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.

Methods And Results: Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed.

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.

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.

Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile.

Bacterial spores of various Bacillus species are impermeable or exhibit low permeability to many compounds that readily penetrate germinated spores, including methylamine. We now show that a lipid probe in the inner membrane of dormant spores of Bacillus megaterium and Bacillus subtilis is largely immobile, as measured by fluorescence redistribution after photobleaching, but becomes free to diffuse laterally upon spore germination. The lipid immobility in and the slow permeation of methylamine through the inner membrane of dormant spores may be due to a significant (1.

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.

Methods And Results: Bacillus subtilis spores were readily killed by Sterilox and spore resistance to this agent was due in large part to the spore coats. Spore killing by Sterilox was not through DNA damage, released essentially no spore dipicolinic acid and Sterilox-killed spores underwent the early steps in spore germination, including dipicolinic acid release, cortex degradation and initiation of metabolism.

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.

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 alpha-beta-) lacking the major DNA-protective alpha/beta-type, small, acid-soluble proteins (SASP) exhibited similar sensitivity to these agents. A recA mutation did not sensitize wild-type or alpha-beta- 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.

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.

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the alpha/beta-type protect spores against this DNA damage.

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed alpha-beta-) lacking the two major alpha/beta-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and alpha-beta- 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 alpha-beta- spores.

Nucleotide sequence of the sspE genes coding for gamma-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.

Effects of inactivation or overexpression of the sspF gene on properties of Bacillus subtilis spores.

Inactivation of the Bacillus subtilis sspF gene had no effect on sporulation, spore resistance, or germination in a wild-type strain or one lacking DNA protective alpha/beta-type small, acid-soluble proteins (SASP). Overexpression of SspF in wild-type spores or in spores lacking major alpha/beta-type SASP (alpha- beta- spores) had no effect on sporulation but slowed spore outgrowth and restored a small amount of UV and heat resistance to alpha- beta- spores. In vitro analyses showed that SspF is a DNA binding protein and is cleaved by the SASP-specific protease (GPR) at a site similar to that cleaved in alpha/beta-type SASP.

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.

Levels of small molecules in dormant spores of Sporosarcina species and comparison with levels in spores of Bacillus and Clostridium species.

Dormant spores of Sporosarcina halophila and Sporosarcina ureae contained no detectable ATP, significant levels of ADP, even higher levels of AMP, and a large pool of 3-phosphoglyceric acid, similar to what is found in dormant spores of Bacillus and Clostridium species. Sporosarcina halophila and S. ureae spores also contained significant pools of free amino acids, in particular glutamic acid, as in the case with spores of Bacillus but not Clostridium species.

Small, acid-soluble, spore proteins and their genes from two species of Sporosarcina.

Small, acid-soluble proteins (SASP) of both the alpha/beta- and gamma-type were present in spores of Sporosarcina ureae and S. halophila, and three genes encoding alpha/beta-type SASP in these species have been cloned and sequenced. The amino acid sequences of the Sporosarcina alpha/beta-type SASP are extremely homologous to those of Bacillus SASP, further indicative of the close evolutionary relationship between these genera.

The expression of a highly expressed Bacillus subtilis gene is not reduced by introduction of multiple codons normally not present in such genes.

Four serine or threonine codons were introduced into a highly expressed Bacillus subtilis gene. The introduced codons were ones either common in highly expressed B. subtilis genes, or never used in such genes.

Cloning and nucleotide sequencing of genes for small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus".

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore germination and which reacted with antibodies raised against B. megaterium SASP.

Genes for Bacillus megaterium small, acid-soluble spore proteins: cloning and nucleotide sequence of three additional genes from this multigene family.

Three genes coding for small, acid-soluble spore proteins (SASP) were cloned from Bacillus megaterium, using previously cloned B. megaterium SASP genes (SASP-C and -C-3) as DNA-DNA hybridization probes. One gene (SASP-A) codes for the A protein, a previously identified major SASP.

Unusual findings related to atypical creatine kinases in two hospitalized patients.

We describe two cases, hospitalized patients, in whom the activity of creatine kinase (EC 2.7.3.

Bacillus megaterium spore protease. Synthesis and processing of precursor forms during sporulation and germination.

The protease which initiates the rapid protein degradation during germination of Bacillus megaterium spores was synthesized during sporulation as a Mr = 46,000 polypeptide (P46) which was found in the developing forespore. P46 was processed during sporulation to a Mr = 41,000 species (P41) 2-3 h after P46 synthesis and at the time of or slightly before accumulation of dipicolinic acid. P41 was the predominant form of the protease in the dormant spore, with smaller amounts of unprocessed P46.

Bacillus megaterium spore protease: purification, radioimmunoassay, and analysis of antigen level and localization during growth, sporulation, and spore germination.

The protease which initiates the massive protein degradation early in bacterial spore germination has been purified from Bacillus megaterium spores. The enzyme has a molecular weight of 160,000 and contains four apparently identical subunits, but only the tetramer is enzymatically active. A radioimmunoassay has been developed for this enzyme and has been used to show that the protease is absent from growing cells, but appears early in sporulation within the developing forespore.

Formation and properties of lactate dehydrogenase inhibitors in NADH.

We describe some characteristics of the mode of formation of inhibitors of lactate dehydrogenase from commercial NADH. Inhibitor formation is time- and concentration-dependent and also varies with the commercial source of NADH. At least two inhibitory components can form in concentrated NADH solutions.