Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Paramagnetism in Bacillus spores: Opportunities for novel biotechnological applications

Spores of Bacillus megaterium, Bacillus cereus, and Bacillus subtilis were found to exhibit intrinsic paramagnetic properties as a result of the accumulation of manganese ions. All three Bacillus species displayed strong yet distinctive magnetic properties arising from differences in manganese quantity and valency. Manganese ions were found to accumulate both within the spore core as well as being associated with the surface of the spore. Bacillus megaterium spores accumulated up to 1 wt.% manganese (II) within, with a further 0.6 wt.% adsorbed onto the surface. At room temperature, Bacillus spores possess average magnetic susceptibilities in the range of 10⁻⁶ to 10⁻⁵. Three spore‐related biotechnological applications—magnetic sensing, magnetic separation and metal ion adsorption—were assessed subsequently, with the latter two considered as having the most potential for development.

     

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores

Aims: To compare physical properties of spores that were produced in broth sporulation media at greater than 10⁸ spores ml⁻¹. Methods and Results: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0·68 ± 0·11 μm wide and 1·21 ± 0·18 μm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0·48 ± 0·38 μm³ and 0·97 ± 0·07 μm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l⁻¹ glutamate and antifoam. Spores were 0·95 ± 0·11 μm wide and 1·31 ± 0·17 μm long. Spore volumes were 0·78 ± 0·61 μm³ and volume-equivalent spherical diameters were 1·14 ± 0·11 μm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. Conclusions: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. Significance and Impact of the Study: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.     

Quantification of magnetic susceptibility in several strains of Bacillus spores: implications for separation and detection

Three strains of Bacillus: Bacillus atrophaeus (formally Bacillus globigii), Bacillus thuringiensis, and Bacillus cereus were tested for their intrinsic magnetic susceptibility. All three strains when sporulated demonstrated significant magnetic susceptibility using an instrument referred to as Cell Tracking Velocimetry. Energy dispersive spectroscopy also confirmed the presence of paramagnetic elements, Fe and Mn, in the spore form of the bacteria. It was demonstrated that this magnetic susceptibility is sufficient to separate and deposit these spores on glass slides in a magnetic deposition system. These results indicate the potential to separate spores with intrinsic magnetic susceptibility directly out of water or air samples.     

Size matters for violent discharge height and settling speed of Sphagnum spores: important attributes for dispersal potential

Background and Aims

Initial release height and settling speed of diaspores are biologically controlled components which are key to modelling wind dispersal. Most Sphagnum (peat moss) species have explosive spore liberation. In this study, how capsule and spore sizes affect the height to which spores are propelled were measured, and how spore size and spore number of discharged particles relate to settling speed in the aspherical Sphagnum spores.

Methods

Spore discharge and spore cloud development were filmed in a closed chamber (nine species). Measurements were taken from snapshots at three stages of cloud development. Settling speed of spores (14 species) and clusters were timed in a glass tube.

Key Results

The maximum discharge speed measured was 3·6 m s⁻¹. Spores reached a maximum height of 20 cm (average: 15 cm) above the capsule. The cloud dimensions at all stages were related positively to capsule size (R² = 0·58–0·65). Thus species with large shoots (because they have large capsules) have a dispersal advantage. Half of the spores were released as singles and the rest as clusters (usually two to four spores). Single spores settled at 0·84–1·86 cm s⁻¹, about 52 % slower than expected for spherical spores with the same diameters. Settling speed displayed a positive curvilinear relationship with spore size, close to predictions by Stokes'' law for spherical spores with 68 % of the actual diameters. Light-coloured spores settled slower than dark spores. Settling speed of spore clusters agrees with earlier studies. Effective spore discharge and small, slowly settling spores appear particularly important for species in forested habitats.

Conclusions

The spore discharge heights in Sphagnum are among the greatest for small, wind-dispersed propagules. The discharge heights and the slow settling of spores affect dispersal distances positively and may help to explain the wide distribution of most boreal Sphagnum species.     

Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line

Aim: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean‐in‐place regimes. Methods and Results: Exposing sessile spores to 1% NaOH at 65°C for 30 min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from ?20 to ?32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l^?1 KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log₁₀ CFU cm^?2) than untreated spores (3·6 log₁₀ CFU cm^?2). Conclusions: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. Significance of Study: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.     

Mass production of spores of lactic acid‐producing Rhizopus oryzae NBRC 5384 on agar plate

Mass production of sporangiospores (spores) of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) on potato‐dextrose‐agar medium was studied aiming at starting its L (+)‐lactic acid fermentation directly from spore inoculation. Various parameters including harvest time, sowed spore density, size of agar plate, height of air space, and incubation mode of plate (agar‐on‐bottom or agar‐on‐top) were studied. Ordinarily used shallow Petri dishes were found out to be unsuitable for the full growth of R. oryzae sporangiophores. In a very wide range of the sowed spore density, the smaller it was, the greater the number of the harvested spores was. It was also interesting to find out that R. oryzae grown downward vertically with a deep air space in an agar‐on‐top mode gave larger amount of spores than in an agar‐on‐bottom mode at 30°C for 7‐day cultivation. Scale‐up of the agar plate culture from 26.4 to 292 cm² was studied, resulting in the proportional relationship between the number of the harvested spores/plate and the plate area in the deep Petri dishes. The number of plates of 50 cm in diameter needed for 100 m³ industrial submerged fermentation started directly from 2 × 10⁵ spores/mL inoculum size was estimated as about 6, from which it was inferred that such a fermentation would be feasible. Designing a 50 cm plate and a method of spreading and collecting the spores were suggested. Bioprocess technological significance of the “full‐scale industrial submerged fermentation started directly from spore inoculation omitting pre‐culture” has been discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:876–881, 2013     

Emission rate of charged spores in basidiomycetous fungi and the relaxation time of their electric charges

Basidiospores are one of the main components of coarse fraction of atmospheric aerosol. Majority of them, the ballistospores of 20,000 species of Basidiomycotina, carry electrostatic charges when getting airborne. To study the polarity and magnitude of primary charges and the hymenial emission rate of charged spores, 128 spore samples of 31 species of Agaricomycetes were collected in natural conditions. A portable device was placed under the fruiting body and the freely falling charged spores were extracted from the air by a horizontal homogeneous electrostatic field. The charge polarity distribution was the same in all intraspecies spore samples; it was unipolar-positive, unipolar-negative, or bipolar, depending on the species. The mean spore charge magnitude was 21–981 e, and it was not related to the emission rate of charged spores. The hymenial emission rate was fluctuating, and the maximum value was 715 charged spores cm^?2 s^?1. To estimate the territorial emission rate of charged spores, area of the hymenial surface per hectare of forest was calculated for three species and the maximum values were 11 m² ha^?1 and 8.6 × 10⁷ charged spores ha^?1 s^?1. Calculations showed that a spore charge diminished sevenfold within 47 min. Ecologists, health and agricultural scientists could be interested in this information. It could be useful by investigating the role of microorganisms in meteorological phenomena and in atmospheric processes in general.     

Fern and lycopod spores rain in a cloud forest of Hidalgo,Mexico

The aim of this study was to determine the composition of the “spore rain” of ferns and lycopods in a cloud forest. We tested whether the canopy impedes spore dispersal to surrounding areas and how spore dispersal is affected by rainfall. The spores were captured with a modified Bush–Gosling trap placed at 30 cm above ground level in forested and non-forested sites from March 2009 to February 2010. We collected 2462 fern spores from 158 morphospecies of which 76 were identified to species level. Thirty-seven species were found exclusively in the spore rain, and 39 were found as sporophytes as well (local component). Mean daily spore density (spores m^?2) was calculated to find the sporulation period for each species. Twenty species showed seasonal patterns of sporulation. The highest spore density was found at the forested site (70 morphospecies and 1856 spores), of which 39 morphospecies (1482 spores) corresponded to the local vegetation. Fifty-five taxa were shared between the forested and non-forested site. In the non-forested site, 605 spores were captured belonging to 64 species. The density of spore rain between sites was significantly different. The rainfall amount was the same at both sites, with a dry period in March, April, and July 2009, and February 2010. There was a negative effect of rainfall on spore rain. The main sporulation occurred in the dry season with strong winds. Although the canopy inhibits airborne dispersal of fern spores, a small amount of spores can disperse beyond the canopy and reach surrounding areas. The rainfall might wash spores to ground and favor the colonization and the establishment of new populations.     

Spatial and temporal distribution of Alternaria spores in the Iberian Peninsula atmosphere, and meteorological relationships: 1993–2009

This paper provides an updated of airborne Alternaria spore spatial and temporal distribution patterns in the Iberian Peninsula, using a common non-viable volumetric sampling method. The highest mean annual spore counts were recorded in Sevilla (39,418 spores), Mérida (33,744) and Málaga (12,947), while other sampling stations never exceeded 5,000. The same cities also recorded the highest mean daily spore counts (Sevilla 109 spores m^?3; Mérida 53 spores m^?3 and Málaga 35 spores m^?3) and the highest number of days on which counts exceeded the threshold levels required to trigger allergy symptoms (Sevilla 38 % and Mérida 30 % of days). Analysis of annual spore distribution patterns revealed either one or two peaks, depending on the location and prevailing climate of sampling stations. For all stations, average temperature was the weather parameter displaying the strongest positive correlation with airborne spore counts, whilst negative correlations were found for rainfall and relative humidity.     

First volumetric record of fungal spores in the atmosphere of Montevideo City,Uruguay: a 2-year survey

In Uruguay, aeromycological studies are restricted to a gravimetric analysis performed from December 1942 to March 1944 in Montevideo where spores of Pucciniaceae, Alternaria and Helminthosporium were the only specimens identified. Daily monitoring of airborne fungal spores was carried out for the first time in Montevideo, from April 2012 to March 2014, using a Rotorod sampler in order to evaluate the seasonal variation and influence of meteorological parameters. A total of 548,309.68 spores/m³ were recorded which belong to anamorphs of Higher Fungi (69.18 %), Phyla Ascomycota (12.62 %), Basidiomycota (8.01 %), Oomycota (0.37 %) and Myxomycota (0.06 %). Airborne spores occurred in Montevideo throughout the whole year. However, a seasonal pattern was revealed, with the highest concentrations recorded in autumn and summer. The most abundant spore types were Cladosporium (53.22 %), Alternaria (6.62 %), Didymella Group (5.86 %), Leptosphaeria Group (4.37 %) and Coprinus (4.3 %). Temperature appeared to be the most influential meteorological factor correlating significantly and positively with total spore, Cladosporium, Alternaria and Didymella Group abundance. Relative humidity influenced positively total spore, Cladosporium and Didymella Group concentrations while a weak negative association was obtained for Alternaria. Wind speed correlated negatively with total spore, Cladosporium, Alternaria and Didymella Group. Precipitation showed a negative influence on Alternaria, while positive correlations were observed for Didymella Group. For the first time, fungal spores considered allergenic were recorded in Montevideo atmosphere and the risk of exposure would have been high from December to June. However, long-term sampling is needed to define seasonal prevalence patterns and the influence of meteorological conditions on spore abundance.     

The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.     

Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase,MnxG, to the exosporium of the marine Bacillus sp. strain SG-1

Dormant spores of the marine Bacillus sp. strain SG-1 catalyze the oxidation of manganese(II), thereby becoming encrusted with insoluble Mn(III,IV) oxides. In this study, it was found that the Mn(II)-oxidizing activity could be removed from SG-1 spores using a French press and recovered in the supernatant following centrifugation of the spores. Transmission electron microscopy of thin sections of SG-1 spores revealed that the ridged outermost layer was removed by passage through the French press, leaving the remainder of the spore intact. Comparative chemical analysis of this layer with the underlying spore coats suggested that this outer layer is chemically distinct from the spore coat. Taken together, these results indicate that this outer layer is an exosporium. Previous genetic analysis of strain SG-1 identified a cluster of genes involved in Mn(II) oxidation, the mnx genes. The product of the most downstream gene in this cluster, MnxG, appears to be a multicopper oxidase and is essential for Mn(II) oxidation. In this study, MnxG was overexpressed in Escherichia coli and used to generate polyclonal antibodies. Western blot analysis demonstrated that MnxG is localized to the exosporium of wild-type spores but is absent in the non-oxidizing spores of transposon mutants within the mnx gene cluster. To our knowledge, Mn(II) oxidation is the first oxidase activity, and MnxG one of the first gene products, ever shown to be associated with an exosporium.     

High-resolution solid-state 13C nuclear magnetic resonance of bacterial spores: identification of the alpha-carbon signal of dipicolinic acid.

Natural-abundance solid-state 13C nuclear magnetic resonance spectra were obtained for bacterial spores for the first time by using the technique of cross-polarization magic-angle-spinning nuclear magnetic resonance spectroscopy. A resonance at about 150 ppm, detectable in spore samples having a Mn content of less than 0.05%, was consistent with an identification as the alpha-carbon signal of calcium dipicolinate; this signal was missing from a spore sample treated with acid to release dipicolinate and from a spore coat preparation. Carbohydrate peaks were particularly intense in spores and coat preparations of Bacillus macerans. Signals ascribable to beta-hydroxybutyrate were prominent in a B. cereus sample.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light

Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10⁷ spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3 × 10⁴ spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3 × 10⁵ spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed.     

Germination proteins in the inner membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster

Dormant bacterial spores are extraordinarily resistant to environmental insults and are vectors of various illnesses. However, spores cannot cause disease unless they germinate and become vegetative cells. The molecular details of initiation of germination are not understood, but proteins essential in early stages of germination, such as nutrient germinant receptors (GRs) and GerD, are located in the spore inner membrane. In this study, we examine how these germination proteins are organized in dormant Bacillus subtilis spores by expressing fluorescent protein fusions that were at least partially functional and observing spores by fluorescence microscopy. We show that GRs and GerD colocalize primarily to a single cluster in dormant spores, reminiscent of the organization of chemoreceptor signalling complexes in Escherichia coli. GRs require all their subunits as well as GerD for clustering, and also require diacylglycerol addition to GerD and GRs' C protein subunits. However, different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. We predict that the clusters represent a functional germination unit or 'germinosome' in the spore inner membrane that is necessary for rapid and cooperative response to nutrients, as conditions known to block nutrient germination also disrupt the protein clusters.     

The source of the heat resistance of bacterial spores: Study of water in spores by NMR

It has been postulated that the heat stabilization of the essential macromolecules in the core of the spore may be produced by dehydration at two levels: (i) the spore is drier at high relative humidity than the vegetative cell and (ii) the core of the spore may be less hydrated than the cortex and the coat. The latter postulate was subjected to experimental testing by ¹H-NMR studies of the water signal in the five species of spores and coat and (coat + cortex) preparations. The transverse relaxation rate $\text{(}\text{1}\text{T}{}_2\text{)}$ was determined in samples equilibrated at constant relative humidity. To allow for the effect of paramagnetic ions on $\text{1}\text{T}{}_2$ a model system (wool keratin) was studied in the presence of known amounts of Ca(II), Mn(II), Cu(II), Ni(II) and Fe(III). Because of the dominant effect of Mn(II) on $\text{1}\text{T}{}_2$, the effect of small amounts of other metal ions in spores was neglected. The relaxation rate of water at a particular relative humidity and manganese concentration was consistently less for intact spores than for coat or coat + cortex, hence the water in the core is more mobile than in the outer integuments. Sorption isotherm studies have shown that at a particular relative humidity there is about as much water in the core as in the cortex and coat. These two results taken together indicate that the hypothesis that the core is drier than the cortex and coat is incorrect, hence the spore is not heat-stabilized in this way. A theory is proposed in which heat stabilization is attributed to immobilization of essential enzymes and nuclei acids by a solid support, calcium dipicolinate, in a similar fashion to the heat stabilization of enzymes in a charged polymer matrix. It is proposed that stabilization is effected by electrostatic and hydrogen bonds between the calcium dipicolinate and the essential macromolecules. Experiments in progress show that enzymes and DNA are heat-stabilized in vitro by calcium dipicolinate.     

Chloroplast activities of dark-imbibed and photoinduced spores of the fernOnoclea sensibilis

Summary Chloroplast activities of dark-imbibed (non-germinating) and photoinduced (germinating) spores of the sensitive fern,Onoclea sensibilis were compared to gain insight into the germination process. There were no changes in the number of chloroplasts or in the chlorophyll contents of the spore during dark-imbibition and during the early phase of germination. Levels of increase in the Chloroplast DNA content of dark-imbibed and photoinduced spores were nearly the same and were associated with autoradiographic incorporation of [³H]thymidine into the cytoplasm. However, incorporation of the label into the nucleus occurred only during photoinduction of spores. Analysis of Chloroplast and nuclear DNA contents by dot-blot hybridization with labeled gene-specific probes has confirmed that chloroplast DNA content of the spore increases during dark-imbibition and photoinduction, while increase in nuclear DNA occurs only in photoinduced spores. Chloroplasts isolated from dark-imbibed and photoinduced spores incorporated [³H]TTP into an acid-insoluble fraction identified as DNA. The results show that physiological activities of chloroplasts of dark-imbibed and photoinduced spores ofO. sensibilis are similar and support an exclusive role for nuclear DNA synthesis in spore germination.     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.