Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus.

The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.

Dynamics of bacterial and fungal communities of mango: From the tree to ready-to-Eat products.

Processing, such as fresh cutting and drying, is essential to enhance profitability; therefore, to limit waste and reduce losses in fruit production such as mangoes. Metabarcoding and microbial enumeration methods were utilized to explore the structure of mango microbiota, as well as their evolution after processing. Two mango ripening stages of cv. Cogshall were selected and processed into fresh-cut pieces or dried slices. Microbiological and physicochemical parameters were monitored during product storage, in order to assess the dynamics of quantitative and qualitative variations of the microbial flora. Proteobacteria was the dominant bacterial phylum of the mango surface and accounted for 73.16%, followed by Actinobacteria (10.16%), Bacteroidetes (7.82%) and Firmicutes (6.68%). Aureobasidium and Cladosporium were the only two genera shared between all types of samples (peel surface, dried slices and mango fresh-cut). However, the bacterial genera Lactobacillus and Pantoea were the most abundant in fresh-cut mango after 14 days of storage. Ascomycota was the dominant fungal phylum in the mango surface and accounted for 90.76% of the total number of detected sequences, followed by Basidiomycota (9.21%). In total, 866 microbial genera were associated with mango surface (562 bacterial and 304 fungal). Among detected yeast genera, Saccharomyces, Candida and Malassezia prevailed in mango flesh and were replaced by Wickerhamomyces after 14 days of storage. Alpha and beta diversity analyzes revealed differences in fungal and bacterial communities on fruit peel, in fresh-cut, dried slices, and during conservation (fresh-cut and dried slices). Mango processing (washing, peeling, cutting and drying) reduced the richness and the microbial diversity (bacterial and fungal) associated to the fruit, and drying limits the development of cultivable microorganisms during storage in comparison to fresh-cuts mangoes.

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures.

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

Short-Chain and Unsaturated Fatty Acids Increase Sequentially From the Lag Phase During Cold Growth of Bacillus cereus.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6-7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus.

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of BacillaceaeIMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.

Terroir Is the Main Driver of the Epiphytic Bacterial and Fungal Communities of Mango Carposphere in Reunion Island.

The diversity of both bacterial and fungal communities associated with mango surface was explored using a metabarcoding approach targeting fungal ITS2 and bacterial 16S (V3-V4) genomic regions. Fruits were collected in Reunion Island from two different orchards according to a sampling method which allowed the effect of several pre-harvest factors such as geographical location (terroir), cultivars, fruit parts, tree position in the plot, fruit position on the tree (orientation and height), as well as the harvest date to be investigated. A total of 4,266,546 fungal and 2,049,919 bacterial reads were recovered then respectively assigned to 3,153 fungal and 24,087 to bacterial amplicon sequence variants (ASVs). Alpha and beta diversity, as well as differential abundance analyses revealed variations in both bacterial and fungal communities detected on mango surfaces depended upon the studied factor. Results indicated that Burkholderiaceae (58.8%), Enterobacteriaceae (5.2%), Pseudomonadaceae (4.8%), Sphingomonadaceae (4.1%), Beijerinckiaceae (3.5%), and Microbacteriaceae (3.1%) were the dominant bacterial families across all samples. The majority of fungal sequences were assigned to Mycosphaerellaceae (34.5%), Cladosporiaceae (23.21%), Aureobasidiaceae (13.09%), Pleosporaceae (6.92%), Trichosphaeriaceae (5.17%), and Microstromatales_fam_Incertae_sedis (4.67%). For each studied location, mango fruit from each cultivar shared a core microbiome, and fruits of the same cultivar harvested in two different locations shared about 80% fungal and bacterial family taxa. The various factors tested in this study affected bacterial and fungal taxa differently, suggesting that some taxa could act as geographical (terroir) markers and in some cases as cultivar fingerprints. The ranking of the factors investigated in the present study showed that in decreasing order of importance: the plot (terroir), cultivar, fruit parts, harvest date and the position of the fruits are respectively the most impacting factors of the microbial flora, when compared to the orientation and the fruit position (height) on the tree. Overall, these findings provided insights on both bacterial and fungal diversity associated with the mango surface, their patterns from intra-fruit scale to local scale and the potential parameters shaping the mango microbiota.

Impact of temperature and oxygen on the fate of Bacillus weihenstephanensis in a food-based medium.

The capacity of the Bacillus weihenstephanensis KBAB4 strain, a psychrotolerant species of the B. cereus sensu lato group, to multiply in carrot broth at 8 °C and 30 °C, in presence or absence of oxygen was determined. In aerobic carrot broth tyndallized in presence of oxygen, at both temperatures, the population of vegetative cells of B. weihenstephanensis inoculated at a level of 103 or 106 CFU/ml dropped immediately. After 16 h at 30 °C, B. weihenstephanensis reached around 103 CFU/ml, indicating that some vegetative cells had survived and multiplied, with lipid inclusions accumulated in cells, indicating possible stressing conditions. At 8 °C, no multiplication of B. weihenstephanensis was observed during 3 days to at least 12 days, depending of carrot broth batches. In anaerobic carrot broth tyndallized without oxygen, the vegetative cells of B. weihenstephanensis were not killed upon inoculation and multiplied in the broth at both 30 °C and 8 °C. Comparison with results from previous studies shows that B. weihenstephanensis behaves differently in carrot broth and in laboratory media at 8 °C with regards to presence or absence of oxygen.

Bacillus cereus cshA Is Expressed during the Lag Phase of Growth and Serves as a Potential Marker of Early Adaptation to Low Temperature and pH.

Bacterial adaptation is characterized by a lag phase during which cells do not multiply or modify their physiology to cope with the constraints of their environment. Our aim was to determine a sequence of events during the lag phase of growth at low temperature and pH for three Bacillus cereus strains. The onsets of expression of two genes, one of which is essential for stress adaptation (cshA, coding for a RNA helicase) and one of which is involved in the transition between lag phase and exponential phase (abrB, coding for a transition regulator), were determined using fluorescent transcriptional reporter systems. Regardless of the stressing conditions and the tested strains, the cshA promoter was active very early, while the biomass increased and always did so before the first cell division. At 12°C and pH 7.0, the onset of cshA promoter activity occurred at between 3 h and 7 h, while the bacterial counts started to increase at between 12 h and 13 h. At pH 5.0 and at 20°C or 30°C, the onset of cshA promoter activity occurred before 1 h and earlier than at pH 7.0. In contrast, the onset of abrB promoter activity depended on the strain and the stressing conditions. In the ATCC 14579 strain, the onset of abrB promoter activity always started at between 30 min and 3 h, before biomass increased and cell division occurred. For the other strains, it took place along with the first cell division at 12°C but did so much later during growth under the other tested conditions.IMPORTANCE The spore-forming bacterium B. cereus is a major cause of foodborne outbreaks in Europe. Some B. cereus strains can grow at low temperatures and low pH in many processed foods. Modeling of the bacterial lag time is hampered by a lack of knowledge of the timing of events occurring during this phase. In this context, the identification of lag phase markers, not currently available, could be a real advance for the better prediction of lag time duration. Currently, no molecular markers of this phase are available. By determining that cshA was always expressed early during the lag phase, we provide a molecular marker of the early adaptation process of B. cereus cells when exposed to low temperature and pH.

Sporulation environment influences spore properties in Bacillus: evidence and insights on underlying molecular and physiological mechanisms.

Bacterial spores are resistant to physical and chemical insults, which makes them a major concern for public health and industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties.

Temperature impacts the sporulation capacities and spore resistance of Moorella thermoacetica.

Temperatures encountered in cannery allow growth of thermophilic spore-forming bacteria, including the strictly anaerobe Moorella thermoacetica, which grows optimally from 55 °C to 65 °C and is the main cause of spoilage of low-acid canned foods (LACFs) at high temperature. Resistance to wet-heat, biocides and UV-C of spores formed at different temperatures was assessed either for a selection of M. thermoacetica strains or for the strain M. thermoacetica ATCC 39073. Spores formed at 45 °C were significantly more sensitive to wet-heat than spores produced at 55 °C, while spores produced at 65 °C were as heat-resistant as spores produced at 55 °C. Spores of M. thermoacetica ATCC 39073 produced at 45 °C were significantly less resistant to peracetic acid than spores formed at 55 °C, while no difference in sensitivity to H2O2 or to UV-C treatment was observed whatever the sporulation temperature. However, both types of treatment enabled at least a 3.3 log CFU/mL reduction of M. thermoacetica ATCC 39073 spores. M. thermoacetica spores thus showed higher resistance properties when sporulation temperature was close to optimal growth temperature. These findings suggest food spoilage due to M. thermoacetica species could be controllable by holding temperatures below optimal growth temperature from the blanching step to the can filling step.

Cereulide production by Bacillus weihenstephanensis strains during growth at different pH values and temperatures.

Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10-15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.

Draft Genome Sequences of 18 Psychrotolerant and 2 Thermotolerant Strains Representative of Particular Ecotypes in the Bacillus cereus Group.

Bacteria from the Bacillus cereus group exhibit genetic and physiological diversity through different ecotypes. Here, we present the draft genome sequences of 20 bacterial strains belonging to the contrasted psychrotolerant and thermotolerant ecotypes.

Heat-resistance of psychrotolerant Bacillus cereus vegetative cells.

Spores of psychrotolerant strains of the foodborne pathogen Bacillus cereus can multiply during storage of cooked or pasteurized, refrigerated foods and can represent a risk if these cells are not eliminated during reheating of food product before consumption. We determined the heat-resistance of psychrotolerant B. cereus vegetative cells at different heating temperatures in laboratory medium and compared it with that of thermotolerant B. cereus vegetative cells. The z values, based on times for a 3 log10 reduction, of the vegetative cells of the three psychrotolerant phylogenetic groups of B. cereus varied between 3.02 °C and 4.84 °C. The temperature at which a 3 log10 reduction was achieved in 10 min varied between 47.6 °C and 49.2 °C for psychrotolerant vegetative cells and it was around 54.8 °C for thermotolerant vegetative cells. Moreover, 0.4 min at 60 °C would be sufficient for a 6 log10 CFU/ml reduction of the most heat resistant psychrotolerant B. cereus vegetative cells. These data clearly showed that psychrotolerant B. cereus vegetative cells can be rapidly eliminated by a mild heat treatment such as food reheating.

Knowledge of the physiology of spore-forming bacteria can explain the origin of spores in the food environment.

Spore-forming bacteria are able to grow under a wide range of environmental conditions, to form biofilms and to differentiate into resistant forms: spores. This resistant form allows their dissemination in the environment; consequently, they may contaminate raw materials. Sporulation can occur all along the food chain, in raw materials, but also in food processes, leading to an increase in food contamination. However, the problem of sporulation during food processing is poorly addressed and sporulation niches are difficult to identify from the farm to the fork. Sporulation is a survival strategy. Some environmental factors are required to trigger this differentiation process and others act by modulating it. The efficiency of sporulation is the result of the combined effects of these two types of factors on vegetative cell metabolism. This paper aims to explain and help identify sporulation niches in the food chain, based on features of spore-former physiology.

Combined effect of anaerobiosis, low pH and cold temperatures on the growth capacities of psychrotrophic Bacillus cereus.

Psychrotrophic strains of the foodborne pathogen Bacillus cereus can multiply during the refrigerated storage of food products. The aim of this study was to determine the impact of anaerobiosis on the growth of two psychrotrophic B. cereus strains exposed to acidic pH at a cold temperature in a laboratory medium. At 10 °C, growth occurred at pH values equal to or higher than 5.7 during anaerobiosis, whereas aerobic growth was observed from pH 5.4. Growth rates during aerobiosis were similar at pH 5.4 and pH 7. No growth was observed for the two tested strains at 8 °C without oxygen regardless of the pH; however, both strains grew at this temperature from pH 5.4 in the presence of oxygen. These pH growth limits in aerobiosis are consistent with those reported for different strains and different foods or media, but no other studies have described anaerobic growth at acidic pH values. The maximal B. cereus concentration was approximately 6.0 log10 CFU/ml for cultures in the absence of oxygen and approximately 8.0 log10 CFU/ml for cultures in the presence of oxygen. In conclusion, we found that the combination of anaerobiosis, pH < 5.7 at 10 °C, or anaerobiosis and temperatures ≤8 °C prevent psychrotrophic B. cereus growth.

Expression of the genes encoding the CasK/R two-component system and the DesA desaturase during Bacillus cereus cold adaptation.

Two-component systems (TCS) allow a cell to elaborate a variety of adaptive responses to environment changes. The recently discovered CasK/R TCS plays a role in the optimal unsaturation of fatty acids necessary for cold adaptation of the foodborne-pathogen Bacillus cereus Here, we showed that the promoter activity of the operon encoding this TCS was repressed during growth at low temperature in the stationary phase in the parental strain when compared to the casK/R mutant, suggesting that CasR negatively regulates the activity of its own promoter in these conditions. The promoter activity of the desA gene encoding the Δ5 fatty acid desaturase, providing unsaturated fatty acids (UFAs) required for low temperature adaptation, was repressed in the casK/R mutant grown at 12°C versus 37°C. This result suggests that CasK/R activates desA expression during B. cereus growth at low temperature, allowing an optimal unsaturation of the fatty acids. In contrast, desA expression was repressed during the lag phase at low temperature in presence of UFAs, in a CasK/R-independent manner. Our findings confirm that the involvement of this major TCS in B. cereus cold adaptation is linked to the upregulation of a fatty acid desaturase.

Agrifood systems and the microbial safety of fresh produce: Trade-offs in the wake of increased sustainability.

Fresh produce has been a growing cause of food borne outbreaks world-wide prompting the need for safer production practices. Yet fresh produce agrifood systems are diverse and under constraints for more sustainability. We analyze how measures taken to guarantee safety interact with other objectives for sustainability, in light of the diversity of fresh produce agrifood systems. The review is based on the publications at the interface between fresh produce safety and sustainability, with sustainability defined by low environmental impacts, food and nutrition security and healthy life. The paths for more sustainable fresh produce are diverse. They include an increased use of ecosystem services to e.g. favor predators of pests, or to reduce impact of floods, to reduce soil erosion, or to purify run-off waters. In contrast, they also include production systems isolated from the environment. From a socio-economical view, sustainability may imply maintaining small tenures with a higher risk of pathogen contamination. We analyzed the consequences for produce safety by focusing on risks of contamination by water, soil, environment and live stocks. Climate change may increase the constraints and recent knowledge on interactions between produce and human pathogens may bring new solutions. Existing technologies may suffice to resolve some conflicts between ensuring safety of fresh produce and moving towards more sustainability. However, socio-economic constraints of some agri-food systems may prevent their implementation. In addition, current strategies to preserve produce safety are not adapted to systems relying on ecological principles and knowledge is lacking to develop the new risk management approaches that would be needed.

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores.

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.