Individual Spores Research Articles

Development and characterization of hybrids from native wine yeasts

For commercial purposes, the winemaking industry is constantly searching for new yeast strains. Historically, this has been achieved by collecting wild strains and selecting the best for industrial use through an enological evaluation. Furthermore, the increasing consumer demands have forced the industry to incorporate new strategies such as genetic engineering to obtain improved strains. In response to the lack of public acceptance of this methodology, alternative strategies based on breeding have gained acceptance in recent years. Through the use of conjugation of individual spores without the support of genetic engineering methods we generated intraspecific hybrids from wild strains with outstanding enological characteristics and interdelta fingerprinting was used to confirm the hybrid condition. A detailed enological characterization of the hybrids in synthetic and natural must indicates that physiological parameters such as sporulation, residual sugar, ethanol yield and total nitrogen uptake are within the levels determined for the parental strains, however, other parameters such as growth rate, lag phase and ethanol production show statistical differences with some parental or commercial strains. These findings allow us to propose these hybrids as new wine-making strains.

Effects of Cortex Peptidoglycan Structure and Cortex Hydrolysis on the Kinetics of Ca 2+ -Dipicolinic Acid Release during Bacillus subtilis Spore Germination

The kinetic parameters of the release of Ca(2+)-dipicolinic acid (CaDPA) during germination of spore populations and multiple individual spores of Bacillus subtilis strains with major alterations in the structure of the spore peptidoglycan (PG) cortex or lacking one or both of the two redundant enzymes involved in cortex hydrolysis (cortex-lytic enzymes [CLEs]) were determined. The lack of the CLE CwlJ greatly slowed CaDPA release with a germinant receptor (GR)-dependent germinant, l-valine, or a non-GR-dependent germinant, dodecylamine. The absence of the cortex-specific PG modification muramic acid-δ-lactam also increased the time needed for full CaDPA release during germination with both types of germinants. In contrast, increased cortex PG cross-linking was associated with faster times for initiation of CaDPA release with both l-valine and dodecylamine but not with faster CaDPA release once this release had been initiated. These data suggest that the precise structure of the spore cortex plays a significant role in determining the timing and the rate of CaDPA release during B. subtilis spore germination and, further, that this effect is independent of effects of GRs.

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Germination of individual wild-type Cl. perfringens spores was followed by monitoring Ca-dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T(lag)) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT(release)) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex-lytic enzyme (CLE) (sleC spores) exhibited the same features during GR-dependent germination, but with longer average T(lag) values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T(lag) of wild-type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild-type and sleC spores had identical average T(lag) and ΔT(release) values in dodecylamine germination that does not utilize GRs. Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE-deficient Bacillus spores, indicating that germination of these species' spores is not completely identical. This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.

Fern spore and pollen airspora profile of Singapore

Monitoring atmospheric fern spore and pollen loads in Singapore was initiated in June 1990. Aside from the more numerous fungal spores, fern spores and pollen grains made up 6.2–8.6% and 4.4–5.4% of the total airspora sampled, respectively. The most frequently encountered fern spores, in descending order, were those of Nephrolepisauriculata, Dicranopterislinearis, Stenochlaenapalustris, Aspleniumnidus, Pteridiumaquilinum, and Dicranopteriscurranii. For pollen grains, the most frequently encountered, in descending order, were Elaeisguineensis, Casuarinaequisetifolia, Acaciaauriculiformis, Kyllingiapolyphylla, Podocarpus, and Poaceae pollen grains. Seasonal patterns for individual fern spore or pollen types were discernible despite the relatively uniform tropical climate in Singapore. The fern spore and pollen calendar for the period 1991–1995 was compared to that of 2005–2006 as a follow-up study to keep abreast with the rapidly changing landscape of Singapore. Diurnal patterns showing a late morning to afternoon peak period were seen in fern spores, while the peak period was in the morning for pollen types studied with the exception to oil palm pollen. Additionally, association between fern spore and pollen counts and local meteorological conditions were also analyzed and found to be highly correlated. This study has thus identified the fern spores and pollen airspora components, and determined the calendars, as well as diurnal profiles of the Singapore airspora and provides invaluable information for allergy studies by highlighting the trigger sources present in the environment.

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.

Quantitative Analysis of Spatial-Temporal Correlations during Germination of Spores of Bacillus Species

Bacteria of Bacillus species sporulate upon starvation, and the resultant dormant spores germinate when the environment appears likely to allow the resumption of vegetative growth. Normally, the rates of germination of individual spores in populations are very heterogeneous, and the current work has investigated whether spore-to-spore communication enhances the synchronicity of germination. In order to do this work, time-lapse optical images of thousands of individual spores were captured during germination, and an image analysis algorithm was developed to do the following: (i) measure the positions and germination rates of many thousands of individual spores and (ii) compute pairwise correlations of their germination. This analysis showed that an individual spore's germination rate was dependent on its distance from other spores, especially at short distances. Thus, spores that were within a few micrometers exhibited an increased synchronicity in germination, suggesting that there is a mechanism for short-range communication between such spores during germination. However, two molecules known to be germinants that are released during germination, l-alanine and the 1:1 chelate of Ca²⁺ and dipicolinic acid, did not mediate spore-to-spore communication during germination.

Monitoring the Wet-Heat Inactivation Dynamics of Single Spores of Bacillus Species by Using Raman Tweezers, Differential Interference Contrast Microscopy, and Nucleic Acid Dye Fluorescence Microscopy

Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca(2+) with dipicolinic acid (CaDPA) was released rapidly at a highly variable time T(lag), the levels of spore nucleic acids remained nearly unchanged, and the T(lag) times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ~50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before T(lag) and reached maximum at a time slightly later than T(release). However, the fluorescence intensities of wet-heat-inactivated spores were ~15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment.

Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers

This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled microscope sample holder containing a germinant solution plus a nucleic acid stain; (ii) capturing a single spore with optical tweezers; (iii) simultaneously measuring phase-contrast images, Raman spectra and fluorescence images of the optically captured spore at 2- to 10-s intervals; and (iv) analyzing the acquired data for the loss of spore refractility, changes in spore-specific molecules (in particular, dipicolinic acid) and uptake of the nucleic acid stain. This information leads to precise correlations between various germination events, and takes 1-2 h to complete. The method can also be adapted to use multi-trap Raman spectroscopy or phase-contrast microscopy of spores adhered on a cover slip to simultaneously obtain germination parameters for multiple individual spores.

Kinetics of Germination of Wet-Heat-Treated Individual Spores of Bacillus Species, Monitored by Raman Spectroscopy and Differential Interference Contrast Microscopy

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores of Bacillus cereus and Bacillus megaterium, as well as of several isogenic Bacillus subtilis strains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca²+ and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time, T(lag), at which spores began release of the great majority of their CaDPA during the germination of B. subtilis spores with different nutrient germinants and also increased the variability of T(lag) values. (iii) The time period, ΔT(release), between T(lag) and the time, T(release), at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values of I(release), the intensity of a spore's DIC image at T(release), than did untreated spores and had much longer time periods, ΔT(lys), for the reduction in I(release) intensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases in T(lag) and ΔT(release) were also observed when wet-heat-treated B. subtilis spores were germinated with the nonnutrient dodecylamine, while the change in I(release) was less significant. (vi) The effects of wet-heat treatment on nutrient germination of B. cereus and B. megaterium spores were generally similar to those on B. subtilis spores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.

Germination of individual Bacillus subtilis spores with alterations in the GerD and SpoVA proteins, which are important in spore germination.

Release of Ca(2+) with dipicolinic acid (CaDPA) was monitored by Raman spectroscopy and differential interference contrast microscopy during germination of individual spores of Bacillus subtilis strains with alterations in GerD and SpoVA proteins. Notable conclusions about germination after the addition of nutrient were as follows. (i) Following L-alanine addition, wild-type and gerD spores and spores with elevated SpoVA protein levels (↑SpoVA spores) slowly released ∼10% of their CaDPA during a variable (6- to 55-min) period ending at T(lag), the time when faster CaDPA release began. (ii) T(lag) times were lower for ↑SpoVA spores than for wild-type spores and were higher for gerD spores. (iii) The long T(lag) times of gerD spores were partially due to slow commitment to germinate. (iv) The intervals between the commitment to germinate and CaDPA release were similar for wild-type and ↑SpoVA spores but longer for gerD spores. (v) The times for rapid CaDPA release, ΔT(release) = T(release) - T(lag) (with T(release) being the time at which CaDPA release was complete), were similar for wild-type, gerD, and ↑SpoVA spores. (vi) Spores with either one of two point mutations in the spoVA operon (spoVA(1) and spoVA(2) spores) exhibited a more rapid rate of CaDPA release beginning immediately after L-alanine addition leading to ∼65% CaDPA release prior to T(lag). (vii) T(lag) times for spoVA(1) and spoVA(2) spores were longer than for wild-type spores. (viii) The intervals between spoVA(1) and spoVA(2) spores' commitment and CaDPA release were similar to those for wild-type spores, but commitment occurred later. In contrast to germination after the addition of nutrient, T(lag) and ΔT(release) times were relatively similar during dodecylamine germination of spores of the five strains. These findings suggest the following. (i) GerD plays no role in CaDPA release during spore germination. (ii) SpoVA proteins are involved in CaDPA release during germination with nutrients, and probably with dodecylamine. (iii) Spores release significant CaDPA before commitment. (iv) CaDPA release during T(lag) and ΔT(release) may signal subsequent germination events.

Orientation and detachment dynamics of Bacillus spores from stainless steel under controlled shear flow: Modelling of the adhesion force

Shear-flow induced spore detachment was performed under well-controlled laminar flow conditions, in a specially-designed shear stress flow chamber. By comparing detachment profiles of a panel of four strains, belonging to the B. cereus group (B. cereus and B. thuringiensis) and to less related Bacillus species (B. pumilus), it was shown that the spore ability of attaching to stainless steel, probed under dynamic conditions, was mainly affected by the presence (and number) of appendages. Adhesion force between the B. cereus 98/4 strain and stainless steel was quantified at nanoscale. To this aim, detachment results were combined with a theoretical modelling, based on the balance of hydrodynamic forces and torque exerted over a simplified spore model with a spherical form. The wall shear stress, required to remove 50% of the spores initially attached to stainless steel, was determined. On this basis, an adhesion force of 930 ± 390 pN was obtained. Real-time re-orientation of B. cereus 98/4 spores was experimentally established, by using a high-speed camera for tracking the motions of individual spores with high temporal and spatial resolution. Even though tethered to stainless steel without any detachment occurring, spores kept mobile on the substratum, probably due to the existence of discrete bonds or local clusters of anchoring sites, and tended to re-orientate in the flow direction, for minimizing hydrodynamic forces and torque exerted by fluid flow. A significant heterogeneity within the population was also observed, with the co-existence of both moving and immobile spores.

Diversity and ecology of tropical African fungal spores from a 25,000-year palaeoenvironmental record in southeastern Kenya

Fossil fungal spores and other non-pollen palynomorphs (NPPs) are powerful environmental proxies in European palaeoecological and archaeological contexts. However, their application on other continents, and particularly in the tropics, is hampered by uncertain equivalence with morphologically similar taxa in Europe, and incomplete knowledge of their ecology in the new local contexts. Here we use fossil NPP assemblages in a 25,000-year sediment record from Lake Challa, a steep-sided crater lake near Mt. Kilimanjaro in southeastern Kenya, to assess NPP diversity in a tropical-African context and the equivalence of African taxa with their European counterparts. We recovered a total of 65 well-defined NPP types, of which 61 are fungal spores, and 42 could be linked to known taxa. We provide diagnoses and illustrations of 61 recovered taxa, 58 of which have not been documented before. Using the Challa pollen record of past regional vegetation dynamics and two independent proxies of past temperature and rainfall, we also assessed the association of individual fungal taxa with particular species and biomes of tropical-African vegetation, and with the history of regional climate change. We often found strong correspondence between the stratigraphic distribution of individual fungal spore taxa and the occurrence of specific vegetation types. Changing climate conditions appear to have had a strong impact on the ability of fungi to play a role in the decomposition of dead plants. For fungal spore assemblages, the most prominent change in regional palaeoenvironments over the past 25,000 years occurred at the start of the wet early Holocene, following Younger Dryas drought. Epicoccum purpurascens is common in the Glacial and Late-Glacial parts of the sequence, but shows a strong decline during the early Holocene. Coniochaeta cf. ligniaria occurs throughout the record but shows dramatic fluctuations that appear to relate to major changes in humidity. Correlation between fungal abundance and humidity is also observed in taxa for which the Challa region provided suitable habitat from ca. 16,500 cal. yr BP (e.g., Curvularia) or from the Late-Glacial to Holocene transition (e.g., Tetraploa aristata, Dictyoarthrinium cf. sacchari, cf. Byssothecium, types HdV-1032 and HdV-1033, cf. Alternaria, cf. Brachysporium, cf. Helminthosporium, Spegazzinia tessarthra and cf. Lasiodiplodia theobromae). Many of these taxa did not occur, or were rare, during both wet and dry phases of the Glacial period, suggesting an additional temperature effect on their occurrence in tropical African environments. A possibly dominant role of temperature is revealed in the stratigraphic distribution of Acrodictys, which appears at the onset of deglacial climate warming ca. 17,500 cal. yr BP and remains common throughout both wet and dry phases of the Holocene. Spores of the dung-inhabiting fungus Sporormiella occur throughout the 25,000-year record without notable fluctuations, suggesting little changes in the overall population density of large herbivores in the region.

Clouding the Issue

Biophysics As anyone who has played table tennis knows, it's hard to throw a table tennis ball very far. Fungal spores, being even smaller and lighter, suffer much more from drag when they are ejected into air; individual Sclerotinia sclerotiorum spores are roughly 10 µm in size and would be predicted to travel only 3 mm after having been launched at a speed of almost 10 m s−1. Streamlining the shape of the spore and sticking to one's brethren do confer some aerodynamic advantages and lead to wider dispersal. Roper et al. show that emerging in a cloud helps, too, and can boost the distance traveled to as much as 10 cm. This enhancement arises from the earliest spores having set the proximal layer of air into motion so that later spores can go with the flow. The authors numerically simulate the spatiotemporal behavior of the spore jet and find a good match with particle-imaging velocimetry measurements. They also find that spores that wait to emerge, so as to board the already moving air mass, can delay their departure only by around 50 ms lest the train leave the station without them. Proc. Natl. Acad. Sci. U.S.A. 107 , 10.1073/pnas.1003577107 (2010).

Monitoring the Kinetics of Uptake of a Nucleic Acid Dye during the Germination of Single Spores of Bacillus Species

Dormant bacterial spores do not take up and bind nucleic acid dyes in the spore core but readily take up such dyes when they are fully germinated. We present a methodology that combines fluorescence microscopy, phase contrast microscopy, and laser tweezers Raman spectroscopy to monitor the kinetics of uptake of the nucleic acid dye SYTO 16 during germination of individual Bacillus cereus and Bacillus subtilis spores. The level of dye bound to nucleic acids of individual spores was measured by fluorescence emission, while changes in spore refractility and the level of the 1:1 chelate of dipicolinic acid and Ca(2+) (CaDPA) were monitored by phase contrast microscopy and Raman spectroscopy, respectively. The results obtained include (1) during nutrient germination, SYTO 16 began to enter the spore core and bind to nucleic acids just when spores had released all CaDPA and continued until hydrolysis of spores' peptidoglycan cortex was complete; (2) during germination with exogenous CaDPA, rapid SYTO 16 uptake began only 2-7 min after complete release of endogenous CaDPA for both B. cereus and B. subtilis spores; (3) the rate but not the timing of dye uptake and the maximum level of dye bound to nucleic acid were increased during nutrient germination of B. subtilis spores lacking ~75% of the DNA binding proteins that normally saturate dormant spore DNA; (4) SYTO 16-DNA binding was not observed during nutrient germination of B. subtilis spores lacking the protease that degrades spores' DNA binding proteins, even after cortex hydrolysis; (5) SYTO 16 uptake by germinating B. subtilis spores lacking the cortex-lytic enzyme (CLE) CwlJ was low, again even after cortex hydrolysis, although SYTO 16 uptake by germinating spores lacking the other redundant CLE SleB was even higher than in germinating wild-type spores; and (6) there was no SYTO 16 uptake by germinating spores that lacked both CwlJ and SleB, even after CaDPA release. These results suggest that during spore germination SYTO 16 uptake is minimal until CaDPA has been released and DNA binding proteins have been degraded and further that CLEs' degradation of the spore cortex plays a crucial role in uptake of this dye.

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore's DPA once rapid DPA release had begun (DeltaT(release)) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for DeltaT(release) were generally 0.5 to 3 min at 25 to 37 degrees C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination > or = 15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T(lag), between mixing spores with nutrient germinants and the beginning of DeltaT(release). (iv) A number of factors decrease spores' T(lag) values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.

Evaluation of a Stochastic Inactivation Model for Heat-Activated Spores of Bacillus spp

Heat activates the dormant spores of certain Bacillus spp., which is reflected in the "activation shoulder" in their survival curves. At the same time, heat also inactivates the already active and just activated spores, as well as those still dormant. A stochastic model based on progressively changing probabilities of activation and inactivation can describe this phenomenon. The model is presented in a fully probabilistic discrete form for individual and small groups of spores and as a semicontinuous deterministic model for large spore populations. The same underlying algorithm applies to both isothermal and dynamic heat treatments. Its construction does not require the assumption of the activation and inactivation kinetics or knowledge of their biophysical and biochemical mechanisms. A simplified version of the semicontinuous model was used to simulate survival curves with the activation shoulder that are reminiscent of experimental curves reported in the literature. The model is not intended to replace current models to predict dynamic inactivation but only to offer a conceptual alternative to their interpretation. Nevertheless, by linking the survival curve's shape to probabilities of events at the individual spore level, the model explains, and can be used to simulate, the irregular activation and survival patterns of individual and small groups of spores, which might be involved in food poisoning and spoilage.

Studies of the Commitment Step in the Germination of Spores of Bacillus Species

Spores of Bacillus species are said to be committed when they continue through nutrient germination even when germinants are removed or their binding to spores' nutrient germinant receptors (GRs) is both reversed and inhibited. Measurement of commitment and the subsequent release of dipicolinic acid (DPA) during nutrient germination of spores of Bacillus cereus and Bacillus subtilis showed that heat activation, increased nutrient germinant concentrations, and higher average levels of GRs/spore significantly decreased the times needed for commitment, as well as lag times between commitment and DPA release. These lag times were also decreased dramatically by the action of one of the spores' two redundant cortex lytic enzymes (CLEs), CwlJ, but not by the other CLE, SleB, and CwlJ action did not affect the timing of commitment. The timing of commitment and the lag time between commitment and DPA release were also dependent on the specific GR activated to cause spore germination. For spore populations, the lag times between commitment and DPA release were increased significantly in spores that germinated late compared to those that germinated early, and individual spores that germinated late may have had lower appropriate GR levels/spore than spores that germinated early. These findings together provide new insight into the commitment step in spore germination and suggest several factors that may contribute to the large heterogeneity among the timings of various events in the germination of individual spores in spore populations.

Characterization of Bacterial Spore Germination Using Integrated Phase Contrast Microscopy, Raman Spectroscopy, and Optical Tweezers

We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus spores in both nutrient (l-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of approximately 2 s. Phase contrast microscopy assesses changes in refractility of individual spores during germination, while Raman spectroscopy gives information on changes in spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant spore is proportional to the level of CaDPA in that spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in spore refractility and complete CaDPA release was observed not only for spores germinating in the well-controlled environment of an optical trap but also for spores germinating when adhered on a microscope coverslip. Using this latter method, we also simultaneously characterized the distribution of the time-to-complete-CaDPA release (T(release)) of hundreds of individual B. cereus spores germinating with both saturating and subsaturating concentrations of l-alanine and with CaDPA.

Spatially Resolved Characterization of Water and Ion Incorporation in Bacillus Spores

We present the first direct visualization and quantification of water and ion uptake into the core of individual dormant Bacillus thuringiensis subsp. israelensis (B. thuringiensis subsp. israelensis) endospores. Isotopic and elemental gradients in the B. thuringiensis subsp. israelensis spores show the permeation and incorporation of deuterium in deuterated water (D(2)O) and solvated ions throughout individual spores, including the spore core. Under hydrated conditions, incorporation into a spore occurs on a time scale of minutes, with subsequent uptake of the permeating species continuing over a period of days. The distribution of available adsorption sites is shown to vary with the permeating species. Adsorption sites for Li(+), Cs(+), and Cl(-) are more abundant within the spore outer structures (exosporium, coat, and cortex) relative to the core, while F(-) adsorption sites are more abundant in the core. The results presented here demonstrate that elemental abundance and distribution in dormant spores are influenced by the ambient environment. As such, this study highlights the importance of understanding how microbial elemental and isotopic signatures can be altered postproduction, including during sample preparation for analysis, and therefore, this study is immediately relevant to the use of elemental and isotopic markers in environmental microbiology and microbial forensics.

The obligate endobacteria of arbuscular mycorrhizal fungi are ancient heritable components related to the Mollicutes

Arbuscular mycorrhizal fungi (AMF) have been symbionts of land plants for at least 450 Myr. It is known that some AMF host in their cytoplasm Gram-positive endobacteria called bacterium-like organisms (BLOs), of unknown phylogenetic origin. In this study, an extensive inventory of 28 cultured AMF, from diverse evolutionary lineages and four continents, indicated that most of the AMF species investigated possess BLOs. Analyzing the 16S ribosomal DNA (rDNA) as a phylogenetic marker revealed that BLO sequences from divergent lineages all clustered in a well-supported monophyletic clade. Unexpectedly, the cell-walled BLOs were shown to likely represent a sister clade of the Mycoplasmatales and Entomoplasmatales, within the Mollicutes, whose members are lacking cell walls and show symbiotic or parasitic lifestyles. Perhaps BLOs maintained the Gram-positive trait whereas the sister groups lost it. The intracellular location of BLOs was revealed by fluorescent in situ hybridization (FISH), and confirmed by pyrosequencing. BLO DNA could only be amplified from AMF spores and not from spore washings. As highly divergent BLO sequences were found within individual fungal spores, amplicon libraries derived from Glomus etunicatum isolates from different geographic regions were pyrosequenced; they revealed distinct sequence compositions in different isolates. Our results show a vertically inherited, monophyletic and globally distributed lineage of endobacteria thriving in AMF cytoplasm. These bacteria split from their sister groups more than 400 Myr ago, colonizing their fungal hosts already before main AMF lineages separated. The BLO-AMF symbiosis can, therefore, be dated back at least to the time when AMF formed the ancestral symbiosis with emergent land plants.