title: Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a SpecificspoVA2mobOperon in Foodborne Strains of Bacillus subtilis.
abstract: Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some naturalBacillus subtilisisolates, encountered in foods, have been attributed to the occurrence of thespoVA2moboperon carried on the Tn1546transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboringspoVA2mobrequired higher HA temperatures for efficient germination than spores lackingspoVA2mobThe optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+(AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers.IMPORTANCEThis study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains ofB. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores.
authors.affiliation: Laboratory of Molecular Genetics, University of Groningen, Groningen, the Netherlands.|Top Institute Food and Nutrition (TIFN), Wageningen, the Netherlands.
authors.forename: Antonina O
authors.initials: AO
authors.lastname: Krawczyk
authors.affiliation: Laboratory of Molecular Genetics, University of Groningen, Groningen, the Netherlands.|Top Institute Food and Nutrition (TIFN), Wageningen, the Netherlands.
authors.forename: Anne
authors.initials: A
authors.lastname: de Jong
authors.affiliation: Laboratory of Molecular Genetics, University of Groningen, Groningen, the Netherlands.|Top Institute Food and Nutrition (TIFN), Wageningen, the Netherlands.
authors.forename: Jimmy
authors.initials: J
authors.lastname: Omony
authors.affiliation: Laboratory of Molecular Genetics, University of Groningen, Groningen, the Netherlands.|Top Institute Food and Nutrition (TIFN), Wageningen, the Netherlands.
authors.forename: Siger
authors.initials: S
authors.lastname: Holsappel
authors.affiliation: Top Institute Food and Nutrition (TIFN), Wageningen, the Netherlands.|NIZO Food Research B.V., Ede, the Netherlands.
authors.forename: Marjon H J
authors.initials: MHJ
authors.lastname: Wells-Bennik
authors.affiliation: Laboratory of Molecular Genetics, University of Groningen, Groningen, the Netherlands o.p.kuipers@rug.nl.|Top Institute Food and Nutrition (TIFN), Wageningen, the Netherlands.
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
authors.affiliation: Laboratory of Molecular Genetics, University of Groningen, Groningen, the Netherlands.|Top Institute Food and Nutrition (TIFN), Wageningen, the Netherlands.|NIZO Food Research B.V., Ede, the Netherlands.
authors.forename: Robyn T
authors.initials: RT
authors.lastname: Eijlander
mesh_terms: D000409:Alanine / Q000494:pharmacology; D001216:Asparagine / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D005516:Food Microbiology; D005519:Food Preservation; D005632:Fructose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006358:Hot Temperature; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009876:Operon; D010641:Phenotype; D013171:Spores, Bacterial / Q000187:drug effects / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; C090772:GerBA protein, Bacillus subtilis; D008565:Membrane Proteins; D005632:Fructose; D001216:Asparagine; D005947:Glucose; D000409:Alanine
keywords: Bacillus subtilis; food spoilage; germinant receptors; spoVA; spore germination; spore heat activation; spore heat resistance