Unknown,Transcriptomics,Genomics,Proteomics

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Study of Bacillus cereus spore germination and outgrowth in a vegetable food model by means of genome-wide transcriptome analysis

ABSTRACT: 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. RNA was isolated from the dormant spores (DS), germinating spores at 40 minutes (GSP), early-log phase at 2h (C2h) and late log phase cells at 12 hours (C12h) after thermal treatment of B. cereus UC10070 previously isolated froma biofilm on a spoiled vegetable-based puree. There were 3 biological replicates (independent cultures) for each condition. Complete genome sequence of B. thuringiensis sv konkukian str. 97-27 (NCBI Reference Sequence: NC_005957.1) was chosen for its high homology with B. cereus s.l. UC10070, to design probes corresponding to 5,197 genes spotted in duplicates onto ElectraSenseH 12K microarrays chip.

ORGANISM(S): Bacillus cereus

SUBMITTER: Daniela Bassi

PROVIDER: E-GEOD-65259 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The members of the genus Clostridium, such as the other spore-forming anaerobic bacteria, have a complex and strictly regulated life-cycle but very little is known about genetic pathways involved at different stages. Clostridium sporogenes, Gram positive bacterium usually involved in food spoilage and frequently isolated from late blowed cheese is genetically indistinguishable from proteolytic Clostridium botulinum and is the non-neurotoxigenic counterpart often used as exemplar for the toxic subtypes. In this work we have performed a microscopy study combined with a custom array-based analysis of C. sporogenes cycle, from dormant spores to early stationary phase. We identified in spores a total of 211 transcripts validating the ipothesis that mRNAs are abundant and strikingly different from those present in growing cells. The spores transcripts included genes responsible of different life-sustaining functions, suggesting the theory of transcripts entrapment or of a basic poly-functional genes activation for future steps. In addition, after three hours from the beginning of the germination process, a 20% of the total up-regulated genes were temporally expressed in germinating spores. The vegetative condition appears to be the more active in terms of gene transcription and protein synthesis and also genes for germination and sporulation factors seem to be expressed at this point. These results suggest that spores are not silent entities and a wider knowledge about genetic pathways involved in Clostridium life cycle could be of help for a better understanding also of pathogenic clostridia types. RNA was isolated from spores, germinating spores (3h), outgrowth/transitional mid-log vegetative cells and early stationary cells of Clostridium sporogenes UC9000 previously isolated from milk. There were 3 biological replicates (independent cultures) for each condition. Probes corresponding to 3400 genomic CDSs of C. botulinum A ATCC 3502 were synthesized in three replicates randomly distributed on the chip.

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Study of Bacillus cereus spore germination and outgrowth in a vegetable food model by means of genome-wide transcriptome analysis

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