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Biomedical subjects

P Setlow

Publications and source records attributed to P Setlow.

At least 199 records · Page 11Linked to original sources
PubMed1982

Noninvolvement of the spore cortex in acquisition of low-molecular-weight basic proteins and UV light resistance during Bacillus sphaericus sporulation.

Two major low-molecular weight, acid-soluble proteins (termed A and B proteins) were purified from Bacillus sphaericus spores and had properties similar to those of the analogous proteins from spores of other Bacillus species. These proteins were accumulated late in sporulation, when the developing spores became resistant to UV light, and were degraded during spore germination by a spore protease. A mutant of B. sphaericus unable to make spore cortex because of a block in diaminopimelic acid (DAP) biosynthesis accumulated and maintained levels of the A and B proteins similar to those in the DAP+ parent or the DAP- strain in which cortex formation was restored by growth with DAP. In addition, the DAP- strain grown without DAP acquired a level of UV light resistance identical to that of wild-type spores and at the time of appearance of the A and B proteins. These findings indicate that formation of little, if any, spore cortex is required for acquisition of UV light resistance or maintenance of high levels of A and B proteins. The data provide further support for a role of the A and B proteins in the spore's UV light resistance.

Bacillus↗
PubMed1982

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.

Antigens, Bacterial↗
PubMed1981

Comparison of various properties of low-molecular-weight proteins from dormant spores of several Bacillus species.

Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared. All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan. The proteins could be subdivided into two groups: group I (B. megaterium A and C proteins, B. cereus A protein, and B. subtilis alpha and beta proteins) and group II (B. cereus and B. megaterium B proteins and B. subtilis gamma protein). Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline. Similarly, proteins in each group were more closely related immunologically. However, antisera against a B. megaterium group I protein cross-reacted more strongly with the B. megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B. megaterium group II protein cross-reacted most strongly with B. megaterium group I proteins. Analysis of the primary sequences at the amino termini and in the regions of the B. cereus and B. subtilis proteins cleaved by the B. megaterium spore protease revealed that the B. cereus A protein was most similar to the B. megaterium A and C proteins, and the B. cereus B protein and the B. subtilis gamma protein were most similar to the B. megaterium B protein. However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.

Amino Acid Sequence↗
PubMed1981

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).

Bacillus megaterium↗
PubMed1980

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.

Amino Acid Sequence↗
PubMed1980

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.

Amino Acid Sequence↗
PubMed1980

In vivo and in vitro synthesis of the spore-specific proteins A and C of bacillus megaterium.

Pulse labeling of cells of Bacillus megaterium followed by cell disruption and immunoprecipitation has shown that the spore-specific Proteins A and C are synthetized only during a discrete time period in sporulation. At its maximum, the synthesis of the A- and C-proteins accounted for 5% of the protein being synthesized in vivo, but the mRNA for the A- and C-proteins had a lifetime no longer than that of other mRNAs translated at that time. No evidence was found for synthesis of Proteins A or C in high molecular weight precursor form, and essentially all of the newly synthesized A- and C-protein was found in the forespore. Isolation of total RNA from cells in various stages of growth and sporulation, translation of this RNA in a cell-free system from vegetative cells, and immunoprecipitation showed that the ability of cellular RNA to promote A- and C-protein synthesis in vitro was directly proportional to the rate at which the cells had been synthesizing Proteins A and C in vivo. These data indicate that synthesis of Proteins A and C during sporulation in B. megaterium is primarily under transcriptional control. The identity of the immunoprecipitated labeled material material synthesized in vitro with the A- and C-proteins was established by: 1) their co-migration on sodium dodecyl sulfate-polyacrylamide gels; 2) co-migration on high performance liquid chromatography of tryptic peptides from an [35S]methionine-labeled immunoprecipitate with the methionine-containing tryptic peptides of the A- and C-proteins; and 3) digestion of the labeled immunoprecipitate with a protease specific for the A- and C-proteins.

Antigens, Bacterial↗
PubMed1980

The amino acid sequence specificity of a protease from spores of Bacillus megaterium.

Previous work has shown that the degradation of 20% of total protein which occurs early in germination of Bacillus megaterium spores is initiated by an endoprotease. This enzyme is found only in the spore and is active only on the spore proteins degraded during germination. Action of the spore protease in vitro on the three major proteins (Proteins A, B, and C) which are degraded in vivo during germination results in cleavage of one (A and C protein) or two (B protein) peptide bonds. The sequences surrounding the cleavage sites are -Tyr-Glu- Ile-Ala-Ser-Glu-Phe- in the A protein, -Phe-Glu- Ile-Ala-Ser-Glu-Phe- in the C protein, and -Thr-Glu- Phe-Gly-Ser-Glu-Thr-, and -Thr-Glu- Phe-Ala-Ser-Glu-Thr- in the B protein, with cleavage taking place at the glutamyl bond noted by the arrow. The similarity of these four sequences suggests the possibility that the specificity of the spore protease may be due to its requirement for a specific pentapeptide sequence of the type -R-Glu-(Phe or Ile)-(Gly or Ala)-Ser-Glu-R- for recognition and cleavage. However, it is also possible that it is the conformation of the A, B, and C proteins which determines their site of cleavage by the spore protease.

Amino Acid Sequence↗
PubMed1980

Measurements of the pH within dormant and germinated bacterial spores.

The pH within the core or central region of dormant spores of Bacillus cereus and B. megaterium is 6.3-6.4 irrespective of the external pH. However, the spore's internal pH rises to 7.3-7.5 upon germination. The low internal pH of the dormant spore may be a contributing factor to its metabolic dormancy.

Bacillus cereus↗
PubMed1979

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

The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.

Amino Acid Sequence↗
PubMed1979

Identification of an NADH-linked disulfide reductase from Bacillus megaterium specific for disulfides containing pantethine 4',4''-diphosphate moieties.

Bacillus megaterium contains an NADH-linked disulfide reductase that is specific for disulfides containing pantethine 4',4''-diphosphate moieties. This reductase is at its highest level in cells late in sporulation and in dormant spores, and could be involved in the formation and cleavage of coenzyme A-protein disulfides which take place late in sporulation and early in spore germination, respectively.

Bacillus megaterium↗
PubMed1979

Purification and properties of phosphoglycerate phosphomutase from spores and cells of Bacillus megaterium.

Phosphoglycerate phosphomutase has been purified to homogeneity from vegetative cells and germinated spores of Bacillus megaterium, and the spore and cell enzymes appear identical. The enzyme is a monomer of molecular weight 61,000. The compound 2,3-diphosphoglyceric acid is not required for activity, but the enzyme has an absolute and specific requirement for Mn2+. The enzyme is inhibited by ethylenediaminetetraacetate and sulfhydryl reagents, has a pH optimum of about 8.0, and has Km values for 3-phosphoglyceric acid and Mn2+ of 5 x 10(-4) and 4 x 10(-5) M, respectively.

Amino Acids↗
PubMed1979

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.

Bacillus megaterium↗
PubMed1979

Regulation of phosphoglycerate phosphomutase in developing forespores and dormant and germinated spores of Bacillus megaterium by the level of free manganous ions.

The large depot of phosphoglyceric acid (PGA) which is accumulated within spores of Bacillus megaterium is greater than 99% 3-phosphoglyceric acid (3-PGA). The 3-PGA depot is stable in forespores and dormant spores, but is utilized rapidly during spore germination. When spores were germinated in KBr plus NaF, the PGA depot was not utilized, but 13% of the 3-PGA was converted to 2-PGA. These data suggest phosphoglycerate phosphomutase as the enzyme which is regulated to allow 3-PGA accumulation during sporulation. Young isolated forespores, in which 3-PGA was normally stable, utilized their 3-PGA rapidly when incubated with Mn2+ plus the divalent cation ionophore X-537A; Mn2+ or ionophore alone or Mg2+ or Ca2+ plus ionophore was without effect. Young forespores contained significant amounts of Mn2+. However, forespore Mn2+ exchanged slowly with exogenous Mn2+ and was removed poorly by toluene treatment. This suggests that much of the forespore Mn2+ is tightly bound to some forespore component. Since phosphoglycerate phosphomutase from B. megaterium has an absolute and specific requirement for Mn2+, these data suggest that the activity of this enzyme in vivo may be regulated to a large degree by the level of free Mn2+. Indeed, the activity of this enzyme in forespore or dormant spore extracts was stimulated greater than 25-fold by Mn2+, whereas comparable extracts from cells or germinated spores were stimulated only two- to fourfold.

Bacillus megaterium↗
PubMed1978

Enolase from spores and cells of Bacillus megaterium: two-step purification of the enzyme and some of its properties.

A simple two-step procedure for purification of enolase from germinated spores or vegetative cells of Bacillus megaterium is described. The procedure resulted in a 1,200-fold purification with production of homogeneous enzyme in approximately 75% yield; the enzymes from spores and cells seemed identical. The molecular weight of the native enzyme was 335,000, with a subunit molecular weight of 42,000. The enzyme required Mg2+ and was inhibited by ethylenediaminetetraacetic acid and fluoride ions. The Michaelis constants for 2-phosphoglyceric acid and Mg2+ were 7.1 X 10(-4) and 4.7 X 10(-4) M, respectively.

Bacillus megaterium↗
PubMed1978

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.

Adenosine Triphosphate↗