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Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.

ABSTRACT: A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that ?(K) is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

SUBMITTER: Kirk DG

PROVIDER: S-EPMC3370484 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.

Kirk David G DG Dahlsten Elias E Zhang Zhen Z Korkeala Hannu H Lindström Miia M

Applied and environmental microbiology 20120427 13

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with dist ...[more]

PMID: 22544236

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Project description: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. Global gene expression analysis on the C.M-BM- botulinum ATCC 3502 strain upon temperature downshift from 37 M-BM-0C to 15M-BM- M-BM-0C 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 already 1 h after the cold shock. At 5 h after the temperature downshift, 199 and 210 genes were up- and down-regulated, respectively. Thus, the cold shock rapidly affected the expression of a gene set of a relatively small size, indicating a targeted acute response to cold shock, whereas extensive metabolic remodeling took place after prolonged exposure to cold. Induction of genes related to fatty acid biosynthesis, oxidative stress response, and iron uptake and storage was observed, in addition to mechanisms previously characterized as cold-tolerance related in bacteria. Furthermore, induction of several uncharacterized DNA-binding transcriptional regulator-encoding genes was observed, suggesting involvement of novel regulatory mechanisms in the cold shock response of C.M-BM- botulinum. The role of such regulatory proteins, CBO0477 and CBO0558A, in cold tolerance of C.M-BM- botulinum ATCC 3502 was demonstrated by the deteriorated growth of mutants of the respective genes at 17M-BM- M-BM-0C. C. botulinum ATCC 3502 wild type cold-shocked vs. pre-cold-shock; 3 replicates; growth at 37C in TPGY broth batch culture and subjected to cold shock to 15C; sampling at mid-log growth phase before cold shock, and 1 h and 5 h after temperature downshift to 15C (= 3 time points). Dye-swapped hybridization.

Dataset Information

Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.

Publications

Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.

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