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P Setlow

Publications and source records attributed to P Setlow.

At least 91 records · Page 5Linked to original sources
PubMed1995

Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from killing by dry heat.

Dry Bacillus subtilis spores lacking their two major DNA-binding proteins (small, acid-soluble proteins [SASP] alpha and beta) were much more sensitive to dry heat than were wild-type spores. Survivors of dry heat treatment of both wild-type and mutant spores exhibited a high frequency of mutations, and the DNA from the heated spores had increased numbers of single-strand breaks. These data indicate that SASP alpha and beta provide significant protection to spore DNA against the damaging effects of dry heat. This DNA damage may be in part depurination, and a purified alpha/beta-type SASP gave significant protection against dry heat-induced DNA depurination in vitro.

Bacillus subtilis↗
PubMed1995

Binding to DNA protects alpha/beta-type, small, acid-soluble spore proteins of Bacillus and Clostridium species against digestion by their specific protease as well as by other proteases.

Binding of alpha/beta-type, small, acid-soluble proteins from Bacillus subtilis and Clostridium bifermentans to DNA protected these proteins against cleavage by their specific protease (GPR) as well as by trypsin and chymotrypsin. These data suggest that alpha/beta-type, small, acid-soluble protein binding to DNA (i) may result in a structural change in these proteins, giving a more compact protein structure, and (ii) may be important in slowing the degradation of these proteins by GPR, in particular during sporulation.

Bacillus subtilis↗
PubMed1995

The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration.

Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB. Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance. The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores. These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration. This defect in spore core dehydration was exaggerated by the additional loss of dacB expression. However, loss of dacB alone did not alter the spore core water content. Spores produced by spmA and spmB mutants germinated faster than did those of the wild type. Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth. Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant. Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants. These results are consistent with spore core water content being the major determinant of spore heat resistance. The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.

Bacillus subtilis↗
PubMed1995

Cloning, nucleotide sequence, and mutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor.

An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight penicillin-binding proteins (PBPs) was used to identify the ponA gene encoding PBP1a and -1b (PBP1) of Bacillus subtilis. The identity of the ponA product was established by (i) the presence of a sequence coding for a peptide generated from PBP1 and (ii) the disappearance of PBP1 in a ponA mutant. DNA sequence analysis revealed that the amino acid sequence of PBP1 was similar to those of other class A high-molecular-weight PBPs and that ponA appeared to be cotranscribed with an upstream gene (termed prfA) of unknown function. Null mutations in ponA resulted in a slight decrease in growth rate and a change in colony morphology but had no significant effect on cell morphology, cell division, sporulation, spore heat resistance, or spore germination. Mutations in prfA which did not effect ponA expression produced a more significant decrease in growth rate but had no other significant phenotypic effects. Deletion of both prfA and ponA resulted in extremely slow growth and a reduction in sporulation efficiency. Studies of expression of transcriptional fusions of ponA and prfA to lacZ demonstrated that these two genes constitute an operon. Expression of these genes was relatively constant during growth, decreased during sporulation, and was induced approximately 15 min into spore germination. The ponA locus was mapped to the 200 degrees region of the chromosomal physical map.

Amino Acid Sequence↗
PubMed1995

Mechanisms for the prevention of damage to DNA in spores of Bacillus species.

The DNA in dormant spores of Bacillus subtilis as well as other Bacillus species is extremely well protected against damage resulting from treatments such as desiccation, heat, oxidizing agents, and UV and gamma radiation. This high degree of DNA protection is a major factor in the survival of spores of these species, not only when subjected to the treatments noted above, but also when incubated under common environmental conditions for many years. Factors that play major roles in overall spore resistance include the low permeability of spores to toxic chemicals and the decreased spore-core water content. However, although decreased spore permeability and water content appear to at least partially protect spore DNA from oxidative damage, these factors seem to play little or no role in protecting spore DNA from heat damage. The major factor preventing damage to spore DNA is the saturation of this DNA with a novel group of small, acid-soluble proteins of the alpha/beta-type whose binding greatly alters DNA's chemical and enzymatic reactivity as well as its UV photochemistry. Binding of these proteins is also a key factor in spore DNA resistance to desiccation, heat, oxidizing agents, and UV radiation.

Amino Acid Sequence↗
PubMed1994

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.

Bacillus cereus↗
PubMed1994

Electron microscopic studies of the interaction between a Bacillus subtilis alpha/beta-type small, acid-soluble spore protein with DNA: protein binding is cooperative, stiffens the DNA, and induces negative supercoiling.

DNA within spores of Bacillus subtilis is complexed with a group of alpha/beta-type small acid-soluble spore proteins (alpha/beta-type SASPs), which have almost identical primary sequences and DNA binding properties. Here electron microscopic and cyclization studies were carried out on alpha/beta-type SASP-DNA complexes. When an alpha/beta-type SASP was incubated with linear DNA, the protein bound cooperatively, forming a helical coating 6.6 +/- 0.4 nm wide with a 2.9 +/- 0.3 nm periodicity. alpha/beta-Type SASP binding to an 890-bp DNA was weakest at an (A+T)-rich region that was highly bent, but binding eliminated the bending. alpha/beta-Type SASP binding did not alter the rise per bp in DNA but greatly increased the DNA stiffness as measured by both electron microscopic and cyclization assays. Addition of alpha/beta-type SASPs to negatively supertwisted DNA led to protein binding without significant alteration of the plectonemically interwound appearance of the DNA. Addition of alpha/beta-type SASPs to relaxed or nicked circular DNA led to molecules that by electron microscopy appeared similar to supertwisted DNA. The introduction of negative supertwists in nicked circular DNA by alpha/beta-type SASPs was confirmed by ligation of these molecules followed by topoisomer analyses using agarose gel electrophoresis.

Bacillus subtilis↗
PubMed1994

Analysis of the expression and regulation of the gerB spore germination operon of Bacillus subtilis 168.

The gerB spore germination operon of Bacillus subtilis 168 is a homologue of the gerA spore germination operon. The expression and regulation of the gerB operon has been examined using a lacZ transcriptional fusion and the transcriptional start defined. The gerB operon is expressed during sporulation under the control of RNA polymerase containing the forespore-specific sigma factor, delta G. This is a further homology to the gerA operon, which is similarly regulated. It is predicted from the localization of expression and the encoded primary sequences that the GerB proteins are located at the inner spore membrane.

Bacillus subtilis↗
PubMed1994

Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from being killed by freeze-drying.

Wild-type spores of Bacillus subtilis were resistant to eight cycles of freeze-drying, whereas about 90% of spores lacking the two major DNA-binding proteins (small, acid-soluble proteins alpha and beta) were killed by three to four cycles of freeze-dryings, with significant mutagenesis and DNA damage accompanying the killing. This role for alpha/beta-type small, acid-soluble proteins in spore resistance to freeze-drying may be important in spore survival in the environment.

Bacillus subtilis↗
PubMed1994

Studies of the processing of the protease which initiates degradation of small, acid-soluble proteins during germination of spores of Bacillus species.

Three mutant forms of the protease (GPR) that initiates degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus species have been generated. In one variant (GPR delta), the putative pro sequence removed in conversion of the GPR zymogen (termed P46) to the active enzyme (termed P41) was deleted. GPR delta was expressed in both Escherichia coli and Bacillus subtilis as a polypeptide of 41 kDa (P41) which was active both in vivo and in vitro. The other two variants had changes in the sequence around the site where the pro sequence is removed, making this sequence even more like that recognized and cleaved by GPR in its SASP substrates. One of these variants (GPRS) was synthesized as P46S in both B. subtilis and E. coli, but P46S was processed to P41S earlier in B. subtilis sporulation than was wild-type P46. The second variant (GPREI) was made as P46EI but underwent extremely rapid processing to P41EI in both E. coli and B. subtilis. Expression of elevated (> 100-fold) levels of GPR delta or GPREI blocked sporulation at the time of synthesis of glucose dehydrogenase. Expression of elevated levels of GPRS or low levels (< 20% of the wild-type level) of GPR delta or GPREI did not retard sporulation, but the SASP level in the resultant spores was greatly reduced. Prolonged incubation of P41 delta, P41EI, or wild-type P41, either in vivo or with purified proteins in vitro, resulted in a second self-cleavage event generating a 39-kDa polypeptide termed P39. The sequence in the P(41)-->P(39) cleavage site was also quite similar to that recognized and cleaved by GPR in SASP. Together, these results strongly support a model in which activation of GPR during sporulation by conversion of P(46) to P(41) is a self-processing event triggered by a change in the spore core environment (i.e., dehydration) which precludes attack of the active P(41) on its SASP substrates. However, in the first minutes of spore germination, rapid spore core hydration allows rapid attack of active GPR on SASP.

Amino Acid Sequence↗
PubMed1994

Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis.

The Bacillus subtilis genes tpi, pgm, and eno, encoding triose phosphate isomerase, phosphoglycerate mutase (PGM), and enolase, respectively, have been cloned and sequenced. These genes are the last three in a large putative operon coding for glycolytic enzymes; the operon includes pgk (coding for phosphoglycerate kinase) followed by tpi, pgm, and eno. The triose phosphate isomerase and enolase from B. subtilis are extremely similar to those from all other species, both eukaryotic and prokaryotic. However, B. subtilis PGM bears no resemblance to mammalian, fungal, or gram-negative bacterial PGMs, which are dependent on 2,3-diphosphoglycerate (DPG) for activity. Instead, B. subtilis PGM, which is DPG independent, is very similar to a DPG-independent PGM from a plant species but differs from the latter in the absolute requirement of B. subtilis PGM for Mn2+. The cloned pgm gene has been used to direct up to 25-fold overexpression of PGM in Escherichia coli; this should facilitate purification of large amounts of this novel Mn(2+)-dependent enzyme. Inactivation of pgm plus eno in B. subtilis resulted in extremely slow growth either on plates or in liquid, but growth of these mutants was enhanced by supplementation of media with malate. However, these mutants were asporogenous with or without malate supplementation.

Amino Acid Sequence↗
PubMed1994

Properties of Bacillus subtilis small, acid-soluble spore proteins with changes in the sequence recognized by their specific protease.

Alpha/beta-type small, acid-soluble proteins (SASP) of dormant spores of Bacillus subtilis bind to DNA and increase its resistance to a variety of damaging agents both in vivo and in vitro. When spores germinate, degradation of alpha/beta-type SASP is rapidly initiated by a sequence-specific protease, which is termed GPR. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the wild-type alpha/beta-type SASP SspCwt; all three mutations change residues in the highly conserved sequence recognized by GPR. In one mutant protein (SspCV), residue 33 (Ser) was changed to Val; in the second (SspCDL), residues 30 and 31 (Glu and Ile) were changed to Asp and Leu, respectively; and in the third mutant protein (SspCDLV), residues 30, 31, and 33 were changed to Asp, Leu, and Val. All three mutant proteins were rapidly degraded by GPR during spore germination, and SspCDL and SspCDLV were degraded by GPR in vitro at rates 8 to 9% of that for SspCwt, although not exclusively at the single site cleaved by GPR in SspCwt. These results indicate (i) that the sequence specificity of GPR is broader than originally imagined and (ii) that GPR can cleave the sequence in SspCDLV. Since the latter sequence is identical to that cleaved during the proteolytic activation of GPR, this result further supports an autoprocessing model for GPR activation during sporulation. The properties of these mutant proteins were also examined, both in vivo in B. subtilis spores and in Escherichia coli and in vitro with purified protein. SspC(v) interacted with DNA similarly to SspC(wt) in vivo, resorting UV and heat resistance to spores lacking major alpha/beta-type SASP to the same extent as SspC(wt). In contrasst, SspC(DL) had much less effect on DNA properties in vivo and bound strongly only to poly(dG) . poly(dC) in vitro; SspC(DLV) exhibited only weak binding to poly(dG).poly(dC) in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the binding of these proteins to spore DNA and alteration of DNA properties and show further that the GRP recognition region in alpha/beta-type SASP plays some role in DNA binding.

Amino Acid Sequence↗
PubMed1994

The zymogen of the protease that degrades small, acid-soluble proteins of spores of Bacillus species can rapidly autoprocess to the active enzyme in vitro.

The zymogen of the protease (GPR) that initiates protein degradation during spore germination in Bacillus species is not activated in vitro under normal physiological conditions. However, there is rapid, acid-pH-dependent, zero-order, proteolytic activation of the purified zymogen in high concentrations of dimethyl sulfoxide. These findings provide further evidence that GPR activates itself during sporulation.

Bacillus↗
PubMed1994

Autoprocessing of the protease that degrades small, acid-soluble proteins of spores of Bacillus species is triggered by low pH, dehydration, and dipicolinic acid.

The sequence-specific protease (termed GPR) that degrades small, acid-soluble proteins (SASP) during germination of spores of Bacillus species is synthesized during sporulation as an inactive precursor termed P46. Approximately 2 h later in sporulation, P46 is converted proteolytically to a smaller form, termed P41, which is active in vitro, but which does not act significantly on SASP in vivo until spore germination is initiated. While it appears likely that P46-->P41 conversion is an autoprocessing event, the mechanisms regulating P46-->P41 conversion in vivo are not clear. In this work we found that P46-->P41 conversion in vitro was stimulated tremendously in an allosteric manner by pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus Ca2+ but not by Ca2+ in combination with a variety of DPA analogs. The processing reaction stimulated by Ca(2+)-DPA was seen at pH 5.1 but not at pH 6.2 or 7, and under these conditions P46-->P41 conversion exhibited a linear time course and was a first-order reaction, indicative of an intramolecular autoprocessing reaction. At pH 5.1, P46-->P41 conversion was stimulated markedly by very high ionic strength. At pHs from 5.1 to 6.6, P46-->P41 conversion also occurred when P46 was dehydrated to approximately 54% relative humidity. This processing was stimulated markedly when dehydration was carried out in the presence of DPA and NaCl but was greatly decreased when dehydration was to 10, 33, or 75% relative humidity. Since previous work has shown that P(46)-->P(41) processing in vivo takes place (i) after a fall in forespore pH to 6.3 to 6.9 and approximately in parallel with (ii) DPA accumulation by the forespore and (iii) dehydration of the forespore, out new finings in vitro suggest that these three changes may synergistically trigger P(46)-->P(41) autoprocessing in the developing forespore. Presumably the conditions in vivo during this authoprocessing preclude significant attack of the P(41) generated on its SASP substrates.

Acids↗
PubMed1994

Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4.

The gene encoding penicillin-binding protein 4 (PBP 4) of Bacillus subtilis, pbpD, was cloned by two independent methods. PBP 4 was purified, and the amino acid sequence of a cyanogen bromide digestion product was used to design an oligonucleotide probe for identification of the gene. An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight PBPs also identified this gene. DNA sequence analysis of the cloned DNA revealed that (i) the amino acid sequence of PBP 4 was similar to those of other class A high-molecular-weight PBPs and (ii) pbpD appeared to be cotranscribed with a downstream gene (termed orf2) of unknown function. The orf2 gene is followed by an apparent non-protein-coding region which exhibits nucleotide sequence similarity with at least two other regions of the chromosome and which has a high potential for secondary structure formation. Mutations in pbpD resulted in the disappearance of PBP 4 but had no obvious effect on growth, cell division, sporulation, spore heat resistance, or spore germination. Expression of a transcriptional fusion of pbpD to lacZ increased throughout growth, decreased during sporulation, and was induced approximately 45 min into spore germination. A single transcription start site was detected just upstream of pbpD. The pbpD locus was mapped to the 275 to 280 degrees region of the chromosomal genetic map.

Amino Acid Sequence↗
PubMed1994

Heat inactivation of Bacillus subtilis spores lacking small, acid-soluble spore proteins is accompanied by generation of abasic sites in spore DNA.

Previous work has shown that lethal heat treatment of Bacillus subtilis spores lacking the major DNA-binding proteins SASP-alpha and -beta (alpha-beta- spores) causes significant DNA damage, including many single-strand breaks. In this work we have used a reagent specific for aldehydes present in abasic sites in DNA to show that DNA from wild-type spores killed by heat treatment to levels of < 0.05% survival had at most two aldehydes (i.e., abasic sites) per 10(4) nucleotides, while DNA from alpha(-)beta- spores killed to similar levels had 7 to 20 times as many abasic sites per 10(4) nucleotides. These data were generally consistent with the level of single-strand breaks in DNA from these heated spores and strongly suggest that a major mechanism responsible for the heat killing of alpha(-)beta- (but not wild-type) spores is DNA depurination followed by strand breakage at the resultant abasic site. In contrast, hydrogen peroxide killing of alpha(-)beta - spores was not accompanied by generation of a high level of DNA aldehydes.

Bacillus subtilis↗