Project leader: Dr Marjon Wells-Bennik
Time frame 2011 – 2014
Project code: SP002
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Many gram-positive bacteria can form spores that ensure survival under extreme conditions. Spores can survive adverse conditions and remain dormant for a long time, but germination and outgrowth can occur under favourable conditions. Current food-preservation techniques can fail to kill sufficient numbers of bacterial spores present in raw ingredients. When viable spores remain and eventually germinate, the vegetative cells can grow out in the final product, resulting in food spoilage, reduced shelf-life and possible food poisoning.

This project aimed to broaden our knowledge of spore resistance and spore germination and outgrowth, using not only model Bacillus strains but also a wide variety of industrially-relevant spore formers. These strains were studied at phenotypic and genomic levels; the genome sequences of 51 spore-forming Bacilli were determined and analysed in relation to key spore properties.

Spore properties and germination behaviour of spores were determined during the sporulation process. Once resistant phase-bright spores are formed, they can remain dormant for prolonged periods of time. Upon sensing germinants in their environment, spores can germinate and vegetative cells can emerge, followed by outgrowth if conditions are favourable. Heterogeneous expression of genes that play important roles in germination were studied at the single-cell level using time-lapse microscopy. This work led to interesting observations, such as premature spore lysis and heterogeneous promoter activity, which were studied in more detail. Importantly, this work also showed that the origin of germination heterogeneity could not be traced back to heterogeneity in gene expression, but that this is the result of heterogeneity in downstream processes, such as translation, modification and/or localisation of germination proteins.

In addition, spore superdormancy was studied in this project. Superdormant spores are defined as dormant spores that fail to respond to germinants/nutrients that normally activate germinant receptors (these receptors play a key role in initiation of germination). Such spores can be present amongst spores that germinate well in heterogeneous spore populations. To quantify spore superdormancy, a pipeline was set up using a novel flow-cytometry approach, in which the responsiveness of superdormant spores to nutrient and non-nutrient germination triggers can be assessed. The phenotypes related to superdormancy were assessed for 20 B. cereus industrial isolates, and significant phenotypical diversity was observed amongst these strains. Linking the genome sequences of these strains with phenotypic trait-relevant spore characteristics provides an excellent opportunity to extend our knowledge of these processes – from a limited number of reference strains to industrial isolates.

When dormant spores sustain damage, repair of such damage upon germination can be required, prior to outgrowth. The contribution of different recovery conditions (which include various food matrices) to the ability of sub-lethally injured spores to grow out, has been identified. In addition, transcriptome analyses, during germination, emergence of the vegetative cell and subsequent outgrowth, have been performed, yielding new target genes that could be involved in these processes. To extrapolate the observations related to the effects of sub-lethal treatments of model strains to industrial isolates, the characterisation of a range of spores of B. cereus industrial isolates was performed.

Another important property of spores is their heat resistance, which was also studied. Within the species B. subtilis, spore heat-resistance was assessed for a large number of strains. Two distinct groups could be identified, with spores from the highly heat-resistant group requiring more-intense heating for inactivation than the low-heat-resistant group. The genome sequences of these strains were analysed and, by performing gene-trait matching, a mobile genetic element was identified which was specifically present in the genomes of strains with high spore heat-resistance. It was demonstrated that this element was responsible for the increased heat-resistance of spores. This element is not restricted to B. subtilis and is found in many other spore-forming Bacilli, indicating a role in evolution and adaptation of these strains. This study provides insight into prediction of spore heat-inactivation processes and the mechanism by which increased spore heat-resistance is mediated and will, ultimately, allow for better detection and control of highly heat-resistant bacterial spores in foods.

In the last part of the project knowledge gained in model spore-forming organisms was translated to wild-type food strains. The genomic content of 47 Bacillus spp. food isolates (which cause problems to industry) was determined via whole-genome sequencing, contig assembly and gene annotation. For selected strains (13 B. subtilis and 5 B. thermoamylovorans) a phenotypic characterisation of germination properties, under selected conditions, was performed and significant differences between the strains of each species were observed. Future work will include studying identified genetic factors to validate their suggested roles in the phenotype with which they are associated.