Validation of DNA microarray results using quantitative real-time reverse-transcription PCR (RT-qPCR).

<p>Log₂ fold-changes of transcript levels measured with DNA microarrays (x-axis) and RT-qPCR (y-axis) in <i>C</i>. <i>botulinum</i> ATCC 3502 continuous culture 10 min (red) and 42 h (black) after temperature up-shift from 39 to 45°C. 16S <i>rrn</i> transcript levels were used as a normalization reference in the RT-qPCR. Linear regression analysis showed an R² correlation value of 0.99 between the microarray and RT-qPCR transcription fold-change results.</p

Minimum and maximum percentage of CDSs in the three reference strains (SM101, ATCC13124, and 13) carried by chromosomal and plasmid-borne <i>cpe</i>-carrying and <i>cpe</i>-negative <i>C. perfringens</i> strains.

<p>Minimum and maximum percentage of CDSs in the three reference strains (SM101, ATCC13124, and 13) carried by chromosomal and plasmid-borne <i>cpe</i>-carrying and <i>cpe</i>-negative <i>C. perfringens</i> strains.</p

Confirmation of DNA microarray results with quantitative real-time reverse-transcription PCR (RT-qPCR).

<p>Log₂ fold changes of transcript levels measured with DNA microarrays (x-axis) and RT-qPCR (y-axis) in <i>C. botulinum</i> ATCC 3502 cultures 1 h after temperature downshift from 37°C to 15°C. 16S <i>rrn</i> transcript levels were used as a normalization reference in the RT-qPCR. Linear regression analysis showed an R² correlation value of 0.93 between the microarray and RT-qPCR transcription fold change results.</p

Genes differentiating the chromosomal <i>cpe</i>-carrying <i>C. perfringens</i> strains from the plasmid-borne <i>cpe</i>-carrying and <i>cpe</i>-negative strains.

<p>The figure was constructed using the MEV software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046162#pone.0046162-Tsoy1" target="_blank">[28]</a>.</p

Distribution of significantly up- or down-regulated genes in functional categories.

<p>The number of significantly up- or down-regulated and unaffected genes 5 h after temperature downshift from 37°C to 15°C in <i>C. botulinum</i> ATCC 3502 divided into functional categories. The bar lengths portray the percentage of affected and unaffected genes of all genes assigned to each functional category. The functional categories were as described in the original annotation of the ATCC 3502 genome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089958#pone.0089958-Sebaihia1" target="_blank">[12]</a>. Genes with no expression data available due to poor array hybridization are assigned to the “Not affected/unknown” category.</p

Projection of genes significantly up- or down-regulated upon cold shock in the <i>C. botulinum</i> ATCC 3502 genome.

<p>From outside to inside: Ring 1, molecular clock of <i>C. botulinum</i> ATCC 3502 genome; ring 2, coding DNA sequences of the forward strand (red); ring 3, coding DNA sequences of the reverse strand (cyan); ring 4, ribosomal and transfer RNA (blue); ring 5, pseudogenes (brown) and prophages (orange); ring 6, genes up-regulated (pink) or down-regulated (green) 1 h after the cold shock; ring 7, genes up-regulated (pink) or down-regulated (green) 5 h after the cold shock.</p

Growth of <i>C. botulinum</i> ATCC 3502 wild type, and <i>cbo0097</i>, <i>cbo0477</i> and <i>cbo0558A</i> mutants at 17°C.

<p>Growth of <i>C. botulinum</i> ATCC 3502 wild type (WT, A-C), <i>cbo0097</i> mutant (A), <i>cbo0477</i> mutant (B), and <i>cbo0558A</i> mutant (C) cultures in TPGY broth at 17°C. Antisense (AS) and sense (S) orientation mutants were constructed for each gene. The error bars represent the minimum and maximum OD₆₀₀ values measured for three independent biological replicates.</p

Chromosomal <i>cpe</i>-carrying <i>C. perfringens</i> strains clustered separately from the plasmid-borne <i>cpe</i>-carrying and <i>cpe</i>-negative strains.

<p>The figure was constructed using the MEV software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046162#pone.0046162-Tsoy1" target="_blank">[28]</a>.</p

Strains and plasmids used in this study.

1<p>ATCC, American Type Culture Collection.</p

Number of CDSs of <i>Clostridium botulinum</i> ATCC 3502 being significantly up-regulated (red), down-regulated (green) or unaffected (grey) after exposure to high temperature at different time points after heat shock.

<p>1: immediately after heat shock, 2: 10 min, 3: 1 h, 4: 18 h, 5: 42 h after heat shock, 6: adapted culture.</p