Barbara Setlow

Characterization of Spores of Bacillus subtilis Which Lack Dipicolinic Acid

Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Deltager3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Deltager3 spores. The Deltager3 spoVF spores lack DPA and have higher levels of core water than Deltager3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to Deltager3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Deltager3 spoVF spores also contain minimal levels of the processed active form (termed P(41)) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P(41) formed in Deltager3 spoVF spores may be at least transiently active on one of this proteases small acid-soluble spore protein (SASP) substrates, SASP-gamma. Analysis of the resistance of wild-type, Deltager3, and Deltager3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Deltager3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.

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.

Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

ABSTRACT Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

Aims:  To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spores inner membrane.

Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid.

Treatment of wild‐type spores of Bacillus subtilis with glutaraldehyde or an iodine‐based disinfectant (Betadine) did not cause detectable mutagenesis, and spores (termed α–β–) lacking the major DNA‐protective α/β‐type, small, acid‐soluble proteins (SASP) exhibited similar sensitivity to these agents. A recA mutation did not sensitize wild‐type or α–β– spores to Betadine or glutaraldehyde, nor did spore treatment with these agents result in significant expression of a recA‐lacZ fusion when the treated spores germinated. Spore glutaraldehyde sensitivity was increased dramatically by removal of much spore coat protein, but this treatment had no effect on Betadine sensitivity. In contrast, nitrous acid treatment of wild‐type and α–β– spores caused significant mutagenesis, with α–β– spores being much more sensitive to this agent. A recA mutation further sensitized both wild‐type and α–β– spores to nitrous acid, and there was significant expression of a recA‐lacZ fusion when nitrous acid‐treated spores germinated. These results indicate that: (a) nitrous acid kills B. subtilis spores at least in part by DNA damage, and α/β‐type SASP protect against this DNA damage; (b) killing of spores by glutaraldehyde or Betadine is not due to DNA damage; and (c) the spore coat protects spores against killing by glutaraldehyde but not Betadine. Further analysis also demonstrated that spores treated with nitrous acid still germinated normally, while those treated with glutaraldehyde or Betadine did not.

Germination of spores of Bacillus subtilis with dodecylamine

Aims: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine‐induced spore germination.

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased ≈2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species. The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.

Role of Dipicolinic Acid in Resistance and Stability of Spores of Bacillus subtilis with or without DNA-Protective α/β-Type Small Acid-Soluble Proteins

Spores of Bacillus species are dormant and extremely resistant to many environmental stresses including, heat, desiccation, radiation, and a variety of toxic chemicals (9, 17, 33). As a consequence, spores can survive for extremely long periods, certainly hundreds of years and perhaps much longer (5, 15, 37). Spore resistance is due to a variety of factors, including the outer spore coats and the relative impermeability of the spores inner membrane (6, 9, 17). There are also novel features of the spores central region or core, the site of spore DNA, which play major roles in spore resistance. These include the relatively low content of core water and the saturation of spore DNA with a group of small acid-soluble proteins (SASP) of the α/β type (9, 11, 23, 31). The low core water protects spores against wet heat, while the α/β-type SASP protect DNA against damage due to wet and dry heat, desiccation, and genotoxic chemicals (6, 17, 31, 33). The binding of the α/β-type SASP also drastically alters the DNAs UV photochemistry, and this change is a major factor in the elevated resistance of dormant spores to UV light (17, 18). Another novel feature of the spore core is the presence of high levels (∼20% of core dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]), that likely exists in the core as a 1:1 chelate with divalent cations, predominantly Ca2+ (11). One role of DPA is to lower the core water content, since DPA loss early in germination is paralleled by its replacement with water, and genetically DPA-less dormant spores have more core water than wild-type dormant spores (10, 21, 32). The role of DPA in spore properties has long been unclear. There are data indicating that increasing spore DPA levels are associated with increased spore wet heat resistance (11, 21). However, mutants have been isolated that produce heat-resistant but DPA-less spores (4, 12). Unfortunately, there has never been a thorough genetic analysis of these strains, and they may well have had multiple mutations. DPA-less spores of B. subtilis can also be generated by mutation of the spoVF operon that encodes DPA synthetase (7, 11, 21). However, the DPA-less spoVF spores are unstable and lyse during sporulation (21, 36). In contrast, when sporulated with exogenous DPA, spoVF spores accumulate near wild-type DPA levels and are stable (21, 36). DPA-less spoVF spores can also be stabilized somewhat, albeit not completely, by deleting the genes encoding the three receptors that trigger spore germination in response to nutrient germinants (termed the ger3 mutations) (21). DPA-less and DPA-replete ger3 spoVF spores have relatively similar resistance to UV radiation, but the DPA-less spores are more sensitive to wet heat and hydrogen peroxide (21). Although the latter differences in resistance between spores with or without DPA are striking, it is not clear whether these differences are due only to the higher core water content of the DPA-less spores or to specific effects of DPA on spore resistance. Indeed, DPA can have striking effects on spore properties, in particular spore DNA properties (8, 26). In particular, UV irradiation of spores lacking DPA is much less efficient in generating the thyminyl-thymine adduct termed the spore photoproduct in DNA than is UV irradiation of spores containing normal DPA levels (8). This difference is even larger in spores with or without DPA that also lack α/β-type SASP (8). These latter studies were carried out with spores of a strain lacking both spoVF and sleB. The latter gene encodes one of the two redundant enzymes responsible for the hydrolysis of the spores peptidoglycan cortex in the first min of germination (32). The second cortex-lytic enzyme, CwlJ, is present in sleB spoVF spores. However, CwlJ requires DPA for its activation (20). Consequently, DPA-less sleB spoVF spores do not germinate either spontaneously or with nutrients and are much more stable than ger3 spoVF spores (20, 21). However, DPA-less sleB spoVF spores can be germinated either by exogenous Ca2+-DPA or by lysozyme treatment of decoated spores in a hypertonic medium (20, 21). Given the extreme stability of the sleB spoVF spores that lack DPA, we have reexamined the role of DPA in the resistance of these spores. We have also examined the survival and resistance of spores that lack both DPA and α/β-type SASP. We report here the results of these studies.

Properties of Spores of Bacillus subtilis Blocked at an Intermediate Stage in Spore Germination

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.

Factors Influencing Germination of Bacillus subtilis Spores via Activation of Nutrient Receptors by High Pressure

ABSTRACT Different nutrient receptors varied in triggering germination of Bacillus subtilis spores with a pressure of 150 MPa, the GerA receptor being more responsive than the GerB receptor and even more responsive than the GerK receptor. This hierarchy in receptor responsiveness to pressure was the same as receptor responsiveness to a mixture of nutrients. The levels of nutrient receptors influenced rates of pressure germination, since the GerA receptor is more abundant than the GerB receptor and elevated levels of individual receptors increased spore germination by 150 MPa of pressure. However, GerB receptor variants with relaxed specificity for nutrient germinants responded as well as the GerA receptor to this pressure. Spores lacking dipicolinic acid did not germinate with this pressure, and pressure activation of the GerA receptor required covalent addition of diacylglycerol. However, pressure activation of the GerB and GerK receptors displayed only a partial (GerB) or no (GerK) diacylglycerylation requirement. These effects of receptor diacylglycerylation on pressure germination are similar to those on nutrient germination. Wild-type spores prepared at higher temperatures germinated more rapidly with a pressure of 150 MPa than spores prepared at lower temperatures; this was also true for spores with only one receptor, but receptor levels did not increase in spores made at higher temperatures. Changes in inner membrane unsaturated fatty acid levels, lethal treatment with oxidizing agents, or exposure to chemicals that inhibit nutrient germination had no major effect on spore germination by 150 MPa of pressure, except for strong inhibition by HgCl2.