Dipicolinic Acid Location in Intact Spores of Bacillus megaterium

Beta-attenuation analysis of intact spores of Bacillus megaterium containing tritium-labeled dipicolinic acid has shown that dipicolinic acid is located in the spore protoplast and not in the cortex.     

Role of Dipicolinic Acid in Survival of Bacillus subtilis Spores Exposed to Artificial and Solar UV Radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Reactivative Action of Ethylenediaminetetraacetic Acid or Dipicolinic Acid on Inactive Glucose Dehydrogenase Obtained from Heated Spores of Bacillus subtilis

Partially purified inactive glucose dehydrogenase obtained from spores which were heated at 87 or 90 C for 30 min is converted to an active from by the addition of ethylenediaminetetraacetic acid, dipicolinic acid, or some salts. The molecular weight of the inactive glucose dehydrogenase in the heated spores is about one-half of that of the active glucose dehydrogenase in the intact resting spores. The possibility is discussed that the active glucose dehydrogenase in the intact resting spores divides into subunits and is converted to stable and inactive form during heating of spores at a particular range of temperature (87 to 90 C).     

Use of Ultraviolet Radiation to Locate Dipicolinic Acid in Bacillus cereus Spores

A novel method is described that allows a direct determination of the location of a portion of the dipicolinic acid (DPA) in spores. Ultraviolet (UV) irradiation is shown to cause cross-linkage of DPA to spore proteins which have the characteristics of membrane proteins. We suggest that DPA resides in the inner forespore membrane (IFSM) and spore cytoplasm (i.e., the spore protoplast). Only that portion of the DPA in the vicinity of the IFSM appeared to form UV-induced DPA-protein adducts.     

Messenger Ribonucleic Acid of Dormant Spores of Bacillus subtilis

Evidence of the presence of messenger ribonucleic acid (mRNA) in dormant spores of Bacillus subtilis has been obtained. The bulk RNA from spores was isolated and labeled in vitro with tritiated dimethyl sulfate. The spore RNA hybridized to 2.4 to 3.2% of the B. subtilis genome. The RNA hybridized to both the complementary heavy and light fractions of deoxyribonucleic acid (DNA). Bulk RNA from log-phase cells competed with virtually all the spore RNA for the heavy DNA fraction and with part of the spore RNA for the light DNA fraction. Bulk RNA from stage IV cells in sporulation also competed with all of the spore RNA for the heavy DNA fraction and with essentially all the spore RNA for the light DNA fraction. These results indicate that dormant spores contain mRNA species present in both log-phase cells and stage IV cells of sporulation. The RNA polymerase in the developing forespore must be able to recognize promotor sites for both log-phase and sporulation genes.     

Synthesis of spoIIA and spoVA mRNA in Bacillus subtilis

The expression of the spoIIA and spoVA sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoVA loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.     

Competence and Deoxyribonucleic Acid Uptake in Bacillus subtilis

The distribution of uninucleate and multinucleate cells of Bacillus subtilis, fractionated by zonal centrifugation, shows that the uninucleate cells are most likely to be competent. Only the competent fraction of the population incorporates deoxyribonucleic acid.     

HtrC Is Involved in Proteolysis of YpeB during Germination of Bacillus anthracis and Bacillus subtilis Spores

Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn²⁺ or Ca²⁺ ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation.     

Relationship of Dipicolinic Acid Content in Spores of Bacillus cereus T to Ultraviolet and Gamma Radiation Resistance

Spores of Bacillus cereus T lacking dipicolinic acid showed a statistically significant reduction in resistance to ultraviolet and gamma radiation as compared with spores with high dipicolinic acid content.     

Amino Acid Enhancement of Recovery in Dry-heat Damaged Spores of Bacillus subtilis

Spores of Bacillus subtilis MD2 and var. niger were dry-heat damaged at 150°, 160° and 170°C and recovered on media of increasing complexity. The greater the heat dose the more marked was the effect of amino acid supplements on recovery. For strain MD2 maximum germination and outgrowth of unheated spores could be obtained on a minimal salts + glucose medium with alanine, aspartic acid, glycine and methionine; the latter three amino acids served to enhance growth, not germination. The recovery of heat-damaged spores was significantly increased by adding valine plus isoleucine or arginine or glutamine. The increase was probably due to the use of valine and isoleucine as substrates of NAD-linked dehydrogenases to generate reducing power and serve as NH₃-donor, initiating germination in spores which were unable to germinate as a result of inactivation of alanine dehydrogenase. Valine or isoleucine added singly suppressed recovery by feedback inhibition of the pathways to both these amino acids during outgrowth.     

Homologues of the Bacillus subtilis SpoVB Protein Are Involved in Cell Wall Metabolism

Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of Escherichia coli MviN, to flip the lipid II peptidoglycan precursor. Bacillus subtilis SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the B. subtilis genome. Mutant strains lacking any or all of these genes (yabM, ykvU, and ytgP) in addition to spoVB are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in B. subtilis. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.The Bacillus subtilis spoVB gene was first identified as a locus in which a mutation could produce a block at a late stage of spore development (14, 30). Analysis of this locus revealed that it encoded an apparent integral membrane protein (33), and a detailed analysis of a spoVB null mutant demonstrated a block at a very early step in synthesis of the spore cortex peptidoglycan (PG) (40). The mutant synthesized essentially no cortex and accumulated cytoplasmic PG precursors, the same phenotype found in other mutant strains blocked in functions known to be directly involved in PG polymerization (40). These results suggested that SpoVB plays a direct role in assembly or function of the spore PG synthesis apparatus.PG synthesis is a highly conserved and complex process that must span the cell membrane (reviewed in reference 38). Soluble nucleotide-linked PG precursors are synthesized in the cytoplasm. N-Acetylmuramic acid with a pentapeptide side chain is then transferred to an undecaprenol lipid carrier to produce lipid I, with subsequent addition of N-acetylglucosamine to produce lipid II, undecaprenyl-pyrophosphoryl-N-acetylmuramic acid (pentapeptide)-N-acetylglucosamine. Lipid II is then flipped across the membrane via an unknown mechanism. Two families of proteins have been postulated to perform this function: the SEDS family of integral membrane proteins, including FtsW, RodA, and SpoVE (13), and, more recently, the COG2244 family (23), which includes SpoVB and the MviN (MurJ) protein of Escherichia coli (35). In both cases, loss of a protein within one of these families has been shown to result in a block in PG synthesis and the accumulation of lipid-linked and/or soluble PG precursors (16, 20, 35, 40).In the standard model of PG synthesis, flippase activity brings the disaccharide-pentapeptide moieties to the penicillin-binding proteins (PBPs), which polymerize the PG macromolecule on the outer surface of the membrane (39). The class A, high-molecular-weight PBPs possess an N-terminal glycosyl transferase domain that polymerizes the disaccharides into polysaccharide chains (38). These chains are cross-linked via the transpeptidase activity within the penicillin-binding, C-terminal domains of both the class A and the class B PBPs. The N-terminal domains of the class A PBPs and the closely related monofunctional glycosyl transferases found in some species are the only defined PG glycan strand polymerases, and in several species the presence of at least one of these enzymes is essential. However, in B. subtilis (26) and Enterococcus faecalis (3), strains lacking all of these known glycosyl transferases are viable and produce PG walls, indicating the presence of another glycosyl transferase capable of this activity. This alternate glycosyl transferase is distinct in that it is relatively resistant to moenomycin (3, 26), an inhibitor of the class A PBP glycosyl transferase activity (6).Given the strong and early block in cortex PG polymerization observed to occur in a spoVB mutant (40), we wished to further analyze the potential role of this class of protein. SpoVB is a member of a relatively large family of proteins, COG2244 (23), some of which are involved in polymerization of other polysaccharides in bacteria, archaea, and eukaryotes. Bioinformatic analysis has generally predicted that these proteins span the membrane 12 to 14 times, and in some cases experimental evidence has supported this structure (7, 24). A role generally ascribed to these proteins is the flipping of lipid-linked oligosaccharides, produced on the inner face of a membrane, to the outside, where the oligosaccharides are then further polymerized or transferred to other substrates. Some prominent members of this family include Wzx, which functions in O-antigen synthesis in gram-negative bacteria (41); TuaB, which functions in teichuronic acid synthesis in B. subtilis (36); and Rft1, which functions in protein glycosylation in eukaryotes (12). MviN is essential in some gram-negative species, including Burkholderia pseudomallei, E. coli, and Sinorhizobium meliloti (22, 34), and has been shown to play a role in E. coli PG synthesis (16, 35). A Rhizobium tropici mutation that truncates mviN approximately 50% into the coding sequence was not lethal (29). However, it is not known whether this was the sole mviN homolog in the genome or whether the truncated gene product might be functional.We have analyzed the phenotypic properties of B. subtilis strains lacking other proteins within the COG2244 family that are most closely related to SpoVB. Results suggest that these proteins also play roles in PG synthesis and that, in one case, this role is in a synthetic system that is relatively moenomycin resistant. We postulate that these proteins function in an alternate pathway for PG synthesis that may involve the flipping of lipid-linked PG oligosaccharides rather than lipid II disaccharides.     

Repair of Radiation Damage to Deoxyribonucleic Acid in Germinating Spores of Bacillus subtilis

The repair of deoxyribonucleic acid (DNA) in germinating spores was studied in comparison with that in vegetative cells. Radiation-induced single-strand breaks in the DNA of spores and of vegetative cells of Bacillus subtilis were rejoined during postirradiation incubation. The molecular weight of single-stranded DNA was restored to the level of nonirradiated cells. The rate of the rejoining of DNA strand breaks in irradiated spores was essentially equal to that in irradiated vegetative cells. The rejoining in spores germinating in nutrient medium occurred in the absence of detectable DNA synthesis. In this state, normal DNA synthesis was not initiated. Very little DNA degradation occurred during the rejoining process. On the other hand, in vegetative cells the rejoining process was accompanied by a relatively large amount of DNA synthesis and DNA degradation in nutrient medium. The rejoining occurred in phosphate buffer in vegetative cells but not in spores in which germination was not induced. Chloramphenicol did not interfere with the rejoining process in either germinating spores or vegetative cells, indicating that the rejoining takes place in the absence of de novo synthesis of repair enzyme. In the radiation-sensitive strain uvs-80, the capacity for rejoining radiation-induced strand breaks was reduced both in spores and in vegetative cells, suggesting that the rejoining mechanism of germinating spores is not specific to the germination process.     

Completed Chromosomes in Thymine-Requiring Bacillus subtilis Spores

Origin:terminus genetic marker ratios (both purA: metB and purA:ilvA) were measured in extracts of spores of Bacillus subtilis strains W23 thy his and 168 thy. For strain W23 thy his, normalized to W23 spore deoxyribonucleic acid, both ratios were equal to unity and were consistent with the presence of only completed chromosomes in the spores. The same ratios in extracts of spores of 168 thy, normalized to strain 168 or the prototroph SB19, were abnormal, i.e., 2.26 +/- 0.10 and 0.71 +/- 0.06 for purA:metB and purA:ilvA, respectively. These values were unaffected by the extent of extraction of the spore deoxyribonucleic acid, the richness of the medium on which they are formed, and the thymine phenotype. The high ratio for purA:metB is in agreement with the results of earlier workers but, because of the low purA:ilvA ratio, cannot be explained simply by the presence of partially replicated chromosomes in spores of strain 168 thy. Furthermore, purA:leuA in such extracts is 1.01 +/- 0.06, consistent with the presence of only completed chromosomes. It is concluded that the abnormal origin:terminus marker ratios are only apparent and result from non-isogenicity between strains 168 thy and 168 in the metB thyB ilvA chromosome region introduced during construction of 168 thy by transformation of strain 168 with W23 thy deoxyribonucleic acid. It is concluded further that the chromosomes of strain 168 thy spores are in a completed form.     

Increasing Activity of Germinating Bacillus subtilis Spores to Incorporate Thymidine Triphosphate into Deoxyribonucleic Acid After Detergent Treatment

The incorporation of (3)H-labeled thymidine triphosphate ((3)H-dTTP) into deoxyribonucleic acid (DNA) of germinated and then Brij 58-treated Bacillus subtilis spores was measured to study DNA replication activity of cells. The dTTP incorporation rate was very low in dormant spores, gradually increased as germination proceeded, and reached a level of the vegetative cell activity approximately 4 hr after the start of germination. This is in contrast to the DNA polymerase activity in the cell extract which remained at the same level throughout the germination period. The increase of the dTTP incorporation activity was inhibited by chloramphenicol or phenethyl alcohol. When these inhibitors were added after germination had proceeded, the elevated dTTP incorporation activity gradually decreased. Permeability to dTTP of spores germinated in the presence of chloramphenicol and then treated with Brij 58 was confirmed by (i) (3)H-dTTP incorporation into the treated spores following either electron or ultraviolet irradiation and (ii) release of radioactivity from the treated spores containing radioactively labeled DNA after deoxyribonuclease I treatment.     

The X-ray Diffraction Pattern of Spores of Bacillus subtilis

ORD and CD of d(+) pantothenic acid, d(+) pantothenyl alcohol and d(?) pantolactone were studied. The acid and the alcohol gave positive Cotton effects with a peak at 227 and 225 mμ, respectively, and the lactone gave a negative Cotton effect with trough at 233 mμ. They gave CD maxima at 214, 213 and 219 mµ corresponding to the inflection points of their ORD curves.

The concentrations were found to be linear to the rotation angles and the possibility of the application to quantitative analysis of the ORD was cited. The ORD showed the quantitative formation of the lactone by acid treatment of the vitamins without any racemization and hence the determination via lactone was suggested.     

Viability of Bacillus subtilis Spores in Rocket Propellants

The sporicidal activity of components used in liquid and solid rocket propellants was tested by use of spores of Bacillus subtilis dried on powdered glass. Liquid propellant ingredients tested were N₂O₄, monomethylhydrazine and 1,1-dimethylhydrazine. N₂O₄ was immediately sporicidal; the hydrazines were effective within several days. Solid propellants consisted of ammonium perchlorate in combination with epoxy resin (EPON 828), tris-1-(2-methyl) aziridinyl phosphine oxide, bis-1-(2-methyl) aziridinyl phenylphosphine oxide, and three modified polybutadiene polymers. There was no indication of appreciable sporicidal activity of these components.