Project description:Bacillus weihenstephanensis is a subspecies of the Bacillus cereus sensu lato group of spore forming bacteria known to cause food spoilage or food poisoning. The key distinguishing phenotype of B. weihenstephanensis is its ability to grow below 7°C or, from a food safety perspective, to grow and potentially produce toxins in a refrigerated environment. In order to gain insight into to the mechanistic basis of its psychrotolerant phenotype, as well as elucidate relevant aspects of its toxigenic profile, the proteome profiles of cells grown at either 6°C or 30°C were compared.
Project description:Bacterial endospores, the transmissible forms of pathogenic bacilli and clostridia, are heterogeneous multilayered structures composed of proteins. These proteins protect the spores against variety of stresses, thus helping spore survival, and assist in germination, by interacting with the environment to form vegetative cells. Owing to the complexity, insolubility, and dynamic nature of spore proteins, it has been difficult to obtain their comprehensive protein profiles. The intact spores of Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile and their vegetative counterparts were disrupted by bead-beating in 6M urea under reductive conditions. The heterogeneous mixture was then double-digested with LysC and trypsin. Next, the peptide mixture was pre-fractionated with Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) followed by reverse phase LC-FT-MS analysis of the fractions. ‘One-pot’ method is a simple, robust method that yields identification of >1000 proteins with high confidence, across all spore layers from Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile. This method can be employed for proteome-wide analysis of non-spore-forming as well as spore-forming pathogens. Analysis of spore protein profile will help to understand the sporulation and germination processes and to distinguish immunogenic protein markers.
Project description:Pathogenic species belonging to Bacillus cereus sensu lato group possess a high evolutionary advantage in the environment and in food matrices thanks to their capacity to survive as silent spores to harsh environmental insults and grow at relatively low temperatures. Ready to re-heat products are at severe risk for contamination by members of Bacillus cereus s.l. group if not stored at proper conditions. In this work, the goal was to assess, by means of a genome-wide transcriptional assay, the isolated strain Bacillus cereus UC10070 gene expression behind the process of spore germination and consequent outgrowth in an artificially contaminated vegetable-based food model. A vegetable food model subjected to a heat treatment was determined to present favourable conditions for spores germination. Microscopic analyses together with OD measurements were applied to select the key steps of B. cereus cell cycle to be used for the microarray analysis. Using this approach we found a total of 1,646 probe sets differentially expressed and modulated during the entire B. cereus life cycle in the vegetable foodstuff.