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. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca2+ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination. CONCLUSIONS: CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.

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.

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. However, these germinated spores never swelled and many had altered permeability properties. CONCLUSIONS: We suggest that Sterilox treatment kills dormant spores by oxidatively modifying the inner membrane of the spores such that this membrane becomes non-functional in the germinated spore leading to spore death. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to and spore killing by a new disinfectant.

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. In contrast, nitrous acid treatment of wild-type and alpha-beta- spores caused significant mutagenesis, with alpha-beta- spores being much more sensitive to this agent. A recA mutation further sensitized both wild-type and alpha-beta- 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 alpha/beta-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 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.

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. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) alpha/beta-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.

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. 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.

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. SspF was also degraded during spore germination and outgrowth, and this degradation was initiated by GPR.

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.

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. Levels of monovalent and divalent inorganic cations were comparable in spores of Sporosarcina, Clostridium, and Bacillus species, and cation levels in spores of the slight halophile S. halophila were similar to those in S. ureae spores. These data suggest that levels of small molecules are generally similar in spores of all Gram-positive organisms, and further suggest that these levels reflect fundamental and conserved features of the sporulation process and dormant spores in these organisms. The data are also consistent with the proposed close evolutionary relationship between Bacillus and Sporosarcina 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. Strikingly, the level and rate of expression of the modified genes containing either type of extra codons was identical. This suggests that in B. subtilis codon usage patterns may play little or no role in effecting the level of gene expression.

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 coding for a B. stearothermophilus and a "T. thalpophilus" SASP as well as for two B. cereus SASP were cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus." This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than simply amino acid storage.

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. The other two (termed genes for SASP-C-4 and -C-5) are extremely similar in much of their nucleotide sequence to the previously cloned B. megaterium SASP-C-2 gene. The proteins coded for by all these SASP genes had extensive sequence homology with each other and with those coded for by the B. megaterium SASP-C, -C-1, -C-2, and -C-3 genes. Their coding sequences are preceded by strong ribosome-binding sites and are followed by regions of dyad symmetry which presumably are transcription stop sites. The SASP-A, -C-4, and -C-5 genes are expressed in parallel during sporulation, and their transcription start points were localized by the size of the mRNAs produced. The sequences localized 10 and 35 base pairs upstream from the transcription start points show significant homology with the analogous regions of the SASP-C, -C-1, -C-2, and -C-3 genes. The identification of seven closely related SASP genes in B. megaterium indicates that the SASP are the products of a very extensive multigene family.

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 2.7.3.2) isoenzyme-MB was above normal. Both are particularly noteworthy in that creatine kinase-BB and a macro creatine kinase form thought to be type II of mitochondrial origin were also present. The macro creatine kinase component in both cases co-migrated electrophoretically with creatine kinase-MM but was easily identified after the latter was removed by precipitation with M-subunit-specific antibodies. In the first case, the patient had a readily diagnosable acute myocardial infarction while under observation in the cardiac intensive care unit: electrocardiographic changes and the rapid increase and decrease in total creatine kinase were as would be expected. In marked contrast, in the second case, we saw no abrupt changes in either of these characteristics. The latter patient's primary disease was a rectal carcinoma with massive metastases to the liver; however, the presence of abnormally high creatine kinase-MB activity raised the question of possible myocardial infarction.

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. In the first minutes of spore germination P41 was processed (t1/2 less than 10 min) to a Mr = 40,000 species (P40), which appeared identical to the subunit of the purified active enzyme. The latter processing reaction did not require metabolic energy, but P40 disappeared completely during further germination (t1/2 approximately 40 min) in a reaction which did require metabolic energy. It seems probable that precursors P46 and P41 of the spore protease are involved in the regulation of the activity of this spore enzyme.

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. In contrast, the protease antigen disappears rapidly during spore germination, in parallel with the loss in enzyme activity.

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. One of these can be separated from NADH by chromatography on either diethylaminoethyl-celluose or diethylaminoethyl-Sephadex; the second cannot. The NADH-associated inhibitor appeared to be present in each of the three commercial NADH preparations studied. The 260 nm/340 nm absorbance ratio was of no help in locating this inhibitor during chromatography.