Charles A. Loshon

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 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.

Analysis of the killing of spores of Bacillus subtilis by a new disinfectant, Sterilox®

Aims: To determine the mechanism whereby the new disinfectant Sterilox® kills spores of Bacillus subtilis.

Role of Dipicolinic Acid in the Germination, Stability, and Viability of Spores of Bacillus subtilis

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

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

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid‐soluble spore proteins of the ααααααα/βββββββ‐type protect spores against this DNA damage

Killing of wild‐type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α−β−) lacking the two major α/β‐type small, acid‐soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild‐type and α−β− spores to formaldehyde treatment, which caused significant expression of a recA‐lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross‐linking in both wild‐type and α−β− spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β‐type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.

Levels of Glycine Betaine in Growing Cells and Spores of Bacillus Species and Lack of Effect of Glycine Betaine on Dormant Spore Resistance

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.

Analysis of the Function of a Putative 2,3-Diphosphoglyceric Acid-Dependent Phosphoglycerate Mutase from Bacillus subtilis

A Bacillus subtilis gene termed yhfR encodes the only B. subtilis protein with significant sequence similarity to 2, 3-diphosphoglycerate-dependent phosphoglycerate mutases (dPGM). This gene is expressed at a low level during growth and sporulation, but deletion of yhfR had no effect on growth, sporulation, or spore germination and outgrowth. YhfR was expressed in and partially purified from Escherichia coli but had little if any PGM activity and gave no detectable PGM activity in B. subtilis. These data indicate that B. subtilis does not require YhfR and most likely does not require a dPGM.

Nucleotide sequence of the sspE genes coding for γ-type small, acid-soluble spore proteins from the round-spore-forming bacteria Bacillus aminovorans, Sporosarcina halophila and S. ureae

The single sspE genes coding for gamma-type small, acid-soluble spore proteins (SASP) of three round-spore-forming bacteria, Bacillus aminovorans, Sporosarcina halophila and S. ureae, have been cloned and sequenced. While the deduced amino acid sequences of these three gamma-type SASP show clear homology to those from six Bacillus species that do not form round spores, there are no residues conserved completely among the 9 sequences known. In addition, the 139 residue B. aminovorans protein is 35 residues larger than any other while the 60 residue S. halophila protein is one of the smallest. These data suggest that the sspE genes have been under little selective pressure in recent evolutionary time.

Cloning and sequencing of the sspF (originally 0.3 kb) genes from Bacillus cereus and Bacillus megaterium

The sspF gene (originally 0.3 kb) of Bacillus cereus and B. megaterium has been cloned and sequenced, and the predicted amino acid sequences of the gene products (SspF) compared to that of B. subtilis SspF. These proteins exhibit an average of 74% sequence identity across species, suggesting they may play some important role in either sporulation or the dormant spore.