The effect of calcium on the transcriptome of sporulating B. subtilis cells.

Bacterial spores formed in the presence of high concentrations of minerals are a major problem in the food industry because of their extreme heat resistance. In order to enhance our insight in the molecular mechanisms underlying this phenomenon we have performed a detailed time-resolved analysis of the genome-wide transcriptome pattern of Bacillus subtilis sporulated both in the absence and presence of high calcium concentrations. The data was analysed in two ways. First, we determined the influence of the presence of high calcium levels during sporulation on the expression of gene groups as defined in Subtilist and KEGG pathways database. Second, we assessed the differential expression at the level of individual genes. When analysing groups and pathways, we found that those annotated as being involved in sporulation were significantly affected. Also, groups and pathways involved in flagella formation and biofilm matrix production were affected by the presence of calcium in the sporulation medium. When we analysed the behaviour of individual genes we found 305 genes influenced by calcium, including all known spore coat polysaccharide biosynthesis genes (10 induced and 1 repressed). A number of the calcium affected genes were also involved in biofilm formation. Minimal overlap with other stress outputs like sigma B activation and weak acid stress response was noted. Those genes that did overlap were unique to that combination which corroborates the notion that the cells sense these conditions differently.

The characterisation of Bacillus spores occurring in the manufacturing of (low acid) canned products.

Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.