Dormant bacterial spores encrypt a long-lasting transcriptional program to be executed during revival.

Sporulating bacteria can retreat into long-lasting dormant spores that preserve the capacity to germinate when propitious. However, how the revival transcriptional program is memorized for years remains elusive. We revealed that in dormant spores, core RNA polymerase (RNAP) resides in a central chromosomal domain, where it remains bound to a subset of intergenic promoter regions. These regions regulate genes encoding for most essential cellular functions, such as rRNAs and tRNAs. Upon awakening, RNAP recruits key transcriptional components, including sigma factor, and progresses to express the adjacent downstream genes. Mutants devoid of spore DNA-compacting proteins exhibit scattered RNAP localization and subsequently disordered firing of gene expression during germination. Accordingly, we propose that the spore chromosome is structured to preserve the transcriptional program by halting RNAP, prepared to execute transcription at the auspicious time. Such a mechanism may sustain long-term transcriptional programs in diverse organisms displaying a quiescent life form.

A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination.

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

A DELAY OF GERMINATION 1 (DOG1)-like protein regulates spore germination in the moss Physcomitrium patens.

DELAY OF GERMINATION 1 is a key regulator of dormancy in flowering plants before seed germination. Bryophytes develop haploid spores with an analogous function to seeds. Here, we investigate whether DOG1 function during germination is conserved between bryophytes and flowering plants and analyse the underlying mechanism of DOG1 action in the moss Physcomitrium patens. Phylogenetic and in silico expression analyses were performed to identify and characterise DOG1 domain-containing genes in P. patens. Germination assays were performed to characterise a Ppdog1-like1 mutant, and replacement with AtDOG1 was carried out. Yeast two-hybrid assays were used to test the interaction of the PpDOG1-like protein with DELLA proteins from P. patens and A. thaliana. P. patens possesses nine DOG1 domain-containing genes. The DOG1-like protein PpDOG1-L1 (Pp3c3_9650) interacts with PpDELLAa and PpDELLAb and the A. thaliana DELLA protein AtRGA in yeast. Protein truncations revealed the DOG1 domain as necessary and sufficient for interaction with PpDELLA proteins. Spores of Ppdog1-l1 mutant germinate faster than wild type, but replacement with AtDOG1 reverses this effect. Our data demonstrate a role for the PpDOG1-LIKE1 protein in moss spore germination, possibly alongside PpDELLAs. This suggests a conserved DOG1 domain function in germination, albeit with differential adaptation of regulatory networks in seed and spore germination.

Proteome plasticity during Physcomitrium patens spore germination - from the desiccated phase to heterotrophic growth and reconstitution of photoautotrophy.

The establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and ß-oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.

Co-evolution at protein-protein interfaces guides inference of stoichiometry of oligomeric protein complexes by de novo structure prediction.

The quaternary structure with specific stoichiometry is pivotal to the specific function of protein complexes. However, determining the structure of many protein complexes experimentally remains a major bottleneck. Structural bioinformatics approaches, such as the deep learning algorithm Alphafold2-multimer (AF2-multimer), leverage the co-evolution of amino acids and sequence-structure relationships for accurate de novo structure and contact prediction. Pseudo-likelihood maximization direct coupling analysis (plmDCA) has been used to detect co-evolving residue pairs by statistical modeling. Here, we provide evidence that combining both methods can be used for de novo prediction of the quaternary structure and stoichiometry of a protein complex. We achieve this by augmenting the existing AF2-multimer confidence metrics with an interpretable score to identify the complex with an optimal fraction of native contacts of co-evolving residue pairs at intermolecular interfaces. We use this strategy to predict the quaternary structure and non-trivial stoichiometries of Bacillus subtilis spore germination protein complexes with unknown structures. Co-evolution at intermolecular interfaces may therefore synergize with AI-based de novo quaternary structure prediction of structurally uncharacterized bacterial protein complexes.

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10 min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Secofumitremorgins C and D, a pair of atropisomers from saltern-derived fungus Aspergillus fumigatus GXIMD00544.

A pair of atropisomers secofumitremorgins C (1a) and D (1b), together with fifteen known alkaloids (2-16), were isolated from a saltern-derived fungus Aspergillus fumigatus GXIMD00544. The structures of atropisomers 1a and 1b were elucidated by the detailed spectroscopic data, chemical reaction and quantum chemical calculations. Compounds 1 and 8 displayed antifungal spore germination effects against plant pathogenic fungus associated with sugarcane Fusarium sp. with inhibitory rates of 53% and 77% at the concentration of 100 µM, repectively. Atropisomers 1 also exhibited antifouling potential against Balanus amphitrite larval settlement with an inhibitory rate of 96% at the concentration of 100 µM.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC.

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.

Genome-wide analysis of the GDSL esterase/lipase family genes in Physcomitrium patens and the involvement of GELP31 in spore germination.

In plants, the ability to produce hydrophobic substances that would provide protection from dehydration was required for the transition to land. This genome-wide investigation outlines the evolution of GDSL-type esterase/lipase (GELP) proteins in the moss Physcomitrium patens and suggests possible functions of some genes. GELP proteins play roles in the formation of hydrophobic polymers such as cutin and suberin that protect against dehydration and pathogen attack. GELP proteins are also implicated in processes such as pollen development and seed metabolism and germination. The P. patens GELP gene family comprises 48 genes and 14 pseudogenes. Phylogenetic analysis of all P. patens GELP sequences along with vascular plant GELP proteins with reported functions revealed that the P. patens genes clustered within previously identified A, B and C clades. A duplication model predicting the expansion of the GELP gene family within the P. patens lineage was constructed. Expression analysis combined with phylogenetic analysis suggested candidate genes for functions such as defence against pathogens, cutin metabolism, spore development and spore germination. The presence of relatively fewer GELP genes in P. patens may reduce the occurrence of functional redundancy that complicates the characterization of vascular plant GELP genes. Knockout lines of GELP31, which is highly expressed in sporophytes, were constructed. Gelp31 spores contained amorphous oil bodies and germinated late, suggesting (a) role(s) of GELP31 in lipid metabolism in spore development or germination. Future knockout studies of other candidate GELP genes will further elucidate the relationship between expansion of the family and the ability to withstand the harsh land environment.

Ger1 is a secreted aspartic acid protease essential for spore germination in Ustilago maydis.

Ustilago maydis expresses a number of proteases during its pathogenic lifecycle. Some of the proteases including both intracellular and extracellular ones have previously been shown to influence the virulence of the pathogen. However, any role of secreted proteases in the sporulation process of U. maydis have not been explored earlier. In this study we have investigated the biological function of one such secreted protease, Ger1 belonging to aspartic protease A1 family. An assessment of the real time expression of ger1 revealed an infection specific expression of the protein especially during late phases of infection. We also evaluated any contribution of the protein in the pathogenicity of the fungus. Our data revealed an involvement of Ger1 in the sporulation and spore germination processes of U. maydis. Ger1 also showed positive influence on the pathogenicity of the fungus and accordingly the ger1 deletion mutant exhibited reduced pathogenicity. The study also demonstrated the protease activity associated with Ger1 to be essential for its biological function. Fluorescence microscopy of maize plants infected with U. maydis cells expressing Ger1-mcherry-HA also revealed that Ger1 is efficiently secreted within maize apoplast.

Host Specificity and Seasonal Variation in the Colonization of Tubakia sensu lato Associated with Evergreen Oak Species in Eastern Japan.

Foliar fungal endophytes are ubiquitous and hyperdiverse, and tend to be host-specific among dominant forest tree species. The fungal genus Tubakia sensu lato is comprised of foliar pathogens and endophytes that exhibit host preference for Quercus and other Fagaceae species. To clarify interspecific differences in ecological characteristics among Tubakia species, we examined the endophyte communities of seven evergreen Quercus spp. at three sites in eastern Japan during summer and winter. Host tree species was the most significant factor affecting endophyte community composition. Tubakia species found at the study sites were divided into five specialists and three generalists according to their relative abundance in each host species and their host ranges. Specialists were dominant on their own host in summer, and their abundance declined in winter. To test the hypothesis that generalists are more widely adapted to their environment than specialists, we compared their spore germination rates at different temperatures. Spores of generalist Tubakia species were more tolerant of colder temperatures than were spores of specialist Tubakia species, supporting our hypothesis. Seasonal and site variations among Tubakia species were also consistent with our hypothesis. Host identity and ecology were significantly associated with endophyte community structure.

Applying the first microcapsule-based self-healing microbial-induced calcium carbonate materials to prevent the migration of Pb ions.

Lead (Pb) accumulation can lead to serious threats to surrounding environments and damage to the liver and kidneys. In the past few years, microbial-induced carbonate precipitation (MICP) technology has been widely applied to achieve Pb immobilization due to its environmentally friendly nature. However, harsh pH conditions can cause the instability of the carbonate precipitation to degrade or dissolve, increasing the potential of Pb2+ migration into nearby environments. In this study, microcapsule-based self-healing microbial-induced calcium carbonate (MICC) materials were applied to prevent Pb migration. The highest sporulation rate of 95.8% was attained at 7 g/L yeast extract, 10 g/L NH4Cl, and 3.6 g/L Mn2+. In the germination phase, the microcapsule not only prevented the bacterial spores from being threatened by the acid treatment but secured their growth and reproduction. Micro analysis also revealed that cerussite, calcite, and aragonite minerals were present, while extracellular polymeric substances (EPSs) were identified via Fourier transform infrared spectroscopy (FTIR). These results confirm their involvement in combining Pb2+ and Ca2+. The immobilization efficiency of above 90% applied to MICC materials was attained, while it of below 5% applied to no MICC use was attained. The findings explore the potential of applying microcapsule-based self-healing MICC materials to prevent Pb ion migration when the calcium carbonate degrades under harsh pH conditions.

Effects of Temperature and Moisture Duration on Spore Germination of Four Fungi that Cause Grapevine Trunk Diseases.

Grapevine trunk diseases (GTDs) are serious threats worldwide and are difficult to control, in part because the environmental requirements for epidemiological processes of the causal fungi are poorly understood. Therefore, we investigated the effects of temperature and moisture duration on spore germination of four fungi associated with two GTDs (esca complex and Eutypa dieback): Phaeomoniella chlamydospora, Phaeoacremonium minimum, Cadophora luteo-olivacea, and Eutypa lata. Conidia of Phaeomoniella chlamydospora, Phaeoacremonium minimum, and C. luteo-olivacea were similar: conidia of these fungi germinated profusely (>90%) between 20 and 30°C; Phaeomoniella chlamydospora and Phaeoacremonium minimum tended to germinate at higher temperatures (up to 40°C for P. minimum), and C. luteo-olivacea at lower temperatures (as low as 5°C). E. lata ascospores germinated between 10 and 30°C. The required duration of moist periods for germination was shortest for C. luteo-olivacea (about 6 h), followed by P. minimum and E. lata (about 12 h) and Phaeomoniella chlamydospora (about 24 h). Further research on the environmental requirements of GTD fungi may increase our ability to predict infection periods and, thereby, improve disease control.

Temperature-Dependent Growth and Spore Germination of Fungi Causing Grapevine Trunk Diseases: Quantitative Analysis of Literature Data.

Grapevine trunk diseases (GTDs) are serious threats in all viticultural areas of the world, and their management is always complex and usually inadequate. Fragmented and inconsistent information on the epidemiology and environmental requirements of the causal fungi is among the reasons for poor disease control. Therefore, we conducted a quantitative analysis of literature data to determine the effects of temperature on mycelial growth and the effects of temperature and moisture duration on spore germination. Using the collected information, we then developed mathematical equations describing the response of mycelial growth to temperature, and the response of spore germination to temperature and moisture for the different species and disease syndromes. We considered 27 articles (selected from a total of 207 articles found through a systematic literature search) and 116 cases; these involved 43 fungal species belonging to three disease syndromes. The mycelial growth of the fungi causing Botryosphaeria dieback (BD) and the esca complex (EC) responded similarly to temperature, and preferred higher temperatures than those causing Eutypa dieback (ED) (with optimal temperature of 25.3, 26.5, and 23.3°C, respectively). At any temperature, the minimal duration of the moist period required for 50% spore germination was shorter for BD (3.0 h) than for EC (17.2 h) or ED (15.5 h). Mathematical equations were developed accounting for temperature-moisture relationships of GTD fungi, which showed concordance correlation coefficients ≥0.888; such equations should be useful for reducing the risk of infection.

An Aniline-Substituted Bile Salt Analog Protects both Mice and Hamsters from Multiple Clostridioides difficile Strains.

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, the rate of CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA (cholic acid substituted with m-aminosulfonic acid) was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active in preventing spore germination by other C. difficile ribotypes, including the hypervirulent strain R20291. The strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, CaPA (cholic acid substituted with phenylamine), was found to be a better antigerminant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs with all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA's efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify antigerminants with broad CDI prophylaxis activity.

Heat Activation and Inactivation of Bacterial Spores: Is There an Overlap?

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has the potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination and then inactivating the less-heat-resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single-spore levels. The heat treatments used were 40 to 80°C and for 0 to 300 min. The results were as follows. (i) Heat activation at 40 to 70°C promoted l-valine- and l-asparagine-glucose-fructose-potassium (AGFK)-induced germination in a time-dependent manner. (ii) The optimal heat activation temperatures for AGFK and l-valine germination via the GerB plus GerK or GerA germinant receptors were 65°C and 50 to 65°C, respectively. (iii) Heat inactivation of dormant spores appeared at 70°C, and the heat damage of molecules essential for germination and growth began at 70 and 65°C, respectively. (iv) Heat treatment at 75°C resulted in both activation of germination and damage to the germination apparatus, and 80°C treatment caused more pronounced heat damage. (v) For the spores that should withstand adverse environmental temperatures in nature, heat activation seemed functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth. IMPORTANCE Bacterial spores are thermal-stress-resistant structures that can thus survive food preservation strategies and revive through the process of spore germination. The more heat resistant spores are, the more heterogeneous their germination upon the addition of germinants. Upon germination, spores can cause food spoilage and food intoxication. Here, we provide new information on both heat activation and inactivation regimes and their effects on the (heterogeneity of) spore germination.

Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores.

The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

Artificial germination of Enterocytozoon hepatopenaei (EHP) spores induced by ions under the scanning electron microscope.

Enterocytozoon hepatopenaei (EHP), is an emerging microsporidian pathogen responsible for hepatopancreatic microsporidiasis (HPM) in shrimps and is associated with severe growth retardation. The disease causes economic losses in shrimp aquaculture. In this study, EHP spore germination was induced and demonstrated with a scanning electron microscope (SEM). The ions (cations and anions) generated by high-energy electrons during frozen water radiolysis in the SEM specimen chamber induce EHP spore germination. This study is the first to demonstrate the induction of a microsporidian spore germination by ions generated under SEM. This study will enhance our understanding of EHP biology, life cycle and lead to the development of prophylactics and therapeutics for EHP control. Also, this method will help standardize the study of germination in other microsporidians.

Inactivation mechanism of slightly acidic electrolyzed water on Bacillus cereus spores.

This study aimed to investigate the inactivation mechanism of Bacillus cereus spores by slightly acidic electrolyzed water (SAEW). Spore inactivation efficacy of SAEW at different available chlorine concentrations (ACC, 20, 60 and 100 mg/L), as well as spore structures change, coat damage, mutagenesis, and inner membrane (IM) properties were examined. The viability of treated spores with lysozyme addition and spore germination induced by germinant was also examined. The results showed that SAEW could reach maximal 5.81 CFU/mL log reduction with ACC of 100 mg/L for 20 min treatment. Scanning and transmission electron photomicrographs indicated that SAEW treatment rendered spore surface ruptured, IM damage and core contents loss. No mutants were generated in survivors of SAEW treated-spores. SAEW significantly weakened spore viability in high salt medium, losing its ability to retain pyridine-2,6-dicarboxylic acid (DPA) at 85 °C. SAEW-treated spores germinated with l-alanine or inosine induction were mostly stained with propidium iodide (PI) but could not recover via lysozyme addition. Furthermore, SAEW treatment inhibited spore germination in the induction of germinant (mixture of l-alanine and inosine or dodecylamine). These findings indicated that SAEW inactivated spore primarily by damaging the spore IM.

Synergistic interaction between pH and NaCl in the limits of germination and outgrowth of Clostridium sporogenes and Group I Clostridium botulinum vegetative cells and spores after heat treatment.

Group I Clostridium botulinum and Clostridium sporogenes are physiologically and genetically closely related. Both are widely distributed in the environment and can cause foodborne botulism. In this work, a physiological study was conducted with 37 isolates from spoiled canned food and five referenced strains of C. sporogenes (three isolates) and Group I C. botulinum (two isolates). Growth limits of vegetative cells were established as a function of pH and NaCl concentration in PYG modified medium (PYGm) at 30 °C for 48 days. The heat resistance of the spores was studied for 2 min and 10 min at 102 °C and 110 °C. This physiological study (pH, NaCl growth limits and heat resistance) allowed the selection of 14 isolates of C. sporogenes (twelve isolates) and Group I C. botulinum (two isolates) representative of the diversity found. This panel of 14 selected isolates (11 isolated from spoiled canned food and three reference strains), were whole genome sequenced, but no association of physiological and genetic characteristics could be detected. Finally, we studied the ability of spores to germinate and grow from 5 isolates (four C. sporogenes and one Group I C. botulinum), under stress conditions generated by pH and NaCl following a low intensity heat treatment. The accumulation of these 3 stresses creates synergies that will strongly reduce the probability of spore growth in pH and salt conditions where they usually proliferate. The effect is progressive as the conditions become drastic: the number of decimal reduction observed increases translating a probability of growth which decreases. This study provides a better understanding of the behaviour of C. sporogenes and Group I C. botulinum isolates and shows how the combination of pH, NaCl and heat treatment can help prevent or minimise foodborne botulism outbreaks.