Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

AIMS: To determine the mechanisms of killing of Bacillus subtilis spores by ethanol or strong acid or alkali. METHODS AND RESULTS: Killing of B. subtilis spores by ethanol or strong acid or alkali was not through DNA damage and the spore coats did not protect spores against these agents. Spores treated with ethanol or acid released their dipicolinic acid (DPA) in parallel with spore killing and the core wet density of ethanol- or acid-killed spores fell to a value close to that for untreated spores lacking DPA. The core regions of spores killed by these two agents were stained by nucleic acid stains that do not penetrate into the core of untreated spores and acid-killed spores appeared to have ruptured. Spores killed by these two agents also did not germinate in nutrient and non-nutrient germinants and were not recovered by lysozyme treatment. Spores killed by alkali did not lose their DPA, did not exhibit a decrease in their core wet density and their cores were not stained by nucleic acid stains. Alkali-killed spores released their DPA upon initiation of spore germination, but did not initiate metabolism and degraded their cortex very poorly. However, spores apparently killed by alkali were recovered by lysozyme treatment. CONCLUSIONS: The data suggest that spore killing by ethanol and strong acid involves the disruption of a spore permeability barrier, while spore killing by strong alkali is due to the inactivation of spore cortex lytic enzymes.SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide further information on the mechanisms of spore killing by various chemicals.     

Mechanism of chemical manipulation of the heat resistance of Clostridium perfringens spores

The mechanism(s) of chemical manipulation of the heat resistance of Clostridium perfringens type A spores was studied. Spores were converted to various ionic forms by base-exchange technique and these spores were heated at 95°C. Of the four ionic forms, i.e. Ca²⁺, Na⁺, H⁺ and native, only hydrogen spores appeared to have been rapidly inactivated at this temperature, when survivors were enumerated on the ordinary plating medium. However, the recovery of the survivors was improved when the plating medium was supplemented with lysozyme, and more dramatically when the heated spores were pretreated with alkali followed by plating in the medium containing lysozyme. In contrast to crucial damage to germination, in particular to spore lytic enzyme, no appreciable amount of DPA was released from the heat-damaged H-spores. These results suggest that a germination system is involved in the thermal inactivation of the ionic forms of spores, and that exchangeable cation load plays a role in protection from thermal damage of the germination system within the spore. An enhancement of thermal stability of spore lytic enzyme in the presence of a high concentration of NaCl was consistent with the hypothesis.     

Germination of Heat- and Alkali-Altered Spores of Clostridium perfringens Type A by Lysozyme and an Initiation Protein

The normal system functioning in the utilization of metabolizable germinants by both heat-sensitive and heat-resistant spores of Clostridium perfringens was inactivated by heat or by treatment of the spores with alkali to remove a soluble coat protein layer. Altered spores were incapable of germination (less than 1%) and outgrowth (less than 0.0005%) in complex media without the addition of either lysozyme or an initiation protein produced by C. perfringens. The addition of either of these agents permitted, in the case of alkali-treated spores, both 90 to 95% germination and outgrowth, as measured by colony formation. In the case of heat-damaged spores, only 50% germination and 2% outgrowth resulted from addition of the initiation protein, whereas lysozyme permitted 85% germination and 8% outgrowth. Alteration of the spores by heat or alkali apparently inactivated the normal lytic system responsible for cortical degradation during germination. Kinetics of production of the initiation protein and conditions affecting both its activity and that of lysozyme on altered spores are described.     

Effect of disruption by sonication under different conditions on the activity of glucose dehydrogenase from resting spores of Bacillus subtilis

The activity of glucose dehydrogenase present in resting spores of Bacillus subtilis varied strikingly with the conditions for disrupting the spores by sonic treatment, namely, the time and strength of sonication, and the type and pH of the solution used for suspending the spores. When the resting spores were sonicated for 30 min at a current of 1.45 A in 100 mM phosphate buffer in the range of pH 6.0 to 6.6 or in deionized water, the enzyme activity of the former suspension was approximately 10 times higher than that of the latter suspension. However, the enzyme activity of the latter was markedly stimulated in the presence of sodium chloride. The glucose dehydrogenase from resting spores disrupted in 100 mM phosphate buffer (pH 6.6) was a salt-independent, active enzyme with a molecular weight of about 120,000, whereas the enzyme from resting spores disrupted in deionized water was a salt-dependent, inactive one with a molecular weight of about 55,000. A high concentration of dipicolinic acid strongly inhibited activation by a salt of inactive glucose dehydrogenase from resting spores in deionized water, suggesting one of its several important roles in vivo.     

Trypsinlike enzymes from dormant and germinated spores of Bacillus cereus T and their possible involvement in germination.

Trypsin-like enzymes were studied in dormant, activated, and germinated spores of Bacillus cereus T. Dormant spores contained two heat-labile enzyme activities. One was extractable with 2 M KCl and hydrolyzed azo-albumin. The second, a trypsinlike activity, was not extractable with 2 M KCl and hydrolyzed benzoyl-L-arginine-p-nitroanilide. Because of their heat instability, these two enzyme activities are probably not involved in the germination of heat-activated spores. Upon germination of heat-treated spores, a trypsinlike protease which was not detected in intact dormant spores was activated or exposed. This enzyme, when measured in intact germinated spores, hydrolyzed benzoyl-DL-arginine-p-nitroanilide but not azo-albumin and was inhibited in situ by sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid and Hg2+. There was a correlation between the inhibition of germination and enzymatic activity by sulfhydryl-blocking reagents. The enzyme was also inhibited by leupeptin, tosyl-L-lysine chromoethyl ketone, and tosyl-L-arginine methyl ester. Good correlation existed between the inhibition of germination and enzymatic activity by these agents. Electron micrographs showed that in the presence of trypsin inhibitors, the spores did not lose their cortex. The protein extracts of the inhibited spores formed a somewhat different electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the protein extracts of dormant or germinated spores.     

Sensitivity of chemically treated spores of Clostridium perfringens type A to an initiation protein.

Extraction of Clostridium perfringens type A spores with dithiothreitol (DTT), DTT plus sodium dodecyl sulphate (DTT-SDS), urea-mercaptoethanol (UME), or alkali, solubilized from 18.6 to 46.5 of the total dry weight of spores. The initiation of germination and lysis of such treated spores with lysozyme and an initiation protein (IP) from the culture supernatant fluid of sporulating cells of C. perfringens was studied under various conditions. The ability of lysozyme and the crude IP to induce germination and lysis of extracted spores was concentration dependent up to 0.5 microgram/ml and 5.6 mg/ml respectively. IP showed an optimum of activity between pH 7 and 8 for DTT-SDS and DTT extracted spores, and between pH 6 and 9 for UME extracted spores. The optimum temperature of activity for IP was 55 degrees C. Dissimilarities in the extent to which lysozyme and the IP initiated germination and lysis of spores extracted by various methods may have been a reflection of the differences in amounts of protein solubilized by each treatment.     

Possible mechanisms for the revival of glutaraldehyde-treated spores of Bacillus subtilis NCTC 8236

Spores of Bacillus subtilis NCTC 8236 were exposed to 2% alkaline glutaraldehyde and subsequently subjected to various treatments in an attempt to revive injured spores. Treatment with alkali (sodium or potassium hydroxide or, to a lesser extent, sodium bicarbonate) proved to be most successful. Some revival was achieved after thermal treatment. No revival was obtained with lysozyme or with various types of coat-removing agents. Experiments designed to distinguish between germination and outgrowth in the revival process established that sodium hydroxide (optimum concentration, 20 mmol/l) added to glutaraldehyde-treated spores increased the potential for germination. In contrast, spores which had been allowed to germinate before exposure to low concentrations of glutaraldehyde and then to sodium hydroxide were inhibited at the outgrowth phase to a much greater extent than germinated spores treated with the dialdehyde without subsequent alkali exposure. The results overall are discussed in terms of the possible mechanism and site of action of glutaraldehyde and the practical implications and significance of its use as a sporicide.     

Possible mechanisms for the revival of glutaraldehydetreated spores of Bacillus subtilis NCTC 8236

Spores of Bacillus subtilis NCTC 8236 were exposed to 2% alkaline glutaraldehyde and subsequently subjected to various treatments in an attempt to revive injured spores. Treatment with alkali (sodium or potassium hydroxide or, to a lesser extent, sodium bicarbonate) proved to be most successful. Some revival was achieved after thermal treatment. No revival was obtained with lysozyme or with various types of coat-removing agents. Experiments designed to distinguish between germination and outgrowth in the revival process established that sodium hydroxide (optimum concentration, 20 mmol/l) added to glutaraldehyde-treated spores increased the potential for germination. In contrast, spores which had been allowed to germinate before exposure to low concentrations of glutaraldehyde and then to sodium hydroxide were inhibited at the outgrowth phase to a much greater extent than germinated spores treated with the dialdehyde without subsequent alkali exposure. The results overall are discussed in terms of the possible mechanism and site of action of glutaraldehyde and the practical implications and significance of its use as a sporicide.     

Protein Synthesis During Fungal Spore Germination II. Aminoacyl-soluble Ribonucleic Acid Synthetase Activities During Germination of Botryodiplodia theobromae Spores

The specific activities of 13 aminoacyl-soluble ribonucleic acid (sRNA) synthetases were measured at various time intervals during the germination of Botryodiplodia theobromae conidiospores. The enzyme activities were low or absent in ungerminated spores, and they increased rapidly as germination proceeded. When extracts of the ungerminated spores were prepared with mortar and pestle, very little or no enzyme activity was detected. When the extracts were prepared with a mechanical homogenizer, however, they exhibited some enzyme activity, although less than did the extracts from germinated spores. Enzyme activities from germinated spores were approximately the same, regardless of the method of preparation. The enzyme fraction from ungerminated spores prepared with a mechanical homogenizer could also stimulate incorporation of phenylalanine into polyphenylalanine in the presence of ribosomes, polyuridylic acid, and sRNA, although the activity was approximately only 15 to 20% that of a similar enzyme fraction from germinated spores. It is concluded that ungerminated spores of B. theobromae contain active aminoacyl-sRNA synthetases and transfer enzymes, although the activities are low when compared to germinated spores.     

Uric acid is a genuine metabolite of Penicillium cyclopium and stimulates the expression of alkaloid biosynthesis in this fungus

On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor L-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor L-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.     

Location of Aryl Sulfatase in Conidia and Young Mycelia of Neurospora crassa

Aryl sulfatase (arylsulfate sulfohydrolase, EC 3.1.6.1) was found to have multiple locations in Neurospora conidia. Some enzyme activity remained in the supernatant when a spore suspension was centrifuged or filtered. Part of the cell-bound activity could be detected by adding the assay ingredients to a suspension of intact spores (patent enzyme), and additional activity was only detectable when the spores were first treated to destroy their permeability barriers (cryptic enzyme). Such treatments include: disruption with an X-press, brief rinsing with chloroform or acetone, incubation at 60 C for 5 min, and incubation with phenethyl alcohol, nystatin, or ascosin. Part of the patent aryl sulfatase was inactivated by briefly acid treating the intact spores (no loss of conidial viability). This enzyme was considered to have a cell surface location. Some enzyme was acid-resistant in intact spores, but all of the enzyme was acid-sensitive in spores whose permeability barriers had been disrupted. The pH dependence, kinetic properties, and p-nitrophenyl sulfate uptake were investigated in acid-treated conidia. No aryl sulfatase was detected in ascospores. Young mycelia contained more aryl sulfatase than did conidia, but little, if any, was secreted into the growth medium. Cryptic activity was demonstrated in young mycelia by brief chloroform treatment or by rinsing the cells with 0.1 m acetate buffer. Enzyme activity in young mycelia was completely labile to acid treatment, as was cell viability.     

Effect of sodium hydroxide and two proteases on the revival of aldehyde-treated spores of Bacillus subtilis

Spores of Bacillus subtilis were subjected to a 24 h exposure (22 C) to vaious commercial and non-commercial aldehyde formulations Subsequent treatment with sodium hydroxide (15 min, 22 C)enabled the recovery of injured spores as determined by enumeration on nutrient agar. Greatest revival was obtained with spores treated with glyoxal, followed by 2% alkaline glutaraldehyde, Sporicidin and 10% Gigasept. Revival was dependentupon neutralization of residual aldehyde with 2% (w/v) glycine prior to alkali treatment. Two percent alkaline glutaradehyde-treated spores were also exposed to to proteases, proteinase K and pronase. No revival was observed. The results are discussed in terms of the mechanism of action of the aldehydes used, with particular emphasis on glutaradehyde.     

Use of yeast spores as a biocatalyst

Vegetative cells of the yeast Saccharomyces cerevisiae 4011 efficiently sporulated at pH 7.7–8.0 in the presence of 1.0–3.0% of potassium acetate. Spores were prepared by lysing them with a lytic enzyme, zymolyase. Alkaline phosphatase (an enzyme selected as a model) in spores exhibited higher stability toward heat and pH than it did in vegetative cells, and was immobilized in a polyacrylamide gel lattice without any appreciable loss of activity. The activity of alkaline phosphatase in spores and immobilized spores was stably maintained during repeated use for the enzyme reactions. These results indicated the usefulness of yeast spores as a biocatalyst.     

Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM.

Two peptidoglycan-lytic enzyme activities were isolated from spores of Bacillus megaterium KM. Surface-bound lytic enzyme was extracted from dormant spores and hydrolysed a variety of peptidoglycan substrates including isolated spore cortex, but did not cause refractility changes in permeabilized spores. Germination-specific lytic enzyme activity appeared early in germination and had minimal activity on isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus species. The germination-specific lytic enzyme was shown to be a heat-sensitive 29 kDa protein with maximal activity at pH 6.5. It catalysed post-commitment muramic acid delta-lactam synthesis and displayed an inhibitor profile similar to that for post-commitment A600 loss. The relationship of the germination-specific enzyme to a recently proposed model of spore germination is discussed.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Immunological Homology Between Crystal and Spore Protein of Bacillus thuringiensis

Spore suspensions containing about 0.3% crystals and crystal suspensions containing about 0.1% spores were obtained from cultures of Bacillus thuringiensis by extraction with a two-phase system. Both preparations were tested for the presence of contaminating material from vegetative cells and were judged to be clean. Solutions of spore protein were obtained by extracting broken spores with phosphate buffer followed by extraction with either alkali- or urea-mercaptoethanol. The alkali spore or urea spore extracts had the same isoelectric point as crystal protein solubilized with these reagents. An antiserum prepared against alkali crystal solution precipitated alkali or urea spore extracts and crystal solutions but not phosphate spore extracts or extracts of whole cells. Lines of identity between spore and crystal precipitates were observed by using the Ouchterlony double-diffusion technique. Absorption of the antiserum with an excess of urea spore extract caused a disappearance of the precipitin bands originating from the spore protein and the homologous bands from the crystal protein. The results suggest that the crystal and endospore contain one or more common proteins.     

Sortase C-mediated anchoring of BasI to the cell wall envelope of Bacillus anthracis

Vegetative forms of Bacillus anthracis replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of B. anthracis peptidoglycan.     

Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity

Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.