Presumably the presence of the spoVA 2mob encoded proteins results in the increased uptake of DPA into the spore core, as was previously found for B. It is known that environmental conditions during sporulation, such as temperature, matrix and medium composition, can influence the heat resistance of spores of the B.
To allow for a direct comparison of the resistance properties of spores of the different strains, the applied sporulation conditions of all B. Furthermore, the temperature during sporulation can influence the heat resistance of B. In line with these findings, the heat resistances of spores of a B. Overall, the environmental conditions during sporulation, the presence or absence of genetic elements such as the spoVA 2mob operon, and the storage conditions of spores will ultimately determine the heat resistance properties of spores.
It is therefore conceivable that spores produced under laboratory conditions do not necessarily reach the same levels of heat resistance as spores found in foods van Zuijlen et al.
It is generally assumed for bacterial spore formers that higher optimal growth temperatures of vegetative cells correlates positively with spore heat resistance Nicholson et al. In this study, some differences were seen between strains with respect to their abilities to grow at different temperatures, but no consistent pattern was seen for strains that contain the Tn element and produce high level heat resistant spores versus the ones that do not harbor the element and produce low-level heat resistant spores.
All strains were able to grow at All genes present on the Tn transposon are under the control of sporulation-specific sigma factors K or G Berendsen et al. It is therefore in line with expectations that the genes on the Tn transposon, which determine spore heat resistance, do not influence the ability of vegetative cells to grow at high temperatures as these genes are not expressed during vegetative growth.
Variation in heat resistance of spores exists between strains of different spore forming species but also within species Oomes et al. In this study, a genomic analysis revealed the presence of Tn transposons in two strains of B. The presence of this transposon, containing the spoVA 2mob operon, correlated with high-level heat resistance of spores. Strains producing low or high level heat resistant spores showed similar temperature ranges for growth.
A functional role of the spoVA 2mob operon in increasing the heat resistance of spores was demonstrated by cloning these operons in B. Clearly, mere identification of the species of spores in food products does not provide information on the heat resistance levels of these spores.
The knowledge obtained in this study on the role of the spoVA 2mob operon in spore heat resistance can be used for specific detection of strains of the B. Multiple DNA based methods can be used for the detection of such genetic elements, such as whole genome sequencing and specific PCR detection, among others Caspers et al.
The ability to detect certain strains of B. EB and RK collected the data. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors would like to thank Patrick Janssen for technical assistance.
The authors have declared that no competing interests exist. The research was funded by TI Food and Nutrition, a public-private partnership on pre-competitive research in food and nutrition. The funding organization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplementary Datasheet 1. Supplementary Table 1. Alkema, W. Microbial bioinformatics for food safety and production. Arthur, M. Characterization of Tn , a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM Baril, E.
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PubMed Abstract Google Scholar. Kort, R. Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release. Kovacs, A. Ubiquitous late competence genes in Bacillus species indicate the presence of functional DNA uptake machineries. Krawczyk, A. Genome sequences of 12 spore-forming Bacillus species, comprising Bacillus coagulans, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus sporothermodurans , and Bacillus vallismortis , isolated from foods.
Kunst, F. The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature , — Li, L. OrthoMCL: identification of ortholog groups for eukaryotic genomes.
Genome Res. Li, Y. Role of a SpoVA protein in dipicolinic acid uptake into developing spores of Bacillus subtilis. Lima, L. Microbiota of cocoa powder with particular reference to aerobic thermoresistant spore-formers. Logan, N. Bacillus and relatives in foodborne illness. Active transposition of the Tn like transposon was not possible as only remnants of the transposase gene tnpA were found in all nine strains that produce high-level heat-resistant spores, suggesting that the active transfer of Tn like transposon is prone to evolutionary decay.
Therefore, natural transfer of this element to B. A transductant was selected that produced spores with significantly higher heat resistance than spores of strain Subsequent deletion of the Tn transposon from strain HR rendered spores that were much more sensitive to heat treatment than those of HR, and similar to those of strain Figure 2a.
A downward arrow means that counts were below the detection limit, i. The third operon on the Tn element, carrying genes that encode SpoVA homologs and four other genes designated spoVA 2mob , was demonstrated to confer high-level heat resistance of spores.
In addition, introduction of the spoVA 2mob operon into the amyE locus of strain rendered a strain amyE ::spoVA 2mob that produces spores with high-level heat resistance.
The impact of the spoVA 2mob element on spore heat resistance is much greater than some of the previously reported factors influencing Bacillus spore heat resistance, such as altered temperature, pH, salts and matrix composition during sporulation, which may lead to up to fold increases in the times required to inactivate spores Cazemier et al.
The other four transcriptional units present on the Tn element were not required for high-level heat resistance as the spores retained high-level heat resistance following deletion of each of these transcriptional units in strain HR Figure 2a. Sporulation-specific expression of all transcriptional units in the Tn transposon except for the fourth transcriptional unit containing the fragmented gene yetF, was seen in strain HR and food isolate B by RNA sequence analysis data not shown.
The encoded products may also determine spore properties other than heat resistance. Some of the encoded proteins were detected in extracts of spores of strain HR using mass spectrometry, revealing peptide fragments of the Mn catalase homolog and of proteins encoded by the first and the last gene on the spoVA 2mob operon Supplementary Table 5.
Additional evidence for the crucial role of spoVA 2mob genes in high-level heat resistance of spores came from detailed genome analysis of the B. The level of spore heat resistance was found to correlate with the number of spoVA 2mob operons present in the chromosome. Of the nine strains that produce high-level heat-resistant spores, one strain B carried one spoVA 2mob operon on a Tn -like transposon element inserted in yitF , and the average time to achieve one decimal reduction of its spores was 1.
In addition to these two Tn -like transposons, three strains B, B and B contained a third s poVA 2mob operon, which was flanked by genes of another mobile genetic element, but further genomic context could not be determined. Spores of these strains required as much as Three strains possess one Tn transposon including the spoVA 2mob operon , and spores of these strains had significantly higher heat resistances than those of the six strains that did not contain this transposon.
Two strains possess at least one Tn transposon including the spoVA 2mob operon , and produce spores with significantly higher heat resistances than the seven strains that did not carry the transposon. Despite extensive studies on many spo genes of B. The spoVA operon of B. Structural analysis of SpoVAD 1 revealed a binding pocket that is important for uptake of pyridine-2,6-dicarboxylic acid known as DPA during sporulation Li et al. SpoVAC 1 was recently shown to function as a mechanosensitive channel during germination, with increased probability of opening at increased membrane tension Velasquez et al.
Given the known roles of SpoVAC 1 and SpoVAD 1 in DPA uptake during sporulation, we hypothesize that proteins encoded by the spoVA 2mob operon have an important auxiliary role in this process, ultimately leading to higher heat resistance of spores. Interestingly, high levels of DPA were previously reported in spores of a B. At present, it has not been established which gene or which combinations of genes on the spoVA 2mob operon are essential and sufficient to convey high-level heat resistance of spores, but we did find that deletion of the last gene of unknown function fully abolished high-level heat resistance of spores Figure 2a , indicating at least its essential role.
The last gene of the spoVA 2mob operon encodes a protein that is predicted to be membrane bound by three transmembrane segments, and contains a DUF domain and a DUF domain. Homologs of this protein were neither found in B. It is not clear at this stage what roles the proteins containing these domains have in heat resistance of spores. It is conceivable that the Tn transposon found in the B. It is not clear whether the presence of the spoVA 2mob operon in B.
Limited sequence variation in key genes in the spoVA 2mob operon found in the B. Maximum likelihood core genome phylogenetic tree of spore-forming Bacillaceae , with indication of the number and type of spoVA operons present in the genomes, and proposed evolutionary scenarios. Three types of spoVA operons were identified in this analysis and are indicated in the tree. Third, a spoVA 2mob operon, which is a duplication of the spoVA 2 operon, but present on a mobile genetic element, e.
Strains of B. Six strains of B. Members of the B. Similarly, spoVA 2mob entered strains of B. Galperin et al. Interestingly, all evaluated strains belonging to the B.
However, some strains gained spoVA 2mob on the Tn transposon Figure 4. The determining role of the spoVA 2mob element in high-level spore heat resistance was experimentally confirmed for strains of B. Interestingly, the genomes of species notorious for very high-level heat resistance of their spores, namely B.
The exact roles of the spoVA 1 , spoVA 2 and spoVA 2mob operons in determining spore properties in these species remains to be established.
Horizontal gene transfer has an important role in bacteria to acquire resistance against selective pressures Ochman et al. The transfer of the spoVA 2mob operon to sporeformers occurs in the vegetative growth phase, subsequently leading to production of highly heat-resistant spores that can survive heat treatments routinely used in food processing.
The acquisition of the spoVA 2mob operon in food isolates may take place during growth in a food-processing environment, but it is also possible that such events occur during growth in other niches, such as soil or compost.
The competitive advantage of acquisition of these genes may also be related to properties other than merely heat resistance of spores. This study shows that horizontal gene transfer can profoundly affect heat resistance characteristics of spores.
Our finding that the spoVA 2mob operon on a Tn -like transposon has an important role in high-level heat resistance of Bacillus spores offers new opportunities for dealing with the problem of highly heat-resistant spores in food and health. Studying phenotypic properties of strains other than the well-studied laboratory strain in conjunction with analysis of their genomes proved to be a powerful approach to match phenotypes with underlying genetic traits. Characterization of Tn, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM J Bacteriol : — Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response.
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A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents. Spore resistance to wet heat is determined largely by the water content of spore core, which is much lower than that in the growing cell protoplast. A lower core water content generally gives more wet heat-resistant spores. However, how wet heat kills spores is not clear, although it is not through DNA damage.
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