, dna2–2; m, mrc1</i>Δ<i>.</i></p><p>Panel 2: segregants from a <i>MRC1/mrc1AQ RAD9/rad9</i>Δ <i>DNA2/dna2–2</i> diploid. <i>d, </i><i>dna2–2; m, mrc1AQ; r, rad9</i>Δ.</p><p>Panel 3: segregants from a <i>DNA2/dna2–1 MRC1/mrc1AQ RAD9/rad9</i>Δ strain. <i>d, dna2–1; m, mrc1AQ; r, rad9</i>Δ.</p><p>Panel 4 (bottom): segregants from a <i>RRM3/rrm3</i>Δ <i>MRC1/mrc1AQ</i> diploid. <i>m, mrc1AQ; r, rrm3</i>.</p></div