Aims To determine the mechanism of killing of spores of Bacillus thuringiensis Al Hakam, a Bacillus anthracis spore surrogate, in a blast environment with or without HIO3 and whether the spores are truly dead. Methods and Results Spores exposed to an aluminium-based blast environment with or without HIO3 with dynamic peak gas phase temperatures near 1000°C persisting for 10's of ms, were killed 97 and 99·99% without and with HIO3 respectively and the spores were truly dead. The survivors of the detonations did not acquire mutations, did not become wet heat sensitive, became sensitive to elevated NaCl but not lack of glucose in recovery media, and many dead spores remained phase bright and retained their Ca-dipicolinic acid. A large fraction of the dead spores could germinate, but most of these germinated spores were dead. Conclusions Most spores exposed to a blast environment are truly dead, and HIO3 increases spore death. The likely mechanism of spore killing in these blast environments is damage to some essential spore protein, although spore inner membrane damage could contribute. Significance and Impact of the Study This work shows that spores of a surrogate for B. anthracis spores are killed in a blast environment without or with HIO3 present, this approach could inactivate up to 99·99% of dry B. anthracis spores, and the spores are likely killed by damage to some essential spore protein.

To determine how the microbicide ceragenin‐13 (CSA‐13) kills Bacillus subtilis spores prepared on growth or sporulation media, and these spores’ properties.

To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs.

AimsThis study was to determine the sources, location and identity of -glucosidases in dormant/germinating/outgrowing spores and growing cells of Geobacillus stearothermophilus ATCC 7953, an enzymatic activity in spores used in rapid tests of steam sterilization.Methods and Results-Glucosidase activity in spores and cells was determined measuring methylumbelliferyl--d-glucoside (-MUG) or -MUG-6-phosphate hydrolysis fluorometrically. While -MUG-6-phosphate was not hydrolysed by cell or spore extracts, assays with -MUG showed that: (1) the -glucosidase activity was inside and outside spores, and the activity outside spores was largely removed by buffer washes or heat activation, whereas -glucosidase activity was only inside vegetative cells; (2) most -glucosidase activity in cells and spores was soluble; (3) Western blots and enzyme inhibition using an anti--glucosidase antiserum identified 2 -glucosidases in spores and growing cells; (4) -glucosidase-specific activities were similar in dormant, germinated and outgrowing spore and growing cell extracts; and (5) significant -glucosidase was synthesized during spore germination and outgrowth and cell growth, this synthesis was not repressed by glucose nor induced by -MUG, but glucose inhibited -MUG uptake.Conclusions-MUG hydrolysis by G.stearothermophilus is by -MUG uptake and hydrolysis by 2 -glucosidases associated with dormant spores and synthesized by germinating and outgrowing spores. The enzyme activity observed by sterilization assurance assays appears likely to come from heat-stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth.Significance and Impact of the StudyThe results of this work provide new insight into the science behind a rapid test for steam sterilization assurance.

Aims: Determine how supercritical CO2 (scCO(2)) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO(2)-PAA, and if spores inactivated by scCO(2)-PAA are truly dead.Methods and Results: Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO(2)-PAA in liquid or dry at 35 degrees C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2-PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85 degrees C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO(2)-PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO(2)-PAA sensitive.Conclusions: These findings suggest that scCO(2)-PAA inactivates spores by damaging spores' inner membrane. The spore coat provided scCO(2)-PAA resistance for both wet and dry spores. DNA protective proteins provided scCO(2)-PAA resistance only for dry spores.Significance and Impact of the Study: These results provide information on mechanisms of spore inactivation of and resistance to scCO(2)-PAA, an agent with increasing use in sterilization applications.

AimsTo determine how hydrated Bacillus anthracis spores are killed in a high-temperature gas environment (HTGE), and how spores of several Bacillus species including B.anthracis are killed by UV radiation, dry heat, wet heat and desiccation.Methods and ResultsHydrated B.anthracis spores were HTGE treated at c. 220 degrees C for 50ms, and the treated spores were tested for germination, mutagenesis, rupture and loss of dipicolinic acid. Spores of this and other Bacillus species were also examined for mutagenesis by UV, wet and dry heat and desiccation. There was no rupture of HTGE-treated B.anthracis spores killed 90-99 center dot 9%, no mutagenesis, and release of DPA and loss of germination were much slower than spore killing. However, killing of spores of B.anthracis, Bacillus thuringiensis and Bacillus subtilis by UV radiation or dry heat, but not wet heat in water or ethanol, was accompanied by mutagenesis.ConclusionsIt appears likely that HTGE treatment kills B.anthracis spores by damage to spore core proteins. In addition, various killing regimens inactivate spores of a number of Bacillus species by the same mechanisms.Significance and Impact of the StudyThis work indicates how hydrated spores treated in a HTGE such as might be used to destroy biological warfare agent stocks are killed. The work also indicates that mechanisms whereby different agents kill spores are similar with spores of different Bacillus species.

More than 95% of individuals in populations of Bacillus subtilis spores killed approximately 95% by hydrogen peroxide or hypochlorite germinated with a nutrient, although the germination of the treated spores was slower than that of untreated spores. The slow germination of individual oxidizing agent-treated spores was due to: (i) 3- to 5-fold longer lag times (T-lag) between germinant addition and initiation of fast release of spores' large dipicolinic acid (DPA) depot (ii) 2- to 10-fold longer times (T-release) for rapid DPA release, once this process had been initiated; and (iii) 3- to 7-fold longer times needed for lysis of spores' peptidoglycan cortex. These results indicate that effects of oxidizing agent treatment on subsequent spore germination are on: (i) nutrient germinant receptors in spores' inner membrane (ii) components of the DPA release process, possibly SpoVA proteins also in spores' inner membrane, or the cortex-lytic enzyme CwlJ; and (iii) the cortex-lytic enzyme SleB, also largely in spores' inner membrane. This study further indicates that rapid assays of spore viability based on measurement of DPA release in spore germination can give false-positive readings.

AIM:To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. METHODS AND RESULTS:Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12h and with temporal resolutions of ≤30s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca-dipicolinic acid (CaDPA) (T(lag) and T(release) times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔT(release) times (T(release) -T(lag) ) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. CONCLUSIONS:Since (i) ΔT(release) times were essentially identical in GR-dependent germination of SD and dormant spores; (ii) rates of GR-independent germination of SD and dormant spores were identical; (iii) large increases in T(lag) times were the major difference in the GR-dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter T(lag) times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. SIGNIFICANCE AND IMPACT OF THE STUDY:This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.

To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.Staining by four dyes and autofluorescence of B. subtilis spores that lack some (cotE, gerE) or most (cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC(6)(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC(6)(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC(6)(3) bound well to all germinated spores.The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.

Aims: To determine if pretreatment with oxidizing agents sensitizes Bacillus subtilis spores to dry heat or desiccation.Methods: Bacillus subtilis spores were killed approx. 90% by oxidizing agents, and the sensitivity of treated and untreated spores to dry heat and desiccation was determined. The effects of pyruvate on spore recovery after oxidizing agent pretreatment and then dry heat or desiccation were also determined.Conclusions: Spores pretreated with Oxone (TM) or hypochlorite were not sensitized to dry heat or freeze-drying. However, hydrogen peroxide or t-butylhydroperoxide pretreatment sensitized spores to dry heat or desiccation, and the desiccation caused mutagenesis in the survivors. Pyruvate increased recovery of spores treated with hydrogen peroxide alone or plus dry heat or desiccation, and with t-butylhydroperoxide and desiccation, but not with t-butylhydroperoxide alone or plus dry heat.Significance and Impact of the Study: Pretreatment with peroxides sensitizes bacterial spores to subsequent stress. This finding may suggest improved regimens for spore inactivation.

Aims: To compare the properties of the spores of Bacillus subtilis prepared in liquid and on plates.Methods and Results: The spores of B. subtilis were prepared at 37 C using a nutrient exhaustion medium either in liquid or on agar plates. The levels of core water, dipicolinic acid (DPA) and small, acid-soluble spore proteins (SASP) were essentially identical in spores made in liquid or on plates. Spores prepared in liquid were killed threefold more rapidly at 90 C in water than the spores prepared on plates, and the spores prepared in liquid were more sensitive to nitrous acid and a diluted stable superoxidized water. Spores prepared in liquid also germinated more rapidly with several agents than those prepared on plates. Pellets of spores prepared on plates were darker than spores prepared in liquid, and spores prepared in liquid had more readily extracted coat protein. However, there were no major differences in the relative levels of individual coat proteins or the cross-linking of the coat protein GerQ in the two types of spores, although the inner membrane of spores prepared on plates had a higher ratio of anteiso- to iso-fatty acids.Conclusions: The preparation in liquid yielded spores with some different properties than those made on agar plates. Spores made in liquid had lower resistance to heat and several chemicals, and germinated more readily with several agents. There were also differences in the composition of the inner membrane of spores prepared under these two conditions. However, there were no major differences in the levels of DPA, core water, SASP and individual coat proteins or the cross-linking of a coat protein in spores made in liquid and on plates.Significance and Impact of the Study: This work demonstrates that the preparation method can affect the resistance and germination properties of bacterial spores, even if an identical medium and temperature are used. Evidence was also obtained consistent with the role of the inner membrane in spore resistance and germination, and that some factor in addition to core water, DPA and SASP content plays a role in spore resistance to wet heat.

Aims: To determine the effectiveness of tert-butyl hydroperoxide (tBHP) plus the cationic surfactant cetyltrimethyl ammonium bromide (CTAB) and a tetraamido macrocyclic ligand (TAML((R))) activator in killing spores of Bacillus subtilis and the mechanisms of spore resistance to and killing by this reagent.Methods and Results: Killing of spores of B. subtilis by tBHP was greatly stimulated by the optimum ratio of concentrations of a TAML activator (1.7 mu mol l(-1)) to tBHP (4.4%, vol/vol) plus a low level (270 mg l(-1)) of CTAB. Rates of killing of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha(-)beta(-) spores) or the major DNA repair protein, RecA, by tBHP plus CTAB and a TAML activator were essentially identical to that of wild-type spore killing. Survivors of wild-type and alpha(-)beta(-) spores treated with tBHP plus CTAB and a TAML activator also exhibited no increase in mutations. Spores lacking much coat protein either because of mutation or chemical decoating were much more sensitive to this reagent than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with this reagent were sensitized to wet heat. The tBHP plus CTAB and TAML activator-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by application of 150 and 500 megaPascals of pressure for 15 min and by lysozyme treatment in hypertonic medium, but these spores lysed shortly after their germination.Conclusions: The combination of tBHP plus CTAB and a TAML activator is effective in killing B. subtilis spores. The spore coat is a major factor in spore resistance to this reagent system, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent system appears to kill spores by damaging the spore's inner membrane in some fashion.Significance and Impact of the Study: This work demonstrates that tBHP plus CTAB and a TAML activator is an effective and mild decontaminant for spores of Bacillus species. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent system.

Aims: To determine the mechanisms of Bacillus subtilis spore resistance to and killing by a novel sporicide, dimethyldioxirane (DMDO) that was generated in situ from acetone and potassium peroxymonosulfate at neutral pH.Methods and Results: Spores of B. subtilis were effectively killed by DMDO. Rates of killing by DMDO of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha(-)beta(-) spores) or the major DNA repair protein, RecA, were very similar to that of wild-type spore killing. Survivors of wild-type and alpha(-)beta(-) spores treated with DMDO also exhibited no increase in mutations. Spores lacking much coat protein due either to mutation or chemical decoating were much more sensitive to DMDO than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with DMDO were sensitized to wet heat. The DMDO-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by very high pressures and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after their germination, and none of these treatments were able to revive the DMDO-killed spores.Conclusions: DMDO is an effective reagent for killing B. subtilis spores. The spore coat is a major factor in spore resistance to DMDO, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent appears to kill spores by damaging the spore's inner membrane in some fashion.Significance and Impact of the Study: This work demonstrates that DMDO is an effective decontaminant for spores of Bacillus species that can work under mild conditions, and the killed spores cannot be revived. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent.

ABSTRACT ytkD and mutT of Bacillus subtilis encode potential 8-oxo-dGTPases that can prevent the mutagenic effects of 8-oxo-dGTP. Loss of YtkD but not of MutT increased the spontaneous mutation frequency of growing cells. However, cells lacking both YtkD and MutT had a higher spontaneous mutation frequency than cells lacking YtkD. Loss of either YtkD or MutT sensitized growing cells to hydrogen peroxide (H 2 O 2 ) and t -butylhydroperoxide ( t -BHP), and the lack of both proteins sensitized growing cells to these agents even more. In contrast, B. subtilis spores lacking YtkD and MutT were not sensitized to H 2 O 2 , t -BHP, or heat. These results suggest (i) that YtkD and MutT play an antimutator role and protect growing cells of B. subtilis against oxidizing agents, and (ii) that neither YtkD nor MutT protects spores against potential DNA damage induced by oxidative stress or heat.

Endospore formation by Bacillus subtilis involves three differentiating cell types, the predivisional cell, the mother cell, and the forespore. Here we report the program of gene expression in the forespore, which is governed by the RNA polymerase sigma factors σF and σG and the DNA-binding proteins RsfA and SpoVT. The σF factor turns on about 48 genes, including the gene for RsfA, which represses a gene in the σF regulon, and the gene for σG. The σG factor newly activates 81 genes, including the gene for SpoVT, which turns on (in nine cases) or stimulates (in 11 cases) the expression of 20 genes that had been turned on by σG and represses the expression of 27 others. The forespore line of gene expression consists of many genes that contribute to morphogenesis and to the resistance and germination properties of the spore but few that have metabolic functions. Comparative genomics reveals a core of genes in the σF and σG regulons that are widely conserved among endospore-forming species but are absent from closely related, but non-spore-forming Listeria spp. Two such partially conserved genes (ykoU and ykoV), which are members of the σG regulon, are shown to confer dry-heat resistance to dormant spores. The ykoV gene product, a homolog of the non-homologous end-joining protein Ku, is shown to associate with the nucleoid during germination. Extending earlier work on gene expression in the predivisional cell and the mother cell, we present an integrated overview of the entire program of sporulation gene expression.

ABSTRACT The Bacillus subtilis enzymes ExoA and Nfo (originally termed YqfS) are endonucleases that can repair apurinic/apyrimidinic (AP) sites and strand breaks in DNA. We have analyzed how the lack of ExoA and Nfo affects the resistance of growing cells and dormant spores of B. subtilis to a variety of treatments, some of which generate AP sites and DNA strand breaks. The lack of ExoA and Nfo sensitized spores (termed α − β − ) lacking the majority of their DNA-protective α/β-type small, acid-soluble spore proteins (SASP) to wet heat. However, the lack of these enzymes had no effect on the wet-heat resistance of spores that retained α/β-type SASP. The lack of either ExoA or Nfo sensitized wild-type spores to dry heat, but loss of both proteins was necessary to sensitize α − β − spores to dry heat. The lack of ExoA and Nfo also sensitized α − β − , but not wild-type, spores to desiccation. In contrast, loss of ExoA and Nfo did not sensitize growing cells or wild-type or α − β − spores to hydrogen peroxide or t -butylhydroperoxide. Loss of ExoA and Nfo also did not increase the spontaneous mutation frequency of growing cells. exoA expression took place not only in growing cells, but also in the forespore compartment of the sporulating cell. These results, together with those from previous work, suggest that ExoA and Nfo are additional factors that protect B. subtilis spores from DNA damage accumulated during spore dormancy.

Fourier transform infrared reflectance microspectroscopy was applied to the classification of bacterial endospores, both untreated and those pretreated with autoclaving. Mid-infrared vibrational spectra were collected following minimal sample preparation from endospores of five species of bacteria: Bacillus cereus, Bacillus globigii, Bacillus megaterium, Bacillus subtilis, and Clostridium perfringens. The amide I and amide II regions were found to provide the best classification of the native spores. For autoclaved bacterial spores, the N-H and C-H stretching regions (2700-3500 cm/sup -1/) were found to be useful for classification. Autoclaving endospores also augmented the classification of untreated endospores in the same region. Speciation of these microorganisms was accomplished by application of principal component analysis (PCA), hierarchical cluster analysis (HCA), and soft independent method of class analogy (SIMCA). The methods described can potentially be applied by homeland security personnel to screen for pathogenic microorganisms.

ABSTRACT The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor α/β- and γ-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (B. cereus T and B. thuringiensis subsp. Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.