Clostridium botulinum presents a significant hazard to the food processing industry. However, the cellular mechanisms and factors that contribute to their regulation utilized by this feared foodborne pathogen in response and adaptation to food processing and storage-induced stress are poorly characterized. Another major aspect of C. botulinum presenting serious implications on food safety is its capability to produce heat-resistant endospores. Nevertheless, the sporulation cascade of C. botulinum has not been characterized. This study sought to investigate the effects of temperature downshift on the global gene expression pattern of Group I C. botulinum type strain ATCC 3502, and further characterize the roles of regulatory mechanisms identified as cold tolerance-related. Furthermore, the role of a major regulatory element, the alternative sigma factor SigK, in the sporulation cascade of C. botulinum was determined. Additionally, its putative function in stress tolerance was investigated.
Transcriptomic analysis of the foodborne pathogen C. botulinum ATCC 3502 upon temperature downshift revealed the induction of several mechanisms previously identified as cold-related in other bacteria, thus suggesting that also C. botulinum utilizes these mechanisms in cold tolerance. Mechanisms with hitherto uncharacterized functions in cold tolerance were also found. The results suggested that secondary oxidative stress was present as a component of cold stress. Additionally, two previously uncharacterized putative DNA-binding regulatory proteins CBO0477 and CBO0558A were shown to play a role in cold tolerance of C. botulinum ATCC 3502.
The two-component system (TCS) CBO0366/CBO0365 was shown to be important in the cold tolerance of C. botulinum ATCC 3502. Expression of this TCS was induced upon temperature downshift, but not under optimal temperature and growth conditions. Disruption of either of the TCS components resulted in deteriorated cold tolerance, whereas over-expression of cbo0366 in a wild-type strain resulted in an increase of growth rate at low temperature.
Inactivation of the TCS response regulator-encoding cbo0365 markedly altered the transcriptome of C. botulinum ATCC 3502. Totals of 150 and 141 chromosomal coding sequences (CDS) were significantly differently expressed in the cbo0365 mutant at either 37 °C or 15 °C, respectively. There was an overlap of 141 common CDSs between the two temperatures. The genes differentially expressed included ones related to acetone-butanol-ethanol (ABE) fermantion, arsenic resistance, phosphate uptake and flagellar rotation. The involvement of CBO0365-regulated metabolic pathways in cold tolerance was demonstrated by the deteriorated cold tolerance of mutants of the respective pathways. Cold-sensitive phenotypes were observed for mutants of the acetone-butanol-ethanol fermentation pathway components bcd, crt, bdh and ctfA, the arsenic detoxifying machinery components arsC and arsR, and the phosphate uptake mechanism component phoT. Electrophoretic mobility shift assays confirmed transcriptional activation or repression as a means for CBO0365 in regulating itself, and the crt, ars, and pho operons.
A dual role for the alternative sigma factor SigK in sporulation and in stress tolerance of C. botulinum ATCC 3502 was demonstrated. Disruption of SigK halted sporulation at an early stage but the phenotype was restored by in trans complementation of the mutation. The expression of sigK was induced upon exposure to low temperature and high salinity, but not upon a downshift in pH. A deteriorated tolerance to low temperature and to high salinity was observed for the sigK mutant strains.
|Tila||Julkaistu - 2013|
|OKM-julkaisutyyppi||G5 Tohtorinväitöskirja (artikkeli)|
- 413 Eläinlääketiede