Transcriptomic analysis of (group I) Clostridium botulinum ATCC 3502 cold shock response

Abstract

Profound understanding of the mechanisms foodborne pathogenic bacteria utilize in adaptation to the environmental stress they encounter during food processing and storage is of paramount importance in design of control measures. Chill temperature is a central control measure applied in minimally processed foods; however, data on the mechanisms the foodborne pathogen Clostridium botulinum activates upon cold stress are scarce. Transcriptomic analysis on the C. botulinum ATCC 3502 strain upon temperature downshift from 37°C to 15°C was performed to identify the cold-responsive gene set of this organism. Significant up- or down-regulation of 16 and 11 genes, respectively, was observed 1 h after the cold shock. At 5 h after the temperature downshift, 199 and 210 genes were up- or down-regulated, respectively. Thus, the relatively small gene set affected initially indicated a targeted acute response to cold shock, whereas extensive metabolic remodeling appeared to take place after prolonged exposure to cold. Genes related to fatty acid biosynthesis, oxidative stress response, and iron uptake and storage were induced, in addition to mechanisms previously characterized as cold-tolerance related in bacteria. Furthermore, several uncharacterized DNA-binding transcriptional regulator-encoding genes were induced, suggesting involvement of novel regulatory mechanisms in the cold shock response of C. botulinum. The role of such regulators, CBO0477 and CBO0558A, in cold tolerance of C. botulinum ATCC 3502 was demonstrated by deteriorated growth of related mutants at 17°C.

Figure 1. Projection of genes significantly up- or down-regulated upon cold shock in the C. botulinum ATCC 3502 genome.

From outside to inside: Ring 1, molecular clock of C. botulinum 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.

Figure 2. Confirmation of DNA microarray results with quantitative real-time reverse-transcription PCR (RT-qPCR).

Log₂ fold changes of transcript levels measured with DNA microarrays (x-axis) and RT-qPCR (y-axis) in C. botulinum ATCC 3502 cultures 1 h after temperature downshift from 37°C to 15°C. 16S rrn 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.

Figure 3. K-means clusters of up- or down-regulated genes 1 h and 5 h after temperature downshift in C. botulinum ATCC 3502.

K-means clustering of significantly up- or down-regulated genes 1 h and 5 h after temperature downshift from 37°C to 15°C in C. botulinum ATCC 3502 into four clusters. The sub-clusters were further subjected to hierarchical clustering.

Figure 4. Distribution of significantly up- or down-regulated genes in functional categories.

The number of significantly up- or down-regulated and unaffected genes 5 h after temperature downshift from 37°C to 15°C in C. botulinum 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 . Genes with no expression data available due to poor array hybridization are assigned to the “Not affected/unknown” category.

Figure 5. Growth of C. botulinum ATCC 3502 wild type, and cbo0097, cbo0477 and cbo0558A mutants at 17°C.

Growth of C. botulinum ATCC 3502 wild type (WT, A-C), cbo0097 mutant (A), cbo0477 mutant (B), and cbo0558A 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.