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Surface Charge Properties of and Cu(II) Adsorption by Spores of the Marine Bacillus sp. Strain SG-1.

Spores of marine Bacillus sp. strain SG-1 are capable of oxidizing Mn(II) and Co(II), which results in the precipitation of Mn(III, IV) and Co(III) oxides and hydroxides on the spore surface. The spores also bind other heavy metals; however, little is known about the mechanism and capacity of this metal binding. In this study the characteristics of the spore surface and Cu(II) adsorption to this surface were investigated. The specific surface area of wet SG-1 spores was 74.7 m per g of dry weight as measured by the methylene blue adsorption method. This surface area is 11-fold greater than the surface area of dried spores, as determined with an N(2) adsorption surface area analyzer or as calculated from the spore dimensions, suggesting that the spore surface is porous. The surface exchange capacity as measured by the proton exchange method was found to be 30.6 mumol m, which is equal to a surface site density of 18.3 sites nm. The SG-1 spore surface charge characteristics were obtained from acid-base titration data. The surface charge density varied with pH, and the zero point of charge was pH 4.5. The titration curves suggest that the spore surface is dominated by negatively charged sites that are largely carboxylate groups but also phosphate groups. Copper adsorption by SG-1 spores was rapid and complete within minutes. The spores exhibited a high affinity for Cu(II). The amounts of copper adsorbed increased from negligible at pH 3 to maximum levels at pH >6. Their great surface area, site density, and affinity give SG-1 spores a high capability for binding metals on their surfaces, as demonstrated by our experiments with Cu(II).

Journal Article↗

A gene encoding alanine racemase is involved in spore germination in Bacillus thuringiensis.

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted L-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of L-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, D-alanine was an effective and competitive inhibitor of L-alanine-induced germination of B. thuringiensis spores. D-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing L-alanine-induced germination by inhibiting alanine racemase. D-alanine also increased the rate of inosine-induced germination of wild-type spores. However, D-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted D-alanine to L-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.

Alanine Racemase↗

Thermal properties of bacterial spores and biopolymers.

Differential scanning calorimetry (DSC) measurements of dormant bacterial spores is traditionally associated with an endothermic transition at around 50 degrees C. This endothermic transition was described as an indicator for two main physico-chemical states in spores. These were a glassy state in the dormant spore core as a model for spore dormancy and a heat-activated state that generally facilitates spore resuscitation. The idea of a glassy state in dormant spores is based on the observation that a similar transition as in dormant spores was observed in low moisture biopolymers that are in a glassy state. Thermal properties of spores of Bacillus subtilis and B. cereus in a dormant and germinated, resuscitated state and of an outer and an inner coatless spore mutant of B. subtilis were investigated. Biopolymers with low moisture (<15%) and high moisture (>30%) contents such as maize starch, pectin, RNA and DNA were further studied. Critical evaluation of results revealed that the low temperature transition in dormant spores has some similarities to those observed in glassy biopolymers, but also to those of fully hydrated proteins and therefore does not necessarily indicate a glassy low moisture state. Its origin can also be attributed to the outer spore coats and it occurred at a lower temperature and for a shorter duration to be of significance for thermal heat activation of spores.

Bacillus cereus↗

Efficacy of heat and ethanol spore treatments for the isolation of psychrotrophic Clostridium spp. associated with the spoilage of chilled vacuum-packed meats.

Psychrotrophic Clostridium spp. associated with chilled meat spoilage are difficult to isolate and culture. In this study the kinetics of heat and ethanol spore inactivation was determined as a first step towards optimising the recovery of psychrotrophic clostridial spores from meat and environmental samples. To determine heat inactivation, spores of nine isolates associated with spoiled chilled or frozen meat and a psychrotrophic reference strain Clostridium algidicarnis NCFB 2931, suspended in phosphate buffer, were exposed to temperatures between 75 degrees C and 95 degrees C for 0 to 120 min using a submerged tube procedure. Survivors after various temperature-time combinations were enumerated on Peptone Yeast Extract Glucose Starch (PYGS) agar containing lysozyme. D-values and z-values for each spore suspension were determined from their respective survival curves. To determine ethanol inactivation, similar phosphate buffer spore suspensions were mixed with equal volumes of absolute ethanol, incubated at 20 degrees C and survivors enumerated on lysozyme-containing PYGS agar after 0 to 300 min. Based on spore heat inactivation, the 10 isolates could be grouped as having either heat-sensitive or heat-resistant spores. For heat-sensitive spore types, 60 min ethanol treatment gave maximum spore recovery whereas for heat-resistant spore types, heat treatment at 80 degrees C for 10 min gave the best recovery. When the spore heat-resistance type is unknown, as would be the case when attempting an isolation from spoiled product, both an ethanol treatment and a separate heat treatment should be used, to ensure maximum spore recovery.

Animals↗

The Smk1p MAP kinase negatively regulates Gsc2p, a 1,3-beta-glucan synthase, during spore wall morphogenesis in Saccharomyces cerevisiae.

Spore formation in Saccharomyces cerevisiae involves the sequential deposition of multiple spore wall layers between the prospore membranes that surround each meiotic product. The Smk1p mitogen-activated protein (MAP) kinase plays a critical role in spore formation, but the proteins that interact with Smk1p to regulate spore morphogenesis have not been described. Using mass spectrometry, we identify Gsc2p as a Smk1p-associated protein. Gsc2p is a 1,3-beta-glucan synthase subunit involved in synthesizing an inner spore wall layer. We find that 1,3-beta-glucan synthase activity is elevated in smk1 mutants, suggesting that SMK1 negatively regulates GSC2. Although deposition of the two inner spore wall layers is normal in smk1 mutants, deposition of the outer layers is aberrant. However, eliminating GSC2 activity restores normal deposition of the third spore wall layer in smk1 mutants, indicating that negative regulation of GSC2 by SMK1 is important for spore wall deposition. Our findings suggest a model for the coordination of spore wall layer deposition in which Smk1p facilitates the transition between early and late phases of spore wall deposition by inhibiting a spore wall-synthesizing enzyme important for early phases of spore wall deposition.

Cell Wall↗

In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody.

Antibodies (Abs) to the protective antigen (PA) component of the anthrax toxins have anti-spore as well as anti-toxin activities. Anti-PA antisera and purified anti-PA Abs enhance the phagocytosis by murine-derived macrophages (MQs) of spores of the Ames and Sterne strains and retard the germination of extracellular spores in vitro. The fate after phagocytosis of untreated and anti-PA-treated spores was further studied in culture medium that supported phagocytosis without stimulating spore germination (Dulbecco's minimal essential medium with horse serum 10%). The spores germinated within cells of primary peritoneal murine MQs (C3H/HeN) and MQs of the RAW264.7 MQ-like cell line; germination was associated with a rapid decline in spore viability. Exposure of MQs to inhibitors of phago-endosomal acidification (bafilomycin A and chloroquine) reduced the efficiency of MQ killing and allowed outgrowth and replication of the organisms. Treatment of spores with anti-PA Abs stimulated their phagocytosis and was associated with enhanced MQ killing of the spores. The enhanced killing of spores correlated with the greater extent of germination of anti-PA-treated spores after phagocytosis. A PA null mutant of the Ames strain exhibited none of the effects associated with anti-PA Ab treatment ofthe parental strain. Thus, the anti-PA Ab-specific immunity induced by vaccines has anti-spore activities and its role in impeding the early stages of infection with Bacillusanthracis needs to be assessed.

Animals↗

Decreased particulate NADH oxidase activity in Bacillus subtilis spores after polymyxin B treatment.

The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed.

Alanine Dehydrogenase↗

CRR1, a gene encoding a putative transglycosidase, is required for proper spore wall assembly in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, sporulation is a developmental process that converts a single cell into four haploid spores. The four haploid nuclei are encapsulated within multilayered spore walls that protect them against stressful conditions. The formation of the spore-specific cell wall is a highly coordinated process that requires the participation of enzymic activities for biosynthesis, degradation, and cross-linking between components. Here the sporulation-specific gene CRR1, encoding a putative transglycosidase that is required for proper spore wall assembly, is described. Both the transcription of CRR1 and the synthesis of Crr1p were induced biphasically under sporulating conditions, with a first expression peak displaying kinetics similar to those of the middle to middle-late sporulation-specific genes, and a second late peak after 24 h under these conditions. Localization studies revealed that Crr1p localized to the spore wall that surrounds each of the four ascospores within the mature asci. Mutation of this gene had no effect on the efficiency of spore formation. However, crr1 mutant spores were sensitive to hydrolytic enzymes such as glusulase and to heat-shock treatments, underscoring the importance of this gene in the proper formation and assembly of the ascospore wall. Moreover, the deletion of CRR1 had additive effects with respect to the sensitivity of cda1 cda2 mutants to these treatments. Interestingly, overexpression of CRR1 not only complemented the phenotype of the crr1 strain but also rendered spores more resistant to the stress conditions than the wild-type. Like other mutants impaired in the formation of the spore outer layer, crr1 mutants were permeable to Calcofluor White. Finally, detailed analysis by electron microscopy of the spore walls in the crr1 mutants revealed a defect in the assembly of the spore wall components, suggesting a role for Crr1p in the cross-linking between the inner (glucan/mannoprotein) and the outer (chitosan/dityrosine) spore layers.

Cell Wall↗

Involvement of calcium in germination of coat-modified spores of Bacillus cereus T.

The effect of calcium on germination of coat-modified Bacillus cereus T spores was investigated. Coat-modified spores produced either by chemical extraction (SDS-DTT-treated spores) or by mutagenesis (10LD mutant spores) were unable to germinate in response to inosine. While SDS-DTT-treated spores could germinate slowly in the presence of L-alanine, 10LD mutant spores could not germinate at all. The lost or reduced germinability of coat-modified spores was restored when exogenous Ca2+ was supplemented to the germination media. The calcium requirement of coat-modified spores for germination was fairly specific. The simultaneous presence of germinant with Ca2+ was also required for germination of coat-modified spores. The optimal recovery of germinability was observed in the presence of 1.0 mM of calcium acetate. The calcium requirement itself was remarkably diminished under the condition in which L-alanine and a certain purine nucleoside analog, adenosine or inosine, coexisted. The lost or diminished germinability observed in SDS-DTT-treated spores or 10LD mutant spores may be attributed to the loss of calcium associated with the spore integuments.

Alanine↗

Assessment of elasticity and topography of Aspergillus nidulans spores via atomic force microscopy.

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA(+) and rodA(-) strains. Tapping-mode AFM images of wild-type and rodA(+) spores in air showed characteristic "rodlet" protein structures covering a granular spore surface. In comparison, rodA(-) spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA(+) spores. Rodlets were removed from rodA(+) spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 +/- 10, 120 +/- 10, and 300 +/- 20 N/m and the elastic moduli to be 6.6 +/- 0.4, 7.0 +/- 0.7, and 22 +/- 2 GPa for wild-type, rodA(+) and rodA(-) spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.

Aspergillus nidulans↗

Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis.

Nucleic acid synthesis was studied during germination and outgrowth of normal spores of Bacillus subtilis, as well as of spores carrying the genome of phage phie. In a system in which development was restricted to the spore-darkening phase, synthesis of ribonucleic acid (RNA), but not deoxyribonucleic acid (DNA), was detected. The extent of RNA synthesis and turnover, during this phase was similar for the two types of spores. In a partially darkened population of spores of either type, there was little RNA degradation, whereas there was considerable turnover in a fully darkened population. The DNA-dependent RNA polymerase of dormant or dark spores was not active in vitro with phi DNA as template, although a sigma-like factor could be separated from the polymerizing activity by zone centrifugation. Within 40 min after resuspension of dark spores in a medium that allows outgrowth, the enzyme acquired the ability to transcribe the phage DNA efficiently. During outgrowth, both normal and carrier spores synthesized DNA, but in carrier spores this DNA was almost entirely phage specific. The pattern of RNA accumulation in normal spores was in two distinct phase (0 to 60 min and 90 to 180 min). The second phase was absent in outgrowing carrier spores. The burst of phage in carrier spores occurred at 160 to 180 min.

Bacillus subtilis↗

Properties of Bacillus cereus temperature-sensitive mutants altered in spore coat formation.

Three conditional Bacillus cereus mutants altered in the assembly or formation of spore coat layers were analyzed. They all grew as well as the wild type in an enriched or minimal medium but produced lysozyme and octanol-sensitive spores at the nonpermissive temperature (35 to 38 degrees C). The spores also germinated slowly when produced at 35 degrees C. Temperature-shift experiments indicated that the defective protein or regulatory signal is expressed at the time of formation of the outer spore coat layers. Revertants regained all wild-type spore properties at frequencies consistent with initial point mutations. Spore coat defects were evident in thin sections and freeze-etch micrographs of mutant spores produced at 35 degrees C. In addition, one mutant contained an extra surface deposit, perhaps unprocessed spore coat precursor protein. A prevalent band of about 65,000 daltons (the same size as the presumptive precursor) was present in spore coat extracts of this mutant and may be incorrectly processed to mature spore coat polypeptides. Another class of mutants was defective in the late uptake of half-cystine residues into spore coats. Such a defect could lead to improper formation of the outer spore coat layers.

Bacillus cereus↗

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination.

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.

Azides↗

Luminometric and differential scanning calorimetry (DSC) studies on heat- and radiation inactivation of Bacillus subtilis luxAB spores.

A bioluminescent derivative of Bacillus subtilis containing a plasmid encoding a luxAB fusion under control of a vegetative promoter and gives bioluminescence upon addition of an exogenous long-chain aldehyde has been used as test organism. Its spore populations have been produced and their heat- and radiation survival curves established. Heat-sensitization effect of pre-irradiation of spores was proven not only by colony counting but also with differential scanning calorimetry. Under a linearly programmed temperature increase, the heat destruction of spores surviving 2.5 kGy gamma irradiation resulted in at a few centigrade lower temperature than that of untreated spores. Heat denaturation endotherms in the DSC-thermogram of irradiated spores were shifted to lower temperatures as well. Comparative turbidimetric, luminometric and phase-contrast microscopic studies of untreated, heat-treated and irradiated spore populations showed that the kinetics of germination and the light emission during germination of radiation-inactivated spores were the same as those of untreated spores, revealing that the pre-formed luciferase enzyme packaged into the spores during sporulation remained intact after an irradiation dose causing 90% decrease in number of colony forming spores. Therefore, in contrast to heat-treated spores, the initial bioluminescence reading upon germination of irradiated spores does not reflect the viable count of their population.

Bacillus subtilis↗

Sexually mediated shedding of Myxobolus fallax spores during spermiation of Litoria fallax (anura).

Myxobolus fallax (Myxosporea) infects the testes of the dwarf green tree frog Litoria fallax without apparently affecting the host's health, behavior, or testicular sperm numbers or quality. We investigated the shedding M. fallax spores and the relationship between M. fallax infection and fertility in L. fallax. During natural spawning, comparisons were made between the prevalence and intensity of M. fallax infection, spore shedding, and fertilization rates. During the hormonal induction of spermiation, comparisons were made between the prevalence and intensity of M. fallax infection, and the number of sperm and spores shed. During natural spawning, the prevalence or intensity of infection or spore shedding did not affect fertility. Spermiation and spore shedding was induced in 10 males by the administration of human chorionic gonadotrophin, with 10 controls. Histology showed that all 10 males were infected by M. fallax. The controls shed no sperm or spores. Nine infected males shed sperm 3 h post-administration (PA), and 3 of these also shed spores. Only the 3 males that shed sperm and spores at 3 h PA also shed sperm at 6 h PA, at which time they also shed spores. In total, the 3 males that shed spores gave higher sperm numbers than the 6 males that did not shed spores. Overall, these results show that the shedding of M. fallax spores is sexually mediated through reproductive hormones without affecting fertility.

Animals↗

Spore dimensions of Puccinia species of cereal hosts as determined by image analysis.

Digital image analysis was used to measure dimensions of spores produced by Puccinia coronata, P. graminis, P. hordei, P. recondita, P. striiformis and P. triticina. Included were teliospores, basidiospores, urediniospores and, except for P. striiformis, pycniospores and aeciospores. Length, width and projection area of spores were measured with NIH Image or Scion software. By using limits on size, spores were automatically selected and measured, except for teliospores, which required manual elimination of the pedicel and separation of images of adhering spores. Length and width were determined as the major and minor axes of the best fitting ellipse for each spore. This procedure gave values for length and width close to results obtained with an ocular micrometer. Projection area was determined as the number of pixels within spore boundaries multiplied by the area represented by each pixel, giving values that are not feasible to obtain accurately with an ocular micrometer. Of the species studied, spores of P. recondita had the largest dimensions, P. triticina had the smallest. The rank of the six species based on increasing width, length or projection area was almost the same, using each spore type except pycniospores. Generally, differences of 5% in a given spore dimension between two species were significant. Differences between species were greater with basidiospores and aeciospores than with other spore types. Teliospores were unique in that length and width were negatively correlated, resulting in less variation in area than in length or width. The results indicate that image analysis is useful for measuring spore dimensions, that projection area of spores is a useful added parameter for characterizing rust species and that dimensions of teliospores, basidiospores, aeciospores and urediniospores each are potentially useful for differentiating species.

Basidiomycota↗

Predictive model for Clostridium perfringens growth in roast beef during cooling and inhibition of spore germination and outgrowth by organic acid salts.

Spores of foodborne pathogens can survive traditional thermal processing schedules used in the manufacturing of processed meat products. Heat-activated spores can germinate and grow to hazardous levels when these products are improperly chilled. Germination and outgrowth of Clostridium perfringens spores in roast beef during chilling was studied following simulated cooling schedules normally used in the processed-meat industry. Inhibitory effects of organic acid salts on germination and outgrowth of C. perfringens spores during chilling and the survival of vegetative cells and spores under abusive refrigerated storage was also evaluated. Beef top rounds were formulated to contain a marinade (finished product concentrations: 1% salt, 0.2% potassium tetrapyrophosphate, and 0.2% starch) and then ground and mixed with antimicrobials (sodium lactate and sodium lactate plus 2.5% sodium diacetate and buffered sodium citrate and buffered sodium citrate plus 1.3% sodium diacetate). The ground product was inoculated with a three-strain cocktail of C. perfringens spores (NCTC 8238, NCTC 8239, and ATCC 10388), mixed, vacuum packaged, heat shocked for 20 min at 75 degrees C, and chilled exponentially from 54.5 to 7.2 degrees C in 9, 12, 15, 18, or 21 h. C. perfringens populations (total and spore) were enumerated after heat shock, during chilling, and during storage for up to 60 days at 10 degrees C using tryptose-sulfite-cycloserine agar. C. perfringens spores were able to germinate and grow in roast beef (control, without any antimicrobials) from an initial population of ca. 3.1 log CFU/g by 2.00, 3.44, 4.04, 4.86, and 5.72 log CFU/g after 9, 12, 15, 18, and 21 h of exponential chilling. A predictive model was developed to describe sigmoidal C. perfringens growth curves during cooling of roast beef from 54.5 to 7.2 degrees C within 9, 12, 15, 18, and 21 h. Addition of antimicrobials prevented germination and outgrowth of C. perfringens regardless of the chill times. C. perfringens spores could be recovered from samples containing organic acid salts that were stored up to 60 days at 10 degrees C. Extension of chilling time to > or =9 h resulted in >1 log CFU/g growth of C. perfringens under anaerobic conditions in roast beef. Organic acid salts inhibited outgrowth of C. perfringens spores during chilling of roast beef when extended chill rates were followed. Although C. perfringens spore germination is inhibited by the antimicrobials, this inhibition may represent a hazard when such products are incorporated into new products, such as soups and chili, that do not contain these antimicrobials, thus allowing spore germination and outgrowth under conditions of temperature abuse.

Animals↗

The MADS-box transcription factor SrfA is required for actin cytoskeleton organization and spore coat stability during Dictyostelium sporulation.

The MADS-box transcription factor SrfA is involved in spore differentiation in Dictyostelium [Development 125 (1998) 3801]. Mutant spores show an altered morphology and loss of viability. A detailed structural analysis of mutant spores has been performed to gain insight into the specific aspects of spore differentiation in which SrfA is involved. Two main structural defects have been observed. One is the formation of high order actin structures, the so-called actin rods. SrfA mutant spores showed the initial stages of rod formation but no mature rods were found in older spores either in the nucleus or the cytoplasm. Moreover, phosphorylation of actin, that is believed to stabilize the actin rods, is strongly reduced in the mutant. The other defect observed was the formation of the spore coat. Young srfA- spores show basically normal trilaminar coat structures suggesting that release of prespore vesicles and basic assembly of the coat takes place in the absence of SrfA. However, the outer layer gets wavier as the spore ages and suffers a progressive degradation suggesting a late defect in the stability of the spore coat. Taken together, these results suggest that SrfA is involved in late events of spore maturation necessary for spore stability.

Actins↗