Induced release of Bacillus spores from sporangia by sodium sulphate

Incubation of sporulating cultures of Bacillus anthracis, B. cereus, B. subtilis and B. thuringiensis in 1°0 mol/l sodium sulphate markedly increased the release of free spores from sporangia. It is postulated that the release of spores is due to activation of latent autolysins which hydrolyse sporangial cell walls. Sodium sulphate-induced lysis of sporangia represents a novel and highly effective method for the recovery of spores from cultures of Bacillus species.     

Parasporal bodies of Bacillus laterosporus sporangia.

Intact colonies of Bacillus laterosporus examined by thin-section transmission electron microscopy revealed sporangia in various stages of development and degeneration as the endospores matured. The sporangia formed a surface layer of hexagonally arranged subunits. The variety of parasporal bodies raised questions of developmental and ecologic utility.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T

S.F. BLOOMFIELD AND M. ARTHUR. 1992. Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortes material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T.

Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortex material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Study of substances released by ultrasonic treatment from Bacillus stearothermophilus spores

P. PALACIOS. J. BURGOS, L. HOZ, B. SANZ AND J.A. ORDÓÑEZ. 1991. Spore heat resistance is reduced when combined ultrasonic and heat treatments are applied. To explain this phenomenon the substances released from Bacillus stearothermophilus spores to the surrounding aqueous medium by the ultrasonic treatment (20 kHz, 120 W, 12°C, 30 min) were studied. Calcium, dipicolinic acid and a glycopeptide of 7 kDa molecular weight were detected in the ultrasonicated medium. Fatty acids, acyl glycerols and glycolipids (but no phospholipids) were also released. The decrease of heat resistance induced by the ultrasonic treatment was attributed to the release of low molecular weight substances from the spore protoplast with consequent modification of its hydration state.     

Induced enzyme release from synaptosomes by halothane

GABA-transaminase has been found to be released from rat brain synaptosomes by halothane in a dose-related manner. The releases of both GABA-transaminase and succinic semialdehyde dehydrogenase were increased with time. The release of other enzymes (creatine kinase, glutamate decarboxylase, aspartate transaminase, lactate dehydrogenase, and malate dehydrogenase) was less in magnitude and not related to the duration of incubation. Such observations suggested a specific event in the halothane-induced release of GABA-catabolizing enzymes. A suggestion linking mode of anesthetic action to a mitochondrial effect of volatile anesthetics was made.     

Inactivation of Bacillus spores by gaseous ozone

The sporicidal activity of ozone in the gas phase was investigated. Spores of six strains of Bacillus species deposited on filter paper or glass fibre filter were conditioned at different relative humidities (r.h.), and then exposed to ozone ranging in concentration from 0.5 to 3.0 mg/1 at different r.h. There was a lag phase in the initial stage of exposure followed by an exponential decrease in the number of survivors with time, although no lag phase was observed with one strain. Inactivation rates increased with increasing exposure r.h. while no significant inactivation was attained at a r.h. of 50% or below. The conditioning r.h. influenced the duration of the lag phase. The D-values (decimal reduction time) in the logarithmic phase varied roughly in inverse proportion to the ozone concentration.     

Sulphate release from keratan sulphate and heparan sulphate by bovine N-acetylglucosamine-6-sulphate sulphatase

The functions of sulphated monosaccharides within glycosaminoglycans(GAGs) and glycoproteins are being studied intensely, but progressis hindered by an inability to selectively desulphate glycoconjugates.We recently identified an N-acetylglucosamine-6-sulphate sulphatase(NG6SS) from bovine kidney that can remove sulphate from N-acetylglucosamine-6-sulphate(GlcNAc-6-SO₄) within oligosaccharides and glycoproteins. However,the potential ‘endosulphatase’ activity of the NG6SStoward GAGs is not known. To test for this possibility, [³H]glucosamine-,[³H]galactose- and ³⁵SO_4- labelled keratan sulphate (KS) wereseparately prepared by metabolic radiolabelling of bovine cornea.NG6SS quantitatively removed sulphate from KS without releaseof sugar fragments. The enzyme had a K_m of 4.7 mM toward freeGlcNAc-6-SO₄, but its K_m for commercially available bovine cornealKS was found to be 9.1 µM. Analyses of both KS and heparansulphate after treatment with NG6SS demonstrated significantloss of sulphate from GlcNAc-6-SO₄ in both GAGs. These findingsmay be relevant for future studies aimed at defining the function(s)of GlcNAc-6-SO₄ residues in GAGs and understanding the catabolismof GAGs, especially in regard to sulphatidoses, such as SanfilippoD syndrome in humans, which involves a deficiency of NG6SS activity catabolism endosulphatase glycosaminoglycans sulphation     

Destruction of Bacillus licheniformis spores by microwave irradiation

Aims:  To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores.
Methods and Results:  We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0·5 kW) was modified to output power at 2·0 kW, which allowed a shorter sterilization cycle. A 2·0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0·5 kW microwave oven. In contrast to boiling and 0·5 kW microwave treatments, the 2·0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2·0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane.
Conclusions:  These results suggest that 2·0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study:  This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.     

Experimental evidence against sexual fusions of spores from unilocular sporangia of Ectocarpus siliculosus (Phaeophyta)

Evidence that sexual fusion does not occur in zoids from unilocular sporangia of Ectocarpus siliculosus is presented as follows: (1) Zoids from various types of unilocular sporangia do not interact with genuine gametes; (2) Aggregates of two zoids, previously claimed to be zygotes, were frequently seen in cultured material but could be demonstrated to be the result of incomplete cell separation during sporogenesis; (3) Culture studies showed that there is no fusion of male and female nuclei in such aggregates.     

Solubilization of coat protein from Bacillus thiaminolyticus spores.

Solubilization of spore coat protein of Bacillus thiaminolyticus was investigated using various reagents, and partial characterization of solubilized protein was carried out. Five per cent of the sodium dodecyl sulphate (SDS) treatment was the most effective for solubilization of coat protein, and 5% SDS + 8 M urea and 0.06 N NaOH were also useful. Acrylamide gel disc electrophoresis indicated that the SDS-soluble fraction mainly consists of a single band of protein and its molecular weight was estimated at about 15,000. The SDS+ urea-soluble fraction comprised two proteins with a molecular weight of 14,500 and 32,000, and an alkali-soluble fraction of 12,000 and 25,000 respectively.     

Recovery of Bacillus sphaericus 2362 spores from growth medium by flocculation/sedimentation

Bacillus sphaericus 2362 spore sedimentation was evaluated through batch settling experiments, using tannin as the flocculating agent. The flocculation was demonstrated to be efficient up to the tannin concentration of 150 ppm. The floc diameter averaged 208 micro m, and its density was found to be 1.09 g/mL. The sedimentation process using tannin as a flocculating agent is promising, and a spore concentrate was obtained without loss of biological activity, presenting a LC(50) equal to 0.22 ppm. The result indicates that the larvicide activity is appropriate for controlling populations of Culex quinquefasciatus.     

Immunostimulatory activity of Bacillus spores

Bacillus species, typically Bacillus subtilis, are being used as probiotics and mounting evidence indicates that Bacillus species are important for development of a robust gut-associated lymphoid system (GALT). We used a number of gut isolates of Bacillus incorporating three species, B. subtilis, Bacillus licheniformis and Bacillus flexus to evaluate the nature of interaction between spores and the GALT. In mice orally administered with spores, evidence of cell proliferation was determined in the germinal centers of Peyer's patches. Stimulation of antigen-presenting cells and T lymphocytes was also markedly enhanced. Cytokines were shown to be induced in spleens and mesenteric lymph nodes of mice including the proinflammatory cytokines, tumour necrosis factor-alpha and IL-6. We also demonstrated that vegetative cells of B. subtilis can stimulate expression of the toll-like receptor (TLR) genes for TLR2 and TLR4. However, we were able to show that spores could not stimulate either and must, by default, interact with another TLR and by this mechanism help activate innate immunity.