[Identification of Clinical Isolates of the Bacillus cereus Group and Their Characterization by Mass Spectrometry and Electron Microscopy].

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

A lab-on-a-chip utilizing microwaves for bacterial spore disruption and detection.

Bacterial spores are problematic in agriculture, the food industry, and healthcare, with the fallout costs from spore-related contamination being very high. Spores are difficult to detect since they are resistant to many of the bacterial disruption techniques used to bring out the biomarkers necessary for detection. Because of this, effective and practical spore disruption methods are desirable. In this study, we demonstrate the efficiency of a compact microfluidic lab-on-chip built around a coplanar waveguide (CPW) operating at 2.45 GHz. We show that the CPW generates an electric field hotspot of ∼10 kV/m, comparable to that of a commercial microwave oven, while using only 1.2 W of input power and thus resulting in negligible sample heating. Spores passing through the microfluidic channel are disrupted by the electric field and release calcium dipicolinic acid (CaDPA), a biomarker molecule present alongside DNA in the spore core. We show that it is possible to detect this disruption in a bulk spore suspension using fluorescence spectroscopy. We then use laser tweezers Raman spectroscopy (LTRS) to show the loss of CaDPA on an individual spore level and that the loss increases with irradiation power. Only 22% of the spores contain CaDPA after exposure to 1.2 W input power, compared to 71% of the untreated control spores. Additionally, spores exposed to microwaves appear visibly disrupted when imaged using scanning electron microscopy (SEM). Overall, this study shows the advantages of using a CPW for disrupting spores for biomarker release and detection.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

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.

Bacterial developmental checkpoint that directly monitors cell surface morphogenesis.

Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous "coat" and an inner peptidoglycan "cortex," separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species.

The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant "Staggered-type" (S-Ena) and the shorter "Ladder-like" type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation.

Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation.

The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Using cryo-electron tomography, genetics and fluorescence microscopy, we found that the organization of the division machinery is different in the two septa. While FtsAZ filaments, the major orchestrators of bacterial cell division, are present uniformly around the leading edge of the invaginating vegetative septa, they are only present on the mother cell side of the invaginating sporulation septa. We provide evidence suggesting that the different distribution and number of FtsAZ filaments impact septal thickness, causing vegetative septa to be thicker than sporulation septa already during constriction. Finally, we show that a sporulation-specific protein, SpoIIE, regulates asymmetric divisome localization and septal thickness during sporulation.

Ultraviolet-C inactivation and hydrophobicity of Bacillus subtilis and Bacillus velezensis spores isolated from extended shelf-life milk.

Bacterial spores are important in food processing due to their ubiquity, resistance to high temperature and chemical inactivation. This work aims to study the effect of ultraviolet C (UVC) on the spores of Bacillus subtilis and Bacillus velezensis at a molecular and individual level to guide in deciding on the right parameters that must be applied during the processing of liquid foods. The spores were treated with UVC using phosphate buffer saline (PBS) as a suspension medium and their lethality rate was determined for each sample. Purified spore samples of B. velezensis and B. subtilis were treated under one pass in a UVC reactor to inactivate the spores. The resistance pattern of the spores to UVC treatment was determined using dipicolinic acid (Ca-DPA) band of spectral analysis obtained from Raman spectroscopy. Flow cytometry analysis was also done to determine the effect of the UVC treatment on the spore samples at the molecular level. Samples were processed for SEM and the percentage spore surface hydrophobicity was also determined using the Microbial Adhesion to Hydrocarbon (MATH) assay to predict the adhesion strength to a stainless-steel surface. The result shows the maximum lethality rate to be 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9% from the sample B. velezensis strain LPL-K103 (B44). The difference in surface hydrophobicity for all isolates was statistically significant (P < 0.05). Flow cytometry analysis of UVC treated spore suspensions clarifies them further into sub-populations unaccounted for by plate counting on growth media. The Raman spectroscopy identified B4002 as the isolate possessing the highest concentration of Ca-DPA. The study justifies the critical role of Ca-DPA in spore resistance and the possible sub-populations after UVC treatment that may affect product shelf-life and safety. UVC shows a promising application in the inactivation of resistant spores though there is a need to understand the effects at the molecular level to design the best parameters during processing.

Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection.

Clostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5ß1 and vitronectin-αvß1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies.

Chromosome Segregation and Peptidoglycan Remodeling Are Coordinated at a Highly Stabilized Septal Pore to Maintain Bacterial Spore Development.

Asymmetric division, a hallmark of endospore development, generates two cells, a larger mother cell and a smaller forespore. Approximately 75% of the forespore chromosome must be translocated across the division septum into the forespore by the DNA translocase SpoIIIE. Asymmetric division also triggers cell-specific transcription, which initiates septal peptidoglycan remodeling involving synthetic and hydrolytic enzymes. How these processes are coordinated has remained a mystery. Using Bacillus subtilis, we identified factors that revealed the link between chromosome translocation and peptidoglycan remodeling. In cells lacking these factors, the asymmetric septum retracts, resulting in forespore cytoplasmic leakage and loss of DNA translocation. Importantly, these phenotypes depend on septal peptidoglycan hydrolysis. Our data support a model in which SpoIIIE is anchored at the edge of a septal pore, stabilized by newly synthesized peptidoglycan and protein-protein interactions across the septum. Together, these factors ensure coordination between chromosome translocation and septal peptidoglycan remodeling to maintain spore development.

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development.

Morphological Changes in Spores during Germination in Bacillus cereus and Bacillus subtilis.

Processes from spore germination to outgrowth were observed in detail using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for Bacillus cereus and Bacillus subtilis. At 15 and 30 min after germination induction, SEM observation and SEM-EDX analysis of Bacillus spores prepared by freeze substitution showed that spherical structures including compounds having the same elemental ratio as that of the spore were observed on the surface of the spores. The results suggested the leakages of the cellular materials from the spores. At 360 min, B. cereus spores in outgrowth phase elongated with hemispherical structures at the end of the long side of the cells. The discoid structures with a hole (20-30 nm diameter) in the center was observed at 360 min. Confocal laser scanning microscopy after staining with fluorescence-labeled anti-spore antibodies showed that the hemispherical and discoid structures originated from the spore coat. These structures broke down after detached from the cells in outgrowth phase.

Clustering, morphology, and treatment resistance of Bacillus globigii spores recovered from a pilot-scale activated sludge system.

This study examines the organization and morphology of Bacillus globigii (BG) spores, a common surrogate for Bacillus anthracis, which were seeded and then recovered at various times from several points within a conventional, pilot-scale activated sludge system. Recovered BG spores were enumerated, microscopically examined, and tested for resistance to chemical (i.e. 5% H2O2 for 8 min), thermal (80 °C for 30 min), and ultraviolet light (8 W, 254 nm UV for 1 min) inactivation. Spores exposed to activated sludge germinated, sporulated, and exhibited unique multilayer clustering patterns and statistically significant changes (p < 0.005) in dimensional morphology. Spores collected in the later experimental stages (i.e., during weeks 6 and 7) were significantly more resistant (p ≤ 0.05) to inactivation than those collected on the first day of testing. These results have direct consequences for sludge treatment requirements at wastewater treatment plants that receive spore-containing waste streams.

Establishing rod shape from spherical, peptidoglycan-deficient bacterial spores.

Chemical-induced spores of the Gram-negative bacterium Myxococcus xanthus are peptidoglycan (PG)-deficient. It is unclear how these spherical spores germinate into rod-shaped, walled cells without preexisting PG templates. We found that germinating spores first synthesize PG randomly on spherical surfaces. MglB, a GTPase-activating protein, forms a cluster that responds to the status of PG growth and stabilizes at one future cell pole. Following MglB, the Ras family GTPase MglA localizes to the second pole. MglA directs molecular motors to transport the bacterial actin homolog MreB and the Rod PG synthesis complexes away from poles. The Rod system establishes rod shape de novo by elongating PG at nonpolar regions. Thus, similar to eukaryotic cells, the interactions between GTPase, cytoskeletons, and molecular motors initiate spontaneous polarization in bacteria.

A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores.

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. Current super-resolution techniques require either the use of special exogenous probes, high illumination doses or multiple image acquisitions with post-processing or combinations of the aforementioned. Here, we describe a new approach by combining annular illumination with rescan confocal microscopy. This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. The lateral resolution is comparable to that of linear structured illumination microscopy and the axial resolution is similar to that of a standard confocal microscope. As a case study, we present super-resolution time-lapse imaging of wild-type Bacillus subtilis spores, which contain low numbers of germination receptor proteins in a focus (a germinosome) surrounded by an autofluorescent coat layer. Here, we give the first evidence for the existence of germinosomes in wild-type spores, show their spatio-temporal dynamics upon germinant addition and visualize spores coming to life.

Development of a gene expression system for the uncommon actinomycete Actinoplanes rectilineatus NRRL B-16090.

The urgent need for discovering new bioactive metabolites prompts exploring novel actinobacterial taxa by developing appropriate tools for their genome mining and rational genetic engineering. One promising source of new bioactive natural products is the genus Actinoplanes, a home to filamentous sporangia-forming actinobacteria producing many important specialized metabolites such as teicoplanin, ramoplanin, and acarbose. Here we describe the development of a gene expression system for a new Actinoplanes species, A. rectilineatus (NRRL B-16090), which is a potential producer of moenomycin-like antibiotics. We have determined the optimal conditions for spore formation in A. rectilineatus and a plasmid transfer procedure for its engineering via intergeneric E. coli-A. rectilineatus conjugation. The φC31- and pSG5-based vectors were successfully transferred into A. rectilineatus, but φBT1- and VWB-based vectors were not transferable. Finally, using the glucuronidase reporter system, we assessed the strength of several heterologous promoters for gene expression in A. rectilineatus.

Surface morphology differences in Clostridium difficile spores, based on different strains and methods of purification.

Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and "pineapple-like" using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (p < 0.001) in relative hydrophobicity suggesting that modifications had occurred but not at a level to be detectable by SEM. In conclusion, we observed significant variation in the spore morphology of clinical isolates of C. difficile due in part to the methods used to produce them. Sonication in particular can markedly change spore appearance and properties. The results of this study highlight the importance of adopting "standard" methods when attempting to compare results between studies and to understand the significance of their differences.

Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth.

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.

The Bacillus subtilis endospore crust: protein interaction network, architecture and glycosylation state of a potential glycoprotein layer.

The endospore of Bacillus subtilis is formed intracellularly upon nutrient starvation and is encased by proteinaceous shells. The outermost layer, the crust, is a postulated glycoprotein layer that is composed of six proteins: CotV, W, X, Y, Z and CgeA. Despite some insight into protein interactions and the identification of players in glycosylation, a clear picture of its architecture is still missing. Here, we report a comprehensive mutational analysis that confirms CotZ as the anchor of the crust, while the crust structure is provided by CotV, CotX and CotY. CotY seems to be the major structural component, while CotV and CotX are polar and co-depend on each other and partially on CotW. CotW is independent of other crust proteins, instead depending on outer coat proteins, indicating a role at the interface of crust and coat. CgeA is co-expressed with putative glycosyltransferases (CgeB and CgeD) and implicated in crust glycosylation. In accordance, we provide evidence that CgeB, CgeCDE, SpsA-L, SpsM and SpsNOPQR (formerly YfnHGFED) contribute to the glycosylation state of the spore. The crust polysaccharide layer consists of functionally linked rhamnose- and galactose-related variants and could contain rare sugars. It may therefore protect the crust against biological degradation and scavenging.

The molecular architecture of engulfment during Bacillus subtilis sporulation.

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.