The role of endospore appendages in spore-spore interactions in the pathogenic Bacillus cereus group.

Species within the Bacillus cereus sensu lato group, known for their spore-forming ability, are recognized for their significant role in food spoilage and food poisoning. The spores of B. cereus are adorned with numerous pilus-like appendages, referred to as S-ENAs and L-ENAs. These appendages are thought to play vital roles in self-aggregation, adhesion, and biofilm formation. Our study investigates the role of S-ENAs and L-ENAs, as well as the impact of various environmental factors on spore-to-spore contacts and the interaction between spores and vegetative cells, using both bulk and single-cell approaches. Our findings indicate that ENAs, especially their tip fibrillae, play a crucial role in spore self-aggregation, but not in the adhesion of spores to vegetative cells. The absence of L-BclA, which forms the L-ENA tip fibrillum, reduced spore aggregation mediated by both S-ENAs and L-ENAs, highlighting the interconnected roles of S-ENAs and L-ENAs. We also found that increased salt concentrations in the liquid environment significantly reduced spore aggregation, suggesting a charge dependency of spore-spore interactions. By shedding light on these complex interactions, our study offers valuable insights into spore dynamics. This knowledge can inform future studies on spore behaviour in environmental settings and assist in developing strategies to manage bacterial aggregation for beneficial purposes, such as controlling biofilms in food production equipment.

EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579.

BACKGROUND: Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished. METHODS: We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar. RESULTS: Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567. CONCLUSION: B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.

Effects of Na+ adaptation on Bacillus cereus endospores inactivation and transcriptome changes.

As Bacillus cereus endospores exist in various vegetables grown in soil, the possibility of contamination in food products with high salt concentrations cannot be ignored. Recent studies revealed that harsh conditions affect the resistance of bacteria; thus, we investigated the developmental aspect of heat resistance of B. cereus after sporulation with high NaCl concentration. RNA sequencing was conducted for transcriptomic changes when B. cereus endospores formed at high salinity, and membrane fluidity and hydrophobicity were measured to verify the transcriptomic analysis. Our data showed that increasing NaCl concentration in sporulation media led to a decrease in heat resistance. Also, endospore hydrophobicity, membrane fluidity, and endospore density decreased with sporulation at higher NaCl concentrations. When the transcript changes of B. cereus sporulated at NaCl concentrations of 0.5 and 7% were analyzed by transcriptome analysis, it was confirmed that the NaCl 7% endospores had significantly lower expression levels (FDR<0.05) of genes related to sporulation stages 3 and 4, which led to a decrease in expression of spore-related genes such as coat proteins and small acid-soluble proteins. Our findings indicated that high NaCl concentrations inhibited sporulation stages 3 and 4, thereby preventing proper cell maturation in the forespores and adequate formation of the coat protein and cortex. This inhibition led to decreased endospore density and hydrophobicity, ultimately resulting in reduced heat resistance.resistanceWe expect that this study will be utilized as a baseline for further studies and enhance sterilization strategies.

[Deficiency in N-acetylglucosamine transport affects the sporulation process and increases the hemolytic activity of the S-layer protein in Lysinibacillus sphaericus ASB13052]. / La deficiencia en el transporte de N-acetilglucosamina en Lysinibacillus sphaericus ASB13052 afecta el proceso de esporulación e incrementa la actividad hemolítica de su S-layer.

Lysinibacillus sphaericus is a bacterium that, along with Bacillus thuringiensis var. israelensis, is considered the best biological insecticide for controlling mosquito larvae and an eco-friendly alternative to chemical insecticides. It depends on peptidic molecules such as N-acetylglucosamine to obtain carbon sources and possesses a phosphotransferase system (PTS) for their incorporation. Some strains carry S-layer proteins, whose involvement in metal retention and larvicidal activity against disease-carrying mosquitoes has been demonstrated. Alterations in the amino sugar incorporation system could affect the protein profile and functionality. Strain ASB13052 and the isogenic mutant in the ptsH gene, which is predominant in the PTS signaling pathway, were used in this study. For the first time, the presence of N-glycosylated S-layer proteins was confirmed in both strains, with a variation in their molecular weight pattern depending on the growth phase. In the exponential phase, an S-layer protein greater than 130 kDa was found in the ptsH mutant, which was absent in the wild-type strain. The mutant strain exhibited altered and incomplete low quality sporulation processes. Hemolysis analysis, associated with larvicidal activity, showed that the ptsH mutant has higher lytic efficiency, correlating with the high molecular weight protein. The results allow us to propose the potential effects that arise as a result of the absence of amino sugar transport on hemolytic activity, S-layer isoforms, and the role of N-acetylglucosamine in larvicidal activity.

Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation.

The transcriptional control of sporulation in Bacillus subtilis is reasonably well understood, but its translational control is underexplored. Here, we use RNA-seq, ribosome profiling and fluorescence microscopy to study the translational dynamics of B. subtilis sporulation. We identify two events of translation silencing and describe spatiotemporal changes in subcellular localization of ribosomes during sporulation. We investigate the potential regulatory role of ribosomes during sporulation using a strain lacking zinc-independent paralogs of three zinc-dependent ribosomal proteins (L31, L33 and S14). The mutant strain exhibits delayed sporulation, reduced germination efficiency, dysregulated translation of metabolic and sporulation-related genes, and disruptions in translation silencing, particularly in late sporulation.

Influence of physicochemical characteristics on the growth and guaiacol production of Alicyclobacillus acidoterrestris in fruit juices.

Alicyclobacillus acidoterrestris is a bacterium known for causing spoilage in the taste and odour of fruit juices due to its thermoacidophilic nature. Its spoilage is attributed to the formation of guaiacol, which requires the presence of suitable precursors in the juices that A. acidoterrestris can metabolize. Therefore, A. acidoterrestris could exhibit different behaviour depending on the physicochemical characteristics the juice. In this study, we aimed to evaluate the behaviour of five A. acidoterrestris strains in seven different fruit juices by monitoring total cell and spore populations and quantifying guaiacol production. Also, physicochemical and phenolic profile, focusing on antimicrobials and guaiacol precursors, were analysed to better understand differences. Results showed growth in orange, apple, and plum juices for all the tested strains, with total cell populations reaching approximately 7 log cfu/mL, except for plum juice. In persimmon juice, growth was only observed in 3 out of 5 strains, for both total cells and spores. In contrast, all strains were inhibited in peach, black grape, and strawberry juices, maintaining a consistent population around 4 log cfu/mL. A strong negative correlation was observed between bacterial population and compounds such as kaempferol (for strains R3, R111, and P1), cyanidin chloride (for strains R111 and P1), and p-coumaric acid (for strain 7094 T). Regarding guaiacol production, orange and persimmon juices exhibited the highest guaiacol levels, with strain P1 (362.3 ± 12.6 ng/mL) and strain EC1 (325.1 ± 1.4 ng/mL) as the top producers, respectively. Plum, black grape, and strawberry juices showed similar guaiacol concentrations (16.9 ± 2.8 to 105.0 ± 33.7 ng/mL). Vanillin was showed positive correlations with guaiacol production in almost all strains (7094 T, R3, R111, and P1), with correlation coefficients of 0.97, 0.99, 0.82, and 0.87, respectively. We have reported different behaviour of A. acidoterrestris strains depending on juice type. Despite growth inhibition observed in some juices, enough guaiacol quantities to spoil the juice can be produced. This highlights the necessity of exploring strategies to prevent guaiacol production, even under growth restriction.

Correlation between sporogenesis and lipopeptide production in Paenibacillus elgii.

Pelgipeptins, tridecaptins, and elgicins are among the antimicrobials produced by Paenibacillus elgii. Growth in complex media is commonly applied to obtain lipopeptides from culture's supernatant, but it requires further purification. This study aimed to improve the yield of pelgipeptins and tridecaptins using chemically defined media. The kinetics of antimicrobial lipopeptide yield in chemically defined media were evaluated in P. elgii AC13. Pelgipeptins were detected in the supernatant and the culture pellet, but tridecaptins were mainly associated with cell debris or endospores. We investigated whether removing Ca2+ would impair P. elgii sporogenesis, consequently improving the yield of tridecaptin. The kinetics of both lipopeptides in the presence and absence of Ca2+ were quantitatively and qualitatively evaluated and further correlated with the cell cycle. The impairment of P. elgii AC13 sporogenesis had no effect on tridecaptin production, which remained undetected in the supernatant of the culture. On the other hand, the yield of pelgipeptin in a Ca2+-free medium increased. We showed for the first time that the removal of Ca2+ interrupted the sporogenesis in P. elgii and improved the yield of pelgipeptins. However, Ca2+ absence had no effect on tridecaptin yield, which is possibly degraded or associated with other cell debris components.

The megaplasmid pCER270 of Bacillus cereus emetic strain affects the timing of the sporulation process, spore resistance properties, and germination.

The Bacillus cereus group includes closely related spore-forming Gram-positive bacteria. In this group, plasmids play a crucial role in species differentiation and are essential for pathogenesis and adaptation to ecological niches. The B. cereus emetic strains are characterized by the presence of the pCER270 megaplasmid, which encodes the non-ribosomal peptide synthetase for the production of cereulide, the emetic toxin. This plasmid carries several genes that may be involved in the sporulation process. Furthermore, a transcriptomic analysis has revealed that pCER270 influences the expression of chromosome genes, particularly under sporulation conditions. In this study, we investigated the role of pCER270 on spore properties in different species of the B. cereus group. We showed that pCER270 plays a role in spore wet heat resistance and germination, with varying degrees of impact depending on the genetic background. In addition, pCER270 ensures that sporulation occurs at the appropriate time by delaying the expression of sporulation genes. This regulation of sporulation timing is controlled by the pCER270-borne Rap-Phr system, which likely regulates the phosphorylation state of Spo0A. Acquisition of the pCER270 plasmid by new strains could give them an advantage in adapting to new environments and lead to the emergence of new pathogenic strains. IMPORTANCE: The acquisition of new mobile genetic elements, such as plasmids, is essential for the pathogenesis and adaptation of bacteria belonging to the Bacillus cereus group. This can confer new phenotypic traits and beneficial functions that enable bacteria to adapt to changing environments and colonize new ecological niches. Emetic B. cereus strains cause food poisoning linked to the production of cereulide, the emetic toxin whose synthesis is due to the presence of plasmid pCER270. In the environment, cereulide provides a competitive advantage in producing bacteria against various competitors or predators. This study demonstrates that pCER270 also regulates the sporulation process, resulting in spores with improved heat resistance and germination capacity. The transfer of plasmid pCER270 among different strains of the B. cereus group may enhance their adaptation to new environments. This raises the question of the emergence of new pathogenic strains, which could pose a serious threat to human health.

Construction nanobiotechnology approach for performance enhancement of microbially induced biomineralization (MIB) using a biopolymer encapsulated spore-based system.

The integration of green construction practices within the built environment has been significantly advanced by biotechnological innovations, among which microbially induced biomineralization (MIB), predominantly facilitated by various strains of spore-forming bacilli, emerges as a pivotal mechanism for the self-healing of concrete. However, the practical deployment of this technology faces challenges, notably the compromised viability of bacterial spores due to germination triggered by severe shear stress during concrete mixing. To address this limitation, a water-insoluble polymer (extracellular polymeric substance) produced by Cellulomonas flavigena was utilized to encapsulate and protect the spores. The encapsulation process was rigorously verified through physicochemical methodologies, including X-ray diffraction (XRD) analysis, which revealed alterations in the interlayer spacings of the extracellular polymeric substance (EPS) structure during the encapsulation process, indicating successful EPS coating of the spores. Furthermore, a proof of concept for the enhanced biomineralization capacity of EPS-coated spores was demonstrated. Standard analytical techniques confirmed the precipitation of calcite and vaterite among other minerals, underscoring the effectiveness of this novel approach. This breakthrough paves the way for the development of innovative, sustainable bioconcrete applications, aligning with broader environmental objectives and advancing the field of green construction technology.IMPORTANCEDevelopment of bioconcrete with self-healing capability through MIB constitutes an important sustainable construction biotechnology approach for restoration and repair of built environment. Like every promising technology, MIB also suffers from certain shortcomings in terms of compromised viability of the microbial cells after premature germination of the spores on exposure to shear stress caused during concrete mixing. In this study, these challenges were adequately addressed by successfully providing a protective coating of indigenously extracted EPS to the bacterial spores and elucidating the interactive mechanisms between them. The results showed stable encapsulation of the spores while providing mechanistic insights of the encapsulation phenomenon. The data also showed enhanced rate of biomineralization by encapsulated microbes when subjected to stress conditions.

Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.

Efficacy, kinetics, inactivation mechanism and application of cold plasma in inactivating Alicyclobacillus acidoterrestris spores.

As spores of Alicyclobacillus acidoterrestris can survive traditional pasteurization, this organism has been suggested as a target bacterium in the fruit juice industry. This study aimed to investigate the inactivation effect of cold plasma on A. acidoterrestris spores and the mechanism behind the inactivation. The inactivation effect was detected by the plate count method and described by kinetic models. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), the detection of dipicolinic acid (DPA) release and heat resistance detection, the detection and scavenging experiment of reactive species, and cryo-scanning electron microscopy were used to explore the mechanism of cold plasma inactivation of A. acidoterrestris. The results showed that cold plasma can effectively inactivate A. acidoterrestris spores in saline with a 3.0 ± 0.3 and 4.4 ± 0.8 log reduction in CFU/mL, for 9 and 18 min, respectively. The higher the voltage and the longer the treatment time, the stronger the overall inactivation effect. However, a lower gas flow rate may increase the probability of spore contact with reactive species, resulting in better inactivation results. The biphasic model fits the survival curves better than the Weibull model. SEM and TEM revealed that cold plasma treatment can cause varying degrees of damage to the morphology and structure of A. acidoterrestris spores, with at least 50 % sustaining severe morphological and structural damage. The DPA release and heat resistance detection showed that A. acidoterrestris spores did not germinate but died directly during the cold plasma treatment. 1O2 plays the most important role in the inactivation, while O3, H2O2 and NO3- may also be responsible for inactivation. Cold plasma treatment for 1 min reduced A. acidoterrestris spores in apple juice by 0.4 ± 0.0 log, comparable to a 12-min heat treatment at 95 °C. However, as the treatment time increased, the survival curve exhibited a significant tailing phenomenon, which was most likely caused by the various compounds in apple juice that can react with reactive species and exert a physical shielding effect on spores. Higher input power and higher gas flow rate resulted in more complete inactivation of A. acidoterrestris spores in apple juice. What's more, the high inactivation efficiency in saline indicates the cold plasma device provides a promising alternative for controlling A. acidoterrestris spores during apple washing. Overall, our study provides adequate data support and a theoretical basis for using cold plasma to inactivate A. acidoterrestris spores in the food industry.

Spore germination and lactic acid combined treatment: A new processing strategy for the shelf-life extension of instant wet noodles.

Bacillus amyloliquefaciens (BAM) was identified as the predominant spoilage bacteria in instant wet noodles (IWNs). The utilization of industrial acid treatment as a long shelf-life strategy resulted in reduced consumer acceptance due to the acidic taste of the products. This study proposed a processing strategy that integrated spore germination (SG) and lactic acid (LA) treatment to effectively reduce the spore survival rate and extend the shelf life of IWNs. L-histidine, d-glucose, and sodium chloride were highly efficient and safe germinants for BAM spores. In IWNs, compound germinants (1.0 % L-histidine, 0.5 % d-glucose, and 1.0 % sodium chloride) boosted the SG rate by 3.61 times. With synergistic LA treatment, the spore lethality increased by 34.41 % -41.68 %. Under the SG and reduced acid-heat conditions of pH 2.30-2.50, the mortality of spores could reach 92.00 %-93.17 %, which was 14.11 %-15.28 % higher than the industrial acid-heat condition of pH 2.10. DPA, ATP, and membrane potential showed that germinants reduced the spore membrane permeability and promoted the occurrence of spore membrane damage under acid-heat conditions. Moreover, this strategy significantly extended the shelf-life of IWNs by 3.00-5.50 times and controlled the pH ≥ 5.50. Additionally, it improved color, texture, and overall sensory evaluation. Accordingly, this strategy solved the contradiction between the long shelf-life of IWNs and the unacceptable acidification in industrial production.

RelQ-mediated alarmone signalling regulates growth, stress-induced biofilm formation and spore accumulation in Clostridioides difficile.

The bacterial stringent response (SR) is a conserved transcriptional reprogramming pathway mediated by the nucleotide signalling alarmones, (pp)pGpp. The SR has been implicated in antibiotic survival in Clostridioides difficile, a biofilm- and spore-forming pathogen that causes resilient, highly recurrent C. difficile infections. The role of the SR in other processes and the effectors by which it regulates C. difficile physiology are unknown. C. difficile RelQ is a clostridial alarmone synthetase. Deletion of relQ dysregulates C. difficile growth in unstressed conditions, affects susceptibility to antibiotic and oxidative stressors and drastically reduces biofilm formation. While wild-type C. difficile displays increased biofilm formation in the presence of sublethal stress, the ΔrelQ strain cannot upregulate biofilm production in response to stress. Deletion of relQ slows spore accumulation in planktonic cultures but accelerates it in biofilms. This work establishes biofilm formation and spore accumulation as alarmone-mediated processes in C. difficile and reveals the importance of RelQ in stress-induced biofilm regulation.

Alanine and glutamate catabolism collaborate to ensure the success of Bacillus subtilis sporulation.

Sporulation as a typical bacterial differentiation process has been studied for decades. However, two crucial aspects of sporulation, (i) the energy sources supporting the process, and (ii) the maintenance of spore dormancy throughout sporulation, are scarcely explored. Here, we reported the crucial role of RocG-mediated glutamate catabolism in regulating mother cell lysis, a critical step for sporulation completion of Bacillus subtilis, likely by providing energy metabolite ATP. Notably, rocG overexpression resulted in an excessive ATP accumulation in sporulating cells, leading to adverse effects on future spore properties, e.g. increased germination efficiency, reduced DPA content, and lowered heat resistance. Additionally, we revealed that Ald-mediated alanine metabolism was highly related to the inhibition of premature germination and the maintenance of spore dormancy during sporulation, which might be achieved by decreasing the typical germinant L-alanine concentration in sporulating environment. Our data inferred that sporulation of B. subtilis was a highly orchestrated biological process requiring a delicate balance in diverse metabolic pathways, hence ensuring both the completion of sporulation and production of high-quality spores.

Unveiling the matrix effect on Bacillus licheniformis and Bacillus subtilis spores heat inactivation between plant-based milk alternatives, bovine milk and culture medium.

This study examined the inactivation of spores of Bacillus licheniformis and Bacillus subtilis in four pea-based milk alternatives, semi-skimmed bovine milk and Brain Heart Infusion (BHI) broth to assess the matrix impact on the thermal inactivation of bacterial spores. Heat inactivation was performed with the method of capillary tubes in temperature range 97-110 °C. A four-parameter non-linear model, including initial level, shoulder duration, inactivation rate and tailing, was fitted to the data obtained. D-values were estimated and secondary ZT-value models were developed for both species. A secondary model for the shoulder length of B. licheniformis in a plant-based milk alternative formulation was built too. Models were validated at a higher temperature, 113.5 °C. D-values in the different matrices ranged between 2.3 and 8.2 min at 97 °C and 0.1-0.3 min at 110 °C for B. licheniformis. D-values for B. subtilis ranged between 3.9 and 6.3 min at 97 °C and 0.2-0.3 min at 110 °C. ZT-values in the different matrices ranged between 7.3 and 8.9 °C and 8.9-10.0 °C for B. licheniformis and B. subtilis, respectively. Significant differences in inactivation parameters were found within the pea-based formulations as well as when compared to bovine milk. Heat resistance was higher in pea-based matrices. Shoulders observed were temperature- and matrix-dependent, while no such trend was found for the tailings. These results provide insights, useful on designing safe thermal processing, limiting spoilage in plant-based milk alternatives and thus, reducing global food waste.

Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - Shedding light on spore superdormancy at 550 MPa.

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency. Bacillus subtilis spores were pressurized for 50 s - 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions. The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43-52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions. The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

How c-di-GMP controls progression through the Streptomyces life cycle.

Members of the antibiotic-producing bacterial genus Streptomyces undergo a complex developmental life cycle that culminates in the production of spores. Central to control of this cell differentiation process is signaling through the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). So far, three proteins that are directly controlled by c-di-GMP in Streptomyces have been functionally and structurally characterized: the key developmental regulators BldD and σWhiG, and the glycogen-degrading enzyme GlgX. c-di-GMP signals through BldD and σWhiG, respectively, to control the two most dramatic transitions of the Streptomyces life cycle, the formation of the reproductive aerial hyphae and their differentiation into spore chains. Later in development, c-di-GMP activates GlgX-mediated degradation of glycogen, releasing stored carbon for spore maturation.

SpoIIQ-dependent localization of SpoIIE contributes to septal stability and compartmentalization during the engulfment stage of Bacillus subtilis sporulation.

During spore development in bacteria, a polar septum separates two transcriptionally distinct cellular compartments, the mother cell and the forespore. The conserved serine phosphatase SpoIIE is known for its critical role in the formation of this septum and activation of compartment-specific transcription in the forespore. Signaling between the mother cell and forespore then leads to activation of mother cell transcription and a phagocytic-like process called engulfment, which involves dramatic remodeling of the septum and requires a balance between peptidoglycan synthesis and hydrolysis to ensure septal stability and compartmentalization. Using Bacillus subtilis, we identify an additional role for SpoIIE in maintaining septal stability and compartmentalization at the onset of engulfment. This role for SpoIIE is mediated by SpoIIQ, which anchors SpoIIE in the engulfing membrane. A SpoIIQ mutant (SpoIIQ Y28A) that fails to anchor SpoIIE, results in septal instability and miscompartmentalization during septal peptidoglycan hydrolysis, when other septal stabilization factors are absent. Our data support a model whereby SpoIIE and its interactions with the peptidoglycan synthetic machinery contribute to the stabilization of the asymmetric septum early in engulfment, thereby ensuring compartmentalization during spore development.IMPORTANCEBacterial sporulation is a complex process involving a vast array of proteins. Some of these proteins are absolutely critical and regulate key points in the developmental process. Once such protein is SpoIIE, known for its role in the formation of the polar septum, a hallmark of the early stages of sporulation, and activation of the first sporulation-specific sigma factor, σF, in the developing spore. Interestingly, SpoIIE has been shown to interact with SpoIIQ, an important σF-regulated protein that functions during the engulfment stage. However, the significance of this interaction has remained unclear. Here, we unveil the importance of the SpoIIQ-SpoIIE interaction and identify a role for SpoIIE in the stabilization of the polar septum and maintenance of compartmentalization at the onset of engulfment. In this way, we demonstrate that key sporulation proteins, like SpoIIQ and SpoIIE, function in multiple processes during spore development.

Biofilm-forming Ability of Bacillus thuringiensis Strains from Biopesticides on Polystyrene and their Attachment on Spinach.

Bacillus thuringiensis-based commercial products as a biopesticide have been used for more than 60 years in agriculture. However, as one of the species in B. cereus group, B. thuringiensis has been considered as an emerging hazard with the potential to cause food toxico-infections. The present study aimed to evaluate the biofilm-forming ability of B. thuringiensis biopesticide strains and their attachment on spinach, compared to foodborne B. cereus strains. Biofilm formations of tested strains were found to be strain-specific and affected by the nutrient conditions more than the incubation time. Nutrient starvation conditions generally reduced the biofilm formation of tested B. thuringiensis and B. cereus strains, particularly B. thuringiensis ABTS-1857 strain was found as the nonbiofilm former in starvation conditions. It is worth mentioning that B. thuringiensis SA-11 strain showed stronger biofilm-forming ability with more air-liquid interface biofilm than the other two B. thuringiensis biopesticide strains, but no such higher attachment of B. thuringiensis SA-11 to spinach was observed. These results indicate that B. thuringiensis SA-11 strain can enter the food processing lines by the attachment on spinach leaves, and it has the potential to form biofilms throughout the processing lines or the production environment when sufficient nutrients are available. However, more biofilm tests of B. thuringiensis biopesticide strains in the vegetable production chain should be performed. The dry formulation of commercial B. thuringiensis biopesticides enhanced their adhesion on spinach leaves, whereas the strength of adhesion was not improved by the formulation. In addition, 1-2 log reductions of spores after the intensive washing of spinach leaves in the lab were detected. However, the log reduction due to the actual washing done by the food processing companies in large-volume washing baths or by consumers at home would be limited and less than this lab simulation.

Endopeptidase activities of Clostridium botulinum toxins in the development of this bacterium.

By-products like CO2 and organic acids, produced during Clostridium botulinum growth, appear to inhibit its development and reduce ATP production. A decrease in ATP production creates an imbalance in the ATP/GTP ratio. GTP activates CodY, which regulates BoNT expression. This toxin is released into the extracellular medium. Its light chains act as a specific endopeptidase, targeting SNARE proteins. The specific amino acids released enter the cells and are metabolized by the Stickland reaction, resulting in the synthesis of ATP. This ATP might then be used by histidine kinases to activate Spo0A, the main regulator initiating sporulation, through phosphorylation.