Functional category annotation of differentially expressed genes (>1.5 fold, <i>p</i><0.05) in the <i>Clostridium difficile</i> strain 630 heat stress transcriptome.

<p>Key: Orange – up regulated; blue – down regulated.</p

Projection of differentially expressed genes on the <i>Clostridium difficile</i> 630 genome.

<p>Those genes showing at least 1.5-fold change with <i>p</i><0.05 were selected. From outside to inside: Ring 1, molecular clock of <i>C. difficile</i> strain 630 genome; Ring 2 (red), coding DNA sequences of the forward strand of the genome; Ring 3 (yellow), coding DNA sequences(CDS) of the opposite strand of the genome; Ring 4 (blue), genes upregulated at 41°C; Ring 5 (green), genes downregulated at 41°C.</p

Comparison of qRT-PCR, iTRAQ proteomics and microarray data for selected <i>Clostridium difficile</i> strain 630 genes.

<p>For each individual gene, expressional fold-change values (up-hatched columns) are shown relative to the 37°C control. Corresponding iTRAQ fold-changes (gray columns) are included for comparison with microarray data (down-hatched columns) and show good correlation between the three data sets. 16S rRNA, <i>tpi</i>, and CD2849, whose expression did not change by more than 1.5-fold, were used as reference genes.</p

Genomic context of Transposon Tn5397 in the genome of <i>Clostridium difficile</i> strain 630.

<p>Key: Arrows represent direction of changes in gene expression; genes lacking an arrow are unchanged. Tn5397 ORF numbers are shown above the genes. CD0506: group II intron. <i>tetM</i>: tetracycline resistance determinant.</p

Proposed model for translational stalling under heat stress in <i>Clostridium difficile</i> strain 630.

<p>Trigger factor (TF) docks with L23 on the ribosome and is the first protein to interact with nascent polypeptides as they emerge from exit site on the ribosome, preventing or reversing premature protein folding. At 37°C there is competitive association of the signal recognition particle (SRP) and TF, both of which interact with L23, with nascent chains emerging from the ribosome: TF inhibits binding of the SRP to proteins destined to remain in the cytoplasm and these are passed to the dnaJ/K/GrpE and GroES/L chaperone systems, resulting in correctly folded mature cytosolic proteins. Upon temperature upshift to 41°C, however, decreased abundance of trigger factor enables non client proteins to be targeted for export by the SRP, and consequently fewer proteins are presented to the dnaJ/K/GrpE and GroES/L systems. Under heat stress, the dnaJ/K/grpE system is titrated by misfolded proteins, resulting in decreased stringency of protein quality control at the ribosome exit port: this prevent nascent chains from emerging cleanly from the ribosome thereby causing translational stalling and decreased growth rates.</p

Organisation of the S10-spx-α region in the genome of <i>Clostridium difficile</i> strain 630.

<p>Black line: Biocyc predicted transcriptional units. Red boxes: Location of autogenous RNA regulatory structures in <i>Escherichia coli</i>. Orange underscore: protein with predicted regulatory function.</p

GeLC/MS-PROVALT workflow used to generate the 37°C and 41°C proteomes in <i>Clostridium difficile</i> strain 630.

<p>GeLC/MS-PROVALT workflow used to generate the 37°C and 41°C proteomes in <i>Clostridium difficile</i> strain 630.</p

Functional category distribution changes (% of proteins identified) in the 37°C and 41°C GeLC/MS proteomes from <i>Clostridium difficile</i> strain 630.

<p>Blue: 37°C proteome; Orange: 41°C proteome.</p

Functional categorisation of identified proteins in the combined <i>Clostridium difficile</i> strain 630 GeLC/MS proteome.

<p>Functional categorisation of identified proteins in the combined <i>Clostridium difficile</i> strain 630 GeLC/MS proteome.</p

Differential expression profile of 65 proteins common to both the 37°C and 41°C <i>Clostridium difficile</i> strain 630 GeLC/MS proteomes whose abundances changed by ≥1.5 fold (dotted lines).

<p>Differential expression profile of 65 proteins common to both the 37°C and 41°C <i>Clostridium difficile</i> strain 630 GeLC/MS proteomes whose abundances changed by ≥1.5 fold (dotted lines).</p