Megaterium Spores Research Articles

Bacillus megaterium spore protein C-3: nucleotide sequence of its gene and the amino acid sequence at its spore protease cleavage site

The nucleotide sequence of the Bacillus megaterium gene coding for spore-specific protein C-3 has been determined. The gene codes for 65 amino acids and the coding sequence is preceded by an efficient ribosome-binding site. The predicted protein C-3 sequence agrees with both the amino acid composition and the amino terminal sequence of protein C-3, and shows homology (approx. 65 % of all residues are identical) with the sequences of the analogous proteins A and C of B. megaterium. Protein C-3 is cleaved by the sequence-specific B. megaterium spore protease, and the amino acid sequence at the new amino-terminus generated is identical to that predicted from the gene sequence, and homologous to the spore protease cleavage sites in the A and C proteins. The protein C-3 gene also shares a number of features with the previously sequenced protein C gene in both upstream and downstream flanking sequence.

Differential scanning calorimetry of membranes isolated from Bacillus megaterium spores

Inner membranes isolated from Bacillus megaterium spores had a broad endothermic phase transition between 6 and 26 °C. Spores grown in isoleucine initiated germination at lower temperatures than control spores grown in glucose and their membrane endotherms were depressed about 8 °C.

Isolation and characterization of two distinct fractions from the inner membrane of dormant Bacillus megaterium spores.

Two distinct membrane bands were obtained after sucrose velocity gradient centrifugation of crude inner membranes from dormant Bacillus megaterium spores disrupted under conditions which minimized endogenous enzyme action. These two inner membrane fractions (termed LD and HD) contained similar amounts of total and individual phospholipid species. However, LD and HD differed significantly in phospholipid/protein ratios (4.3 and 0.47 mg/mg, respectively), equilibrium densities (1.12 and 1.18 g/cm3), NADH oxidase specific activity (less than 0.01 and 0.13 mumol/min X mg), and content of specific proteins. In contrast, crude membranes prepared in identical fashion from germinated spores gave only a single inner membrane band (termed G) on sucrose velocity gradients. G had a phospholipid/protein ratio of 0.98 mg/mg, an equilibrium density of 1.16 g/cm3, and an NADH oxidase specific activity of 2.1 mumol/min X mg. Essentially all of the proteins present in LD or HD or both were found in G, consistent with the latter membrane being derived from a mixture of LD and HD. No evidence was found suggesting that there is significant degradation of dormant spore inner membrane protein upon spore germination.

Cloning of a new low-molecular-weight spore-specific protein gene from Bacillus megaterium.

Three EcoRI fragments of Bacillus megaterium DNA hybridized only under nonrestrictive conditions on Southern blots to a probe containing the previously cloned gene for protein C, a small, acid-soluble spore protein (SASP) from B. megaterium. All three fragments were cloned in Escherichia coli cells in plasmid pBR325, and after being transferred to an E. coli expression vector, one of the fragments (C-3) directed the synthesis of a new small, acid-soluble spore protein (termed C-3) immunologically related to protein C. As previously observed with the protein C gene, protein C-3 gene expression in E. coli required an external promoter and suppression of termination of transcription. Protein C-3 was purified from induced E. coli cells, and its immunological properties, electrophoretic mobility, amino acid composition, and amino-terminal sequence were determined. These data indicated that protein C-3 was related, but not identical, to either protein C or the closely related protein A--two of the major small, acid-soluble spore proteins of B. megaterium. Detailed examination of acid extracts of B. megaterium spores showed that they contained a minor protein which comigrated with C-3 on acrylamide gel electrophoresis at low pH and reacted immunologically like C-3.

Combination of misonidazole and lipoic acid on the radiation sensitivity of Bacillus megaterium spores.

The radiation sensitizing action of a combination of misonidazole and lipoic acid was investigated in Bacillus megaterium spores at various oxygen concentrations. Lipoic acid and misonidazole were applied in two concentrations (0.1 and 1.0 mmol/l) and four combinations were prepared from them. No uniform correlation was found, neither to the combination of the compounds nor to the gas conditions. The combination of two radiation sensitizing compounds with in all probability different action mechanisms does not unequivocally enhance the radiation sensitizing effect under anoxic and hypoxic conditions.

Expression of a Bacillus megaterium sporulation-specific gene during sporulation of Bacillus subtilis.

The gene for the Bacillus megaterium spore C protein, a sporulation-specific gene, has been transferred into Bacillus subtilis. The B. megaterium gene was expressed little, if at all, during log-phase and early-stationary-phase growth, but was expressed during sporulation with the same kinetics as and at a level similar to that of the analogous B. subtilis genes. This finding is most consistent with the regulation of this class of genes by a mechanism of positive control.

Steady-state fluorescence anisotropy changes of 1,6-diphenyl-1,3,5,-hexatriene in membranes from Bacillus megaterium spores

The steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene incorporated into isolated Bacillus megaterium spore membranes was measured. Compounds capable of triggering spore germination in vivo caused an increase in the anisotropy of diphenylhexatriene. These increases in anisotropy of diphenylhexatriene in spore membranes are likely to represent at least a portion of the trigger mechanism for spore germination based on the following observations. First, there was an exceptional positive correlation between compounds that both triggered germination in vivo and caused changes in anisotropy in vitro. Second. the capacity of membranes to respond to germinants by increases in anisotropy was unique to membranes from spores but disappeared after germination. Third, alteration of spores chemically or genetically to block the in vivo triggering of germination by l-proline also blocked the in vitro anisotropy change with l-proline but not d-glucose. Finally, there was no correlation between the transport activities of specific compounds and the ability of these compounds to either trigger germination or alter the anisotropy of diphenylhexatriene in the membranes. Although we do not known the nature of the molecular interactions giving rise to the anisotropy changes, we hypothesize that they are due to changes in protein conformation that alter protein-protein and/or protein-lipid interactions. Such modifications of membrane structures could account for the rapid release of small molecular weight compounds such as K + and Ca 2+ early in germination.

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.

Acid-soluble spore proteins of Bacillus subtilis.

Acid-soluble spore proteins (ASSPs) comprise about 5% of the total protein of mature spores of different Bacillus subtilis strains. They consist of three abundant species, alpha, beta, and gamma, four less abundant species, and several minor species, alpha, beta, and gamma make up about 18, 18 and 36%, respectively, of the total ASSPs of strain 168, have molecular weights of 5,900, 5,9000, and 11,000, respectively, and resemble the major (A, C, and B) components of Bacillus megaterium ASSPs in several respects, including sensitivity to a specific B. megaterium spore endopeptidase. However, they have pI's of 6.58, 6.67, and 7.96, all lower than those of any of the B. megaterium ASSPs. Although strains varied in the proportions of different ASSPs, to overall patterns seen on gel electrophoresis are constant. ASSPs are located interior to the cortex, presumably in the spore cytoplasm, and are synthesized during sporulation and degraded during germination.

The complete covalent structure of protein B. The third major protein degraded during germination of Bacillus megaterium spores.

The complete covalent structure of Protein B, the third major protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with the specific B. megaterium spore protease into three peptides, residues 1 to 31 (B-III), 32 to 66 (B-I), and 67 to 96 (B-II). Cleavage of the intact protein with trypsin allowed isolation of the peptide encompassing residues 61 to 77 (T-11) as well as the COOH-terminal peptide, residues 94 to 96 (T-4). Cleavage of Peptide B-I with trypsin or chymotrypsin allowed isolation of peptides encompassing residues 53 to 60 (B-I-T-2) and residues 52 to 66 (B-I-C-4), respectively. Subtractive Edman degradation of Peptide T-4, automated sequenator analysis of Peptides B-I, B-II, T-11, B-I-T-2, and B-I-C-4, previously published partial sequence data on the intact B-protein and carboxypeptidase V digestion of the intact protein provided the data from which the following unique sequence was deduced: NH2-Ala-Lys-Gln-Thr-Asn-Lys-Thr-Ala-Ser-Gly-Thr-Ser-Thr-Gln-His-15 Val-Lys-Gln-Gln-Asp-Ala-Gln-Ala-Ser-Lys-Asn-Asn-Phe-Gly-Thr-30 Glu-Phe-Gly-Ser-Glu-Thr-Asn-Val-Gln-Glu-Val-Lys-Gln-Gln-Asn-45 Ala-Gln-Ala-Ala-Asn-Lys-Ser-Gln-Asn-Ala-Gln-Ala-Ser-Lys-60 Asn-Asn-Phe-Gly-Thr-Glu-Phe-Ala-Ser-Glu-Thr-Ser-Ala-Gln-Glu-75 Val-Arg-Gln-Gln-Asn-Ala-Gln-Ala-Gln-Lys-Lys-Asn-Gln-Asn-90 Ser-Gly-Lys-Tyr-Gln-Gly-COOH. The primary sequence of the B-protein contains a large internal duplication (residues 17 to 50 and 52 to 85), and shows significant sequence homology with the A- and C-proteins, the other major proteins degraded during B. megaterium spore germination.

Covalent structure of protein C. A second major low molecular weight protein degraded during germination of Bacillus megaterium spores.

The complete covalent structure of protein C, a protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 30 and 31 to 71. The intact protein was also cleaved by cyanogen bromide into two peptides, residues 1 to 27 and 28 to 71. Cleavage of the larger cyanogen bromide peptide with trypsin allowed isolation of the COOH-terminal peptide, residues 59 to 71. Automated sequenator analysis of the intact protein and peptide fragments, together with previously published partial sequence data on this protein and carboxypeptidase A digestion of the intact protein provided data from which the following unique sequence was deduced: (formula: see text). The primary sequence of the C protein shows an extremely high degree of homology with that of the A protein--another protein degraded during germination of B. megaterium spores.

Bacillus megaterium spore protease. Action of the enzyme on peptides containing the amino acid sequence cleaved by the enzyme in vivo.

Bacillus megaterium spore protease. Action of the enzyme on peptides containing the amino acid sequence cleaved by the enzyme in vivo.

Dielectric properties of native and decoated spores of Bacillus megaterium.

A general model for use in interpreting dielectric data obtained with bacterial endospores is developed and applied to past results for Bacillus cereus spores and new results for Bacillus megaterium spores. The latter were also subjected to a decoating treatment to yield dormant cells with damaged outer membranes that could be germinated with lysozyme. For both spore types, core ions appeared to be completely immobilized, and decoating of B. megaterium spores did not affect this extreme state of electrostasis in the core. The cortex of B. megaterium appeared to contain a high level of mobile ions, in the cortex of B. cereus. The outer membrane-coat complex of B. megaterium acted dielectrically as an insulating layer around the cortex, so that native dormant spores showed a Maxwell-Wagner dispersion over the frequency range from about 1 to 20 MHz. The decoating treatment resulted in a shift in the dispersion to frequencies below the range of observation. Increases in cell conductivity in response to increases in environmental ionic strength indicated that the coats. of B. megaterium could be penetrated by environmental ions and that they had an inherent fixed charge concentration of about 10 to 20 milliequivalents per liter. In contrast, the dispersion for B. cereus spores was very sensitive to changes in environmental ion concentration, and it appeared that some 40% of the spore volume could be penetrated by environmental ions and that these ions traversed a dielectrically effective layer, either the exosporium or the outer membrane. It appears that dormancy is associated with extreme electrostasis of core ions but not necessarily of ions in enveloping structures and that the coat-outer membrane complex is dielectrically effective but not required for maintenance of extreme electrostasis in the core.

Localization of low-molecular-weight basic proteins in Bacillus megaterium spores by cross-linking with ultraviolet light.

Two low-molecular-weight basic proteins, termed A and B proteins, comprise about 15% of the protein of dormant spores of Bacillus megaterium. Irradiation of intact dormant spores with ultraviolet light results in covalent cross-linking of the A and B proteins to other spore macromolecules. The cross-linked A and B proteins are precipitated by ethanol and can be solubilized by treatment with deoxyribonuclease (75%) or ribonuclease (25%). Irradiation of complexes formed in vitro between deoxyribonucleic acid (DNA) or ribonucleic acid and a mixture of the low-molecular-weight basic proteins from spores also resulted in cross-linking of A and B proteins to nucleic acids. The dose-response curves for formation of covalent cross-links were similar for irradiation of both a protein-DNA complex in vitro and intact spores. However, if irradiation was carried out in vitro under conditions where DNA-protein complexes were disrupted, no covalent cross-links were formed. These data suggest that significant amounts of the low-molecular-weight basic proteins unique to bacterial spores are associated with spore DNA in vivo.

Production of large amounts of acetate during germination of Bacillus megaterium spores in the absence of exogenous carbon sources

When Bacillus megaterium spores germinate in the absence of an exogenous carbon source, the first minutes of germination are accompanied by production of large amounts (approximately 70 nmol/mg of dry spores) of acetate and much smaller amounts of pyruvate and lactate. The majority of these compounds are excreted into the medium. Exogenous pyruvate and alanine are also converted to CO2 and acetate by germinating spores, presumably by using the pyruvate dehydrogenase that is present in dormant spores. These data suggest that the 3-phosphoglyceric acid stores in the dormant spore and alanine generated by proteolysis early in germination can be catabolized to acetate during germination with production of large amounts of reduced nicotinamide adenine dinucleotide, acetyl coenzyme A, and adenosine 5'-triphosphate.

Purification and properties of a specific proteolytic enzyme present in spores of Bacillus magaterium

A proteolytic enzyme with high activity on the specific, low molecular weight dormant spore proteins (termed A and B proteins) degraded during spore germination has been purified approximately 1000-fold from Bacillus megaterium spores. This enzyme accounts for greater than 85% of the proteolytic activity toward the A and B proteins in crude spore extracts. The protease has a pH optimum of approximately 7.5, is inactivated by phenylmethylsulfonyl fluoride and EDTA (10 mM), and is inhibited approximately 70% by NaCl (1 M). The enzyme is unstable and requires glycerol, divalent cations, and high enzyme concentrations for maximum stability. The protease shows a high degree of specificity for the A and B proteins, since high levels of enzyme catalyze no detectable bond cleavage on a variety of amide, ester, peptide, or other protein substrates. The enzyme is an endoprotease and digestion of the A and B proteins in vitro generates a number of peptide fragments at least one of which appears identical with a fragment isolated from lysates of spores carrying out hydrolysis of endogenous A and B proteins. In vitro, the peptide products can be rapidly degraded to amino acids by an aminopeptidase which has also been partially purified from spores. Although high levels of the protease are present in extracts of dormant spores, and of spores germinated for a few minutes, the enzyme is undetectable in log phase and early stationary phase cells. Furthermore, the protease disappears rapidly (t1/2 approximately 30 min) and completely (greater than 90%) as the process of germination proceeds.

Radiation Sensitization of Bacterial Spores by cis-Dichlorodiammineplatinum(II)

The x irradiation of B. megaterium spores in aqueous suspension in the presence of cis-dichlorodiammineplatinum(II) (CPCA) results in biphasic survival curves showing pronounced increases in radiation sensitivity in N/sub 2/ , O/sub 2/, and in N/sub 2/O in the low-dose portion of the response. In N/sub 2/, the degree of CPCA-induced sensitization fully equals that due to O/sub 2/ alone. In the presence of O/sub 2/ and N/sub 2/O, the added component of sensitization is half that obtained under N/sub 2/ saturation. Additional results are also obtained in the presence of hydroxyl radical scavengers, and in the presence of N/sub 2/O. These results are additionally significant considering that CPCA has been successfully used in animal studies as an antitumor drug.

Additive Effects in the Radiosensitization of B. megaterium Spores by p-Nitroacetophenone and Norpseudopelletierine-N-Oxyl

Norpseudopelletierine-N-oxyl (NPPN) has been shown to increase the sensitivity of B. megaterium spores when irradiated in anoxic buffered suspensions. The maximum sensitization achieved by NPPN alone corresponded to a 58% increase over the simple anoxic response. This compares with a 40% increase produced by paranitroacetophenone (PNAP) and 115% increase shown by oxygen. Like PNAP, NPPN is capable of producing a small measure of sensitization by interfering with water radiolysis intermediates (indirect affects) when used at low concentrations. At high concentrations, at which NPPN exerts its maximum degree of sensitization, it is concluded that the compound operates by increasing the lethality of the direct effects of radiation on the cellular target. The use of mixtures of NPPN and PNAP produces a greater degree of sensitization than can be achieved by either alone, at least a 91% increase over the anoxic sensitivity being observed. It is concluded that these two sensitizers, chosen as representatives of...

Modification of Radiation Sensitivity of Bacillus megaterium Spores by N 2 O and p-Nitroacetophenone

The basic anoxic radiosensitivity of B. megaterium spores irradiated in buffered aqueous suspension can be increased by up to 40% by the addition of PNAP to the suspension. At the highest concentrations, above 6× 10-4 M PNAP, this increased sensitivity is constant and cannot be reduced by the addition of t-butanol as an OH scavenger. This does not hold, however, at lower concentrations at which PNAP exerts a submaximal sensitizing action since in this range t-butanol can eliminate the effect of PNAP. Nitrous oxide produces radiosensitization in the spore system that can be completely reversed by the presence of t-butanol. PNAP, however, can apparently only partially remove the sensitization produced by nitrous oxide. These results are explained in terms of indirect, or water radical-mediated, sensitization by nitrous oxide or low concentrations of PNAP. High concentrations of PNAP, however, are believed to enhance the lethality of the direct effects of radiation on the cellular target and this effect is i...