Intact Spores Research Articles

Thioflavin-T does not report on electrochemical potential and memory of dormant or germinating bacterial spores.

Bacillus and Clostridium spores cause food spoilage and disease because of spores' dormancy and resistance to microbicides. However, when spores "come back to life" in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of "germinate to eradicate" strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores "remember" germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable.

Impact of weather on the behaviour of Alternaria spore and Alt a 1 concentration in the air of Ankara (Turkey)

Intact fungal spores and smaller fungal fragments such as hyphae are sources of airborne allergens. Information on the distribution of sub-spore allergenic particles in the atmosphere is however limited. Therefore, the main aim of this study was to explore the behaviour of Alternaria aeroallergens in the atmosphere of Ankara, central Turkey. Aerobiological monitoring was carried out in the 2020 season. A Burkard volumetric spore trap was used for fungal spore collection, and a high-volume cascade impactor for allergen collection. The Alt a 1 levels in two air fractions (particulate matter (PM) > 10 µm and 10 > PM > 2.5 µm) were measured by an enzyme-linked immunosorbent assay. The seasonal spore integral (SSIn) was 1846 spore day/m3. In the same period, a total of 95.71 pg/m3 Alt a 1 concentration was recorded, of which 93% was recorded in PM>10 and 7% in PM10–2.5 air fraction. The amount of allergen per spore was 5.18 × 10−2 pg/spore/m3. The highest concentration for both spores and allergens was observed on 30 June, i.e. before the local crop harvest. Four days with highest spore levels occurred about one week after the rainfall (> 1 mm). Hot and humid weather probably contributed to the intensive development of fungi and an increase in the concentration of spores in the atmosphere. Our findings suggest that in areas with semi-arid climate, such as central Turkey, the weather conditions have a greater effect on behaviour of the distribution of Alternaria aeroallergens than agricultural practices.

Biobased Spore Microcapsules for Asphalt Self-Healing.

Asphalt pavements and bituminous composites are majorly damaged by bitumen aging and fatigue cracking by traffic load. To add, maintenance and reparation of asphalt pavements is expensive and also releases significant amounts of greenhouse gases. These issues can be mitigated by promoting asphalt self-healing mechanisms with encapsulated rejuvenators. The ability of the required microcapsules to be resilient against high temperatures, oxidation, and mechanical stress is essential to promote such self-healing behavior without compromising the field performance of the asphalt pavement. This work proposes, for the first time, the use of extremely resistant biobased spores for the encapsulation of recycled oil-based rejuvenators to produce more resilient self-healing pavements. Spore encapsulants were obtained from natural spores (Lycopodium clavatum) by applying different chemical treatments, which enabled the selection of the best morphologically intact and clean spore encapsulant. The physical, morphological, and physicochemical changes were examined using fluorescence images, ATR-FTIR, SEM, size distribution, XRD, TGA and DSC analyses. Sunflower oil was used as the encapsulated rejuvenator with an optimal sol colloidal mixture for sporopollenin-oil of 1:5 (gram-to-gram). Vacuum, passive, and centrifugal encapsulation techniques were tested for loading the rejuvenator inside the clean spores and for selecting the best encapsulation technology. The encapsulation efficiency and the profiles of the accelerated release of the rejuvenator from the loaded spores over time were studied, and these processes were visualized with optical and inverted fluorescence microscopy. Vacuum encapsulation was identified as the best loading technique with an encapsulation efficiency of 93.02 ± 3.71%. The rejuvenator was successfully encapsulated into the clean spores, as observed by optical and SEM morphologies. In agreement with the TGA and DSC, the microcapsules were stable up to 204 °C. Finally, a self-healing test was conducted through fluorescence tests to demonstrate how these biobased spore microcapsules completely heal a crack into an aged bitumen sample in 50 min.

Characterization of sub-pollen particles in size-resolved atmospheric aerosol using chemical tracers.

Pollen grains may contain allergens that exacerbate allergic respiratory diseases like asthma and rhinitis. In the presence of water, pollen grains (10–100 μm) can rupture to produce sub-pollen particles (SPP) with diameters <2.5 μm, which in comparison to intact pollen grains, have longer atmospheric lifetimes and greater penetration to the lower lung. The current study examines SPP, fungal spores, and bacteria in size-resolved atmospheric particulate matter (PM) using chemical and biological tracers. During springtime tree pollen season in Iowa City, Iowa, fine particle (PM2.5) concentrations of fructose (a pollen chemical tracer) increased on rainy sampling periods, especially during severe thunderstorms, and peaked when a tornado struck nearby. Submicron fluorescent particles, measured by single-particle fluorescence spectroscopy, were also enhanced during rain events, particularly thunderstorms in agreement with the chemical tracer measurements. PM2.5 sucrose (a pollen chemical tracer) concentrations were higher in early spring when nighttime temperatures were closer to freezing, while fructose concentrations were higher in late spring with warmer temperatures, consistent with chemical tracers being sensitive to seasonal temperature influences. The first co-located measurements of fructose and Bet v 1 (birch pollen allergen), indicated that SPP ranged in diameter from <0.25 to 2.5 μm during rainy sampling periods and that allergens and carbohydrates exhibited distinct size distributions. Meanwhile, mannitol (a fungal spore tracer) peaked on warm, dry days following rain and was primarily in supermicron particles (>1.0 μm), which is consistent with intact fungal spore diameters (1–30 μm). Bacterial endotoxins in PM also increased during extreme weather events, primarily in supermicron particles. While the concentrations of fructose, mannitol, and endotoxin all increased in PM2.5 μm during thunderstorms, the greatest relative increase in concentration was observed for fructose. Together, these observations suggest that SPP containing starch granules and allergens (Bet v 1) were released during rainy sampling periods. This study advances the use of chemical tracers to track SPP and other bioaerosols in the atmosphere, by providing new insight to their size distribution and response to extreme weather conditions.

Squirrel consuming “poisonous” mushrooms

The representative toadstool Amanita muscaria plays an important role in maintaining forest ecosystems by forming mutualistic associations with a variety of trees. A muscaria is also known for the poisonous properties of its hallucinogenic constituents, namely, ibotenic acid, muscimol, and muscarin. Serious cases of poisoning in humans may involve deliria, hallucinations, seizures, and (rarely) death. A typical symptom is visual distortion of the size of objects, a phenomenon that formed the basis of the mind-altering events described in Lewis Carroll’s 1865 novel Alice’s Adventures in Wonderland. Interestingly, we observed a Japanese squirrel (Sciurus lis) routinely feed not only on A muscaria but also on other Amanita species that are also poisonous to humans. This squirrel returned to feed on Amanita fruiting bodies for several days, indicating that it could safely consume the “poisonous” mushrooms. The ecological roles of fungal toxins remain largely unexplored. One possible role is to deter fungivores. However, fungivores are not always disadvantageous to Amanita, because they may disperse intact spores via their digestive tracts. Perhaps Amanita mushrooms facilitate mutualisms with toxin-resistant squirrels, which can disperse viable spores, while deterring toxin-susceptible enemies, which could negatively impact spore survival. If so, potential dispersal of fungal spores by squirrels is analogous to a seed dispersal mutualism, in which a plant offers a reward to an animal as a seed disperser. How do squirrels safely eat Amanita mushrooms? What role do squirrels play in Amanita spore dispersal? How would squirrel-assisted spore dispersal contribute to the establishment of ectomycorrhizal relationships within these ecosystems? These questions warrant further investigation.

Novel Mouse Models of Fungal Asthma.

Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development of asthma. Inhalation of intact spores is the primary cause of human exposure to fungal allergen. However, allergen-rich cultured fungal filtrates are oftentimes used in the current models of fungal sensitization that do not fully reflect real-life exposures. Thus, establishing novel spore exposure models is imperative. In this study, we established novel fungal exposure models of both adult and neonate to live spores. We examined pathophysiological changes in the spore models as compared to the non-exposure controls and also to the conventional filtrate models. While both Alternaria filtrate- and spore-exposed adult BALB/c mice developed elevated airway hyperresponsiveness (AHR), filtrates induced a greater IgE mediated response and higher broncholavage eosinophils than spores. In contrast, the mice exposed to Alternaria spores had higher numbers of neutrophils. Both exposures induced comparable levels of lung tissue inflammation and mucous cell metaplasia (MCM). In the neonatal model, exposure to Alternaria spores resulted in a significant increase of AHR in both adult and neonatal mice. Increased levels of IgE in both neonatal and adult mice exposed to spores was associated with increased eosinophilia in the treatment groups. Adult demonstrated increased numbers of lymphocytes that was paralleled by increased IgG1 production. Both adults and neonates demonstrated similarly increased eosinophilia, IgE, tissue inflammation and MCM.

Isolation of protein-free chitin spore coats of Nosema ceranae and its application to screen the interactive spore wall proteins.

Nosema ceranae is the pathogen of nosemosis in the honey bee, which can bring great economic loss to apiculture. Chitin acts as a major component of the endospore of microsporidia and plays an essential role to form the bridges across the endospore. Here, Chitin Spore Coats (CSCs) of N. ceranae were successfully extracted by optimized hot alkaline treatment. SDS-PAGE and Calcofluor White Stain (CWS) staining indicated that the obtained CSCs were protein-free and the transmission electron microscopy analysis showed that CSCs performed the intact and loose chitin spore coats. Western blotting and indirect immunofluorescence analysis (IFA) demonstrated that CSCs could interact with three spore wall proteins (rNcSWP7, rNcSWP8, and rNcSWP12). Our method was effective to extract CSCs of N. ceranae and this could be very useful for screening spore wall proteins involved in endospore composition, which could be helpful to uncover the biological structure and pathogenesis of microsporidia.

Highly sensitive protein detection using recombinant spores and lateral flow immunoassay.

Lateral flow immunoassays (LFIs) can be used to detect intact bacteria or spores; when gold nanoparticles (AuNPs) are used as the signal reporters, the detection limits are very low. Spore-based surface display has been widely studied for enzyme immobilization and live-nontoxic oral vaccines. In this study, recombinant spores were used to improve the sensitivity of a LFI. We developed a test kit that combines streptavidin-displayed spores with a LFI assay for rapid protein detection. The recombinant spores served as a signal amplifier and AuNPs were used as the signal reporters. For detection of β-galactosidase, which was used as the model protein, the detection limit was about 10-15mol, while that of the conventional LFI is about 10-12mol. In both methods, nanogold was used as the colorimetric signal and could be observed with the naked eye. This method improved LFI sensitivity without sacrificing its advantages. Furthermore, enhanced green fluorescent protein (eGFP) was also displayed on the surface of the streptavidin-displayed spores. Without AuNPs, the fluorescent recombinant spores acted as the signal, which could be detected by a fluorescence detector, such as a fluorescence microscope. The detection limit was 10-16mol under fluorescence microscopy whose magnification was 25-fold. Therefore, in conclusion, in this proof of concept study, the detection limits of both proposed methods were far superior to those of traditional LFI assay.

Analysis of 5'-NAD capping of mRNAs in dormant spores of Bacillus subtilis.

Spores of Gram-positive bacteria contain 10s-1000sof different mRNAs. However, Bacillus subtilis spores contain only ∼ 50 mRNAs at >1 molecule/spore, almost all transcribed only in the developing spore and encoding spore proteins. However, some spore mRNAs could be stabilized to ensure they are intact in dormant spores, perhaps to direct synthesis of proteins essential for spores' conversion to a growing cell in germinated spore outgrowth. Recent work shows that some growing B. subtilis cell mRNAs contain a 5'-NAD cap. Since this cap may stabilize mRNA in vivo, its presence on spore mRNAs would suggest that maintaining some intact spore mRNAs is important, perhaps because they have a translational role in outgrowth. However, significant levels of only a few abundant spore mRNAs had a 5'-NAD cap, and these were not the most stable spore mRNAs and had likely been fragmented. Even higher levels of 5'-NAD-capping were found on a few low abundance spore mRNAs, but these mRNAs were present in only small percentages of spores, and had again been fragmented. The new data are thus consistent with spore mRNAs serving only as a reservoir of ribonucleotides in outgrowth.

Killing of bacterial spores by dodecylamine and its effects on spore inner membrane properties.

The objective of this study was to determine the effects of Ca-dipicolinic acid (CaDPA), cortex-lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. Bacillus subtilis spores, wild-type, CaDPA-less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine-treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild-type and CaDPA-less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores±CaDPA, with up to 99% killing with 1moll-1 dodecylamine in 4h at 45°C with spores at ~108 ml-1 . Dodecylamine killing of decoated wild type and CLE-less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA-less spores, with ≥99% killing in 5min. Propidium iodide stained intact spores±CaDPA minimally, decoated CaDPA-replete spores or dodecylamine-killed CLE-less spores peripherally, and cores of decoated CaDPA-less spores and dodecylamine-killed intact spores with CLEs. The IM of some decoated CaDPA-less spores was greatly reorganized. Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. This work gives new information on killing bacterial spores by dodecylamine, and how spore IM's relative impermeability is maintained.

Identification of the Wzx flippase, Wzy polymerase and sugar-modifying enzymes for spore coat polysaccharide biosynthesis in Myxococcus xanthus.

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N-acetylgalactosamine-1-P to undecaprenyl-phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.

Particle size distribution of the major Alternaria alternata allergen, Alt a 1, derived from airborne spores and subspore fragments

Fungal fragments are abundant immunoreactive bioaerosols that may outnumber the concentrations of intact spores in the air. To investigate the importance of Alternaria fragments as sources of allergens compared to Alternaria spores, we determined the levels of Alternaria spores and Alt a 1 (the major allergen in Alternaria alternata spores) collected on filters within three fractions of particulate matter (PM) of different aerodynamic diameter: (1) PM>10, (diameter>10 μm); (2) PM2.5-10 (2.5–10μm); (3) PM2.5 (0.12–2.5 μm). The airborne particles were collected using a three stage high-volume ChemVol cascade impactor during the Alternaria sporulation season in Poznań, Poland (30 d between 6 July and 22 September 2016). The quantification of Alt a 1 was performed using the enzyme-linked immunosorbent assay. High concentrations of Alt a 1 were recorded during warm and dry d characterized by high sunshine duration, lack of clouds and high dew point values. Atmospheric concentrations of Alternaria spores correlated significantly (r = 0.930, p < 0.001) with Alt a 1 levels. The highest Alt a 1 was recorded in PM2.5-10 (66.8 % of total Alt a 1), while the lowest in PM2.5 (<1.0 %). Significantly more Alt a 1 per spore (>30 %) was observed in PM2.5-10 than in PM>10. This Alt a 1 excess may be derived from sources other than spores, e.g. hyphal fragments. Overall, in outdoor air the major source of Alt a 1 are intact Alternaria spores, but the impact of other fungal fragments (hyphal parts, broken spores, conidiophores) cannot be neglected, as they may increase the total atmospheric Alt a 1 concentration.

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection.

In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.

Mold inhalation causes innate immune activation, neural, cognitive and emotional dysfunction

Individuals living or working in moldy buildings complain of a variety of health problems including pain, fatigue, increased anxiety, depression, and cognitive deficits. The ability of mold to cause such symptoms is controversial since no published research has examined the effects of controlled mold exposure on brain function or proposed a plausible mechanism of action. Patient symptoms following mold exposure are indistinguishable from those caused by innate immune activation following bacterial or viral exposure. We tested the hypothesis that repeated, quantified doses of both toxic and nontoxic mold stimuli would cause innate immune activation with concomitant neural effects and cognitive, emotional, and behavioral symptoms. We intranasally administered either 1) intact, toxic Stachybotrys spores; 2) extracted, nontoxic Stachybotrys spores; or 3) saline vehicle to mice. As predicted, intact spores increased interleukin-1β immunoreactivity in the hippocampus. Both spore types decreased neurogenesis and caused striking contextual memory deficits in young mice, while decreasing pain thresholds and enhancing auditory-cued memory in older mice. Nontoxic spores also increased anxiety-like behavior. Levels of hippocampal immune activation correlated with decreased neurogenesis, contextual memory deficits, and/or enhanced auditory-cued fear memory. Innate-immune activation may explain how both toxic mold and nontoxic mold skeletal elements caused cognitive and emotional dysfunction.

Fungi and Atopy.

Atopy and fungi have a long associative history. Fungal spores were among the first substances to which humans were noted to be sensitized. Humans contact fungal spores in the outdoor, indoor, and occupational environments. As organisms, fungi have their own kingdom and are found in all environmental niches on earth. Currently, fungal exposure in the indoor environment especially related to wet housing conditions is of particular concern. Sensitization rates to fungi typically exceed 5% of the general public with higher rates among the atopic population. Alternaria is the best studied of the allergic fungi; however, cross sensitization to multiple fungi is well documented. Recent advances in understanding mechanisms of the innate immune system are beginning to explain why the fungal atopy relationship is unique and why fungal sensitivity seems to extend to many non-atopic individuals. Evidence has been accumulated that indicates fungal allergen exposure can be via intact spores as well as spore and mycelial fragments. Germinating spores produce a different and often increased allergen picture. Much evidence has been developed through animal studies that extends the mechanisms surrounding long-term low-level fungal exposure. However, it should be emphasized that the presence of fungi in the air does not necessarily equate with illness. Indeed, in the absence of an atopic individual and/or a significant immune response against fungi, there is little evidence suggesting pathology. Allergists frequently deal with patients who have concerns about indoor fungal exposure and respiratory disease in those patients with an allergic response.

"One-Pot" Sample Processing Method for Proteome-Wide Analysis of Microbial Cells and Spores.

PurposeBacterial endospores, the transmissible forms of pathogenic bacilli and clostridia, are heterogeneous multilayered structures composed of proteins. These proteins protect the spores against a variety of stresses, thus helping spore survival, and assist in germination, by interacting with the environment to form vegetative cells. Owing to the complexity, insolubility, and dynamic nature of spore proteins, it has been difficult to obtain their comprehensive protein profiles.Experimental designThe intact spores of Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile and their vegetative counterparts were disrupted by bead beating in 6 m urea under reductive conditions. The heterogeneous mixture was then double digested with LysC and trypsin. Next, the peptide mixture was pre‐fractionated with zwitterionic hydrophilic interaction liquid chromatography (ZIC‐HILIC) followed by reverse‐phase LC‐FT‐MS analysis of the fractions.Results"One‐pot" method is a simple, robust method that yields identification of >1000 proteins with high confidence, across all spore layers from B. subtilis, B. cereus, and P. difficile.Conclusions and medical relevanceThis method can be employed for proteome‐wide analysis of non‐spore‐forming as well as spore‐forming pathogens. Analysis of spore protein profile will help to understand the sporulation and germination processes and to distinguish immunogenic protein markers.

Dominikia emiratia and Rhizoglomus dunense, two new species in the Glomeromycota

The morphological, histochemical, and molecular properties of two new species of arbuscular mycorrhizal fungi (AMF; Glomeromycota) have been characterized. The first species is distinguished by spores that are orange to brownish orange, small, and formed only in clusters and mainly by having two laminate layers in a three-layered spore wall, with layer three staining dark in Melzer’s reagent. Despite the morphological similarity to some Septoglomus spp., phylogenetic analyses of sequences of the SSU–ITS–LSU nrDNA region and the RPB1 gene accommodated the fungus in the genus Dominikia, hence it was named Dominika emiratia. Intact spores of the second species, named Rhizoglomus dunense, closely resemble colourless isolates of R. clarum, but their spore wall layer three never becomes coloured with age, as does that in most R. clarum spores, and most importantly, the two fungi are separated by a large molecular distance. Dominikia emiratia was originally extracted from the rhizosphere of three plant species cultivated in two fields in a sandy desert in the Emirate of Abu Dhabi of the United Arab Emirates. Rhizoglomus dunense was found in a trap culture inoculated with the rhizosphere soil and root fragments of Ammophila arenaria, which had colonized sand dunes of the Mediterranean Sea, located near Thessalonica, Greece.

The preference of Morulina alata (Collembola: Neanuridae) feeding on some fungal sporocarps and the effects of passage through the gut on spores

Some soil invertebrates, such as springtails, feed on fungal sporocarps, which produce spores for reproduction. These soil invertebrates are likely to carry spores to fungal substrates in soil, even for fungal species for which the spores are believed to be primarily wind-dispersed. Few studies have examined the possibility of endozoochory for springtail species without molar plates. In Japan, Morulina alata (Neanuridae), which lacks molar plates, has been observed on sporocarps. This study determined 1) on which fungal species M. alata occurs most frequently and abundantly in the field, 2) whether it feeds on spores in the field, and 3) whether spores are damaged during gut passage in the laboratory. We observed the feeding habits of M. alata on Cortinarius anomalus, Inocybe fastigiata, and Mycena pura. In the field, M. alata was found on I. fastigiata and M. pura sporocarps, but not on C. anomalus. Spores of I. fastigiata and M. pura were also found in the gut of M. alata. Feeding experiments showed that almost all C. anomalus and I. fastigiata spores, and some M. pura spores, appeared to be intact after passage through the gut. Our results suggest that dispersal of intact spores of I. fastigiata and M. pura after ingestion by M. alata is possible. The movement of M. alata remains to be studied.

Identification of Bremia lactucae and Oidium neolycopersici proteins extracted for intact spore MALDI mass spectrometric biotyping

Several proteomic approaches were applied to identify protein markers providing typical signals during intact cell/spore (IC/IS) MALDI-TOF MS of two plant pathogens, namely Bremia lactucae (a downy mildew) and Oidium neolycopersici (a powdery mildew). First, proteins were extracted from intact spores of the microorganisms under conditions simulating their treatment prior to the mass spectrometric analysis. After a separation by electrophoresis and tryptic digestion, 198 and 140 proteins were identified in the B. lactucae and O. neolycopersici extracts, respectively. A large portion of them were found to be involved in the process of protein biosynthesis. For the first time, some proteins were assigned to characteristic signals in MS profiles of the investigated pathogens based on an agreement in the molecular mass. There were 9 and 10 proteins recognized, respectively, which could contribute significantly to the spectral patterns. These proteins were assigned tentatively to the following peaks in the MS profiles: (i) m/z 7828; 8593; 10456; 11312; 12450; 12763; 14756 and 16854 for B. lactucae; (ii) m/z 7709; 8895; 9504; 9952; 11317; 14082 and 14839 for O. neolycopersici. We demonstrated the presence of ribosomal proteins and histones, which could be employed as markers in biotyping analyses for pathogen identification.

Unraveling the predator-prey relationship of Cupriavidus necator and Bacillus subtilis

Cupriavidus necator is a non-obligate bacterial predator of Gram-negative and Gram-positive bacteria. In this study, we set out to determine the conditions, which are necessary to observe predatory behavior of C. necator. Using Bacillus subtilis as a prey organism, we confirmed that the predatory performance of C. necator is correlated with the available copper level, and that the killing is mediated, at least in part, by secreted extracellular factors. The predatory activity depends on the nutrition status of C. necator, but does not require a quorum of predator cells. This suggests that C. necator is no group predator. Further analyses revealed that sporulation enables B. subtilis to avoid predation by C. necator. In contrast to the interaction with predatory myxobacteria, however, an intact spore coat is not required for resistance. Instead resistance is possibly mediated by quiescence.