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P Setlow

Publications and source records attributed to P Setlow.

At least 37 records · Page 2Linked to original sources
PubMed2002

Studies on the mechanisms of the sporicidal action of ortho-phthalaldehyde.

AIMS: To determine the mechanism of killing of spores of Bacillus subtilis by ortho-phthalaldehyde (OPA), an aromatic dialdehyde currently in use as an antimicrobial agent. METHODS AND RESULTS: OPA is sporicidal, although spores are much more OPA resistant than are vegetative cells. Bacillus subtilis mutants deficient in DNA repair, spore DNA protection and spore coat assembly have been used to show that (i) the coat appears to be a major component of spore OPA resistance, which is acquired late in sporulation of B. subtilis at the time of spore coat maturation, and (ii) B. subtilis spores are not killed by OPA through DNA damage but by elimination of spore germination. Furthermore, OPA-treated spores that cannot germinate are not recovered by artificial germinants or by treatment with NaOH or lysozyme. CONCLUSIONS: OPA appears to kill spores by blocking the spore germination process. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to, and spore killing by, the disinfectant, OPA.

Bacillus subtilis↗
PubMed2002

Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures.

AIMS: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition. METHODS AND RESULTS: Bacillus subtilis spores prepared at temperatures from 22 to 48 degrees C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures. As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures. Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox. However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures. In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased. However, this latter change was not due to a reduction in cotA gene expression at higher temperatures. CONCLUSIONS: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The precise conditions for the formation of B. subtilis spores have a large effect on many spore properties.

Bacillus subtilis↗
PubMed2002

Studies on the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide.

AIMS: To determine the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide. METHODS AND RESULTS: Killing of spores of B. subtilis with hydrogen peroxide caused no release of dipicolinic acid (DPA) and hydrogen peroxide-killed spores were not appreciably sensitized for DPA release upon a subsequent heat treatment. Hydrogen peroxide-killed spores appeared to initiate germination normally, released DPA and hydrolysed significant amounts of their cortex. However, the germinated killed spores did not swell, did not accumulate ATP or reduced flavin mononucleotide and the cores of these germinated spores were not accessible to nucleic acid stains. CONCLUSIONS: These data indicate that treatment with hydrogen peroxide results in spores in which the core cannot swell properly during spore germination. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide further information on the mechanism of killing of spores of Bacillus species by hydrogen peroxide.

Adenosine Triphosphate↗
PubMed2001

Resistance of spores of Bacillus species to ultraviolet light.

Dormant spores of the various Bacillus species, including B. subtilis, are 5 to 50 times more resistant to UV radiation than are the corresponding growing cells. This elevated spore UV resistance is due to: a) the photochemistry of DNA within spores, as UV generates few if any cyclobutane dimers, but rather a photoproduct (Fig. 1) called spore photoproduct (SP; 5-thyminyl-5,6-dihydrothymine); and b) DNA repair, in particular SP-specific repair, during spore germination. The novel UV photochemistry of spore DNA is largely due to its saturation with a group of small, acid-soluble proteins (SASP), which are unique to spores and whose binding alters the DNA conformation and thus its photochemistry. SP-specific repair is also unique to spores and is carried out by a light-independent SP-lyase, an iron-sulfur protein that utilizes S-adenosylmethionine to catalyze SP monomerization without DNA backbone cleavage.

Amino Acid Sequence↗
PubMed2001

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.

Bacillus subtilis↗
PubMed2001

A cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus is actually a broad specificity phosphatase.

The distribution of phosphoglycerate mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor-dependent and a cofactor-independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor-independent PGM, genes homologous to those encoding cofactor-dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity. Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor-dependent PGM homologs from B. stearothermophilus and B. subtilis, and show that these enzymes are phosphatases with broad substrate specificity. Homologs from other gram-positive bacteria are also likely to possess phosphatase activity. These studies clearly show that the exploration of genomic sequences through three-dimensional modeling is capable of producing useful predictions regarding function. However, significant methodological improvements will be needed before such analysis can be carried out automatically.

Amino Acid Sequence↗
PubMed2001

Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores.

Dormant Bacillus subtilis spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the gerA family of operons, of which the gerB operon is a member. The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane. In this study we have used Western blot analysis to determine the precise location of GerBA, a gerB-encoded receptor protein, in various spore fractions. GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles. GerBA was also solubilized from the P100 fraction with detergent but not with high salt. These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane. Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane. Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore. We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of gerB operon expression and with the putative receptor function of the proteins encoded by the gerB operon.

Bacillus subtilis↗
PubMed2001

Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate.

Dormant Bacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA). Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown. As Ca(2+)-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination. We found that Ca(2+)-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core. Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex. Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca(2+)-DPA. Our findings thus define a mechanism for Ca(2+)-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.

Amidohydrolases↗
PubMed2001

Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination.

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.

Bacillus subtilis↗
PubMed2001

Heat shock proteins do not influence wet heat resistance of Bacillus subtilis spores.

Spores of Bacillus subtilis are significantly more resistant to wet heat than are their vegetative cell counterparts. Analysis of the effects of mutations in and the expression of fusions of a coding gene for a thermostable beta-galactosidase to a number of heat shock genes has shown that heat shock proteins play no significant role in the wet heat resistance of B. subtilis spores.

Bacillus subtilis↗
PubMed2001

An alpha/beta-type, small, acid-soluble spore protein which has very high affinity for DNA prevents outgrowth of Bacillus subtilis spores.

A derivative of SspC, a minor alpha/beta-type, small, acid-soluble spore protein (SASP) from Bacillus subtilis, was generated that has a very high affinity for DNA. This protein (SspC(Delta11-D13K)) was able to confer UV resistance on spores lacking alpha/beta-type SASP, and spores with SspC(Delta11-D13K) triggered germination normally. However, SspC(Delta11-D13K) blocked outgrowth of > or = 90% of germinated spores, and SspC(Delta11-D13K) persisted in these germinated spores, whereas wild-type SspC was almost completely degraded. The outgrowth phenotype of spores with SspC(Delta11-D13K) is proposed to be due to the high stability of the SspC(Delta11-D13K)-DNA complex, which prevents rapid degradation of this alpha/beta-type SASP early in germination. The persistence of this protein on spore DNA then interferes with transcription during spore outgrowth.

Amino Acid Sequence↗
PubMed2000

Equilibrium and kinetic binding interactions between DNA and a group of novel, nonspecific DNA-binding proteins from spores of Bacillus and Clostridium species.

Binding of alpha/beta-type small acid-soluble spore proteins (SASP) is the major determinant of DNA resistance to damage caused by UV radiation, heat, and oxidizing agents in spores of Bacillus and Clostridium species. Analysis of several alpha/beta-type SASP showed that these proteins have essentially no secondary structure in the absence of DNA, but become significantly alpha-helical upon binding to double-stranded DNAs or oligonucleotides. Folding of alpha/beta-type SASP induced by a variety of DNAs and oligonucleotides was measured by CD spectroscopy, and this allowed determination of a DNA binding site size of 4 base pairs as well as equilibrium binding parameters of the alpha/beta-type SASP-DNA interaction. Analysis of the equilibrium binding data further allowed determination of both intrinsic binding constants (K) and cooperativity factors (omega), as the alpha/beta-type SASP-DNA interaction was significantly cooperative, with the degree of cooperativity depending on both the bound DNA and the salt concentration. Kinetic analysis of the interaction of one alpha/beta-type SASP, SspC(Tyr), with DNA indicated that each binding event involves the dimerization of SspC(Tyr) monomers at a DNA binding site. The implications of these findings for the structure of the alpha/beta-type SASP.DNA complex and the physiology of alpha/beta-type SASP degradation during spore germination are discussed.

Bacillus↗
PubMed2000

N-terminal amino acid residues mediate protein-protein interactions between DNA-bound alpha /beta -type small, acid-soluble spore proteins from Bacillus species.

The binding of alpha/beta-type small, acid-soluble spore proteins (SASP) to DNA of spores of Bacillus species is the major determinant of DNA resistance to a variety of damaging treatments. The primary sequence of alpha/beta-type SASP is highly conserved; however, the N-terminal third of these proteins is less well conserved than the C-terminal two-thirds. To determine the functional importance of residues in the N-terminal region of alpha/beta-type SASP, variants of SspC (a minor alpha/beta-type SASP from Bacillus subtilis) with modified N termini were generated and their structural and DNA binding properties studied in vitro and in vivo. SspC variants with deletions of up to 14 residues ( approximately 20% of SspC residues) were able to bind DNA in vitro and adopted similar conformations when bound to DNA, as determined by circular dichroism spectroscopy and protein-protein cross-linking. Progressive deletion of up to 11 N-terminal residues resulted in proteins with progressively lower DNA binding affinity. However, SspC(Delta)(14) (in which 14 N-terminal residues have been deleted) showed significantly higher affinity for DNA than the larger proteins, SspC(Delta)(10) and SspC(Delta)(11). The affinity of these proteins for DNA was shown to be largely dependent upon the charge of the first few N-terminal residues. These results are interpreted in the context of a model for DNA-dependent alpha/beta-type SASP protein-protein interaction involving the N-terminal regions of these proteins.

Acids↗
PubMed2000

Mechanism of catalysis of the cofactor-independent phosphoglycerate mutase from Bacillus stearothermophilus. Crystal structure of the complex with 2-phosphoglycerate.

The structure of the complex between the 2, 3-diphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus and its 3-phosphoglycerate substrate has recently been solved, and analysis of this structure allowed formulation of a mechanism for iPGM catalysis. In order to obtain further evidence for this mechanism, we have solved the structure of this iPGM complexed with 2-phosphoglycerate and two Mn(2+) ions at 1. 7-A resolution. The structure consists of two different domains connected by two loops and interacting through a network of hydrogen bonds. This structure is consistent with the proposed mechanism for iPGM catalysis, with the two main steps in catalysis being a phosphatase reaction removing the phosphate from 2- or 3-phosphoglycerate, generating an enzyme-bound phosphoserine intermediate, followed by a phosphotransferase reaction as the phosphate is transferred from the enzyme back to the glycerate moiety. The structure also allowed the assignment of the function of the two domains of the enzyme, one of which participates in the phosphatase reaction and formation of the phosphoserine enzyme intermediate, with the other involved in the phosphotransferase reaction regenerating phosphoglycerate. Significant structural similarity has also been found between the active site of the iPGM domain catalyzing the phosphatase reaction and Escherichia coli alkaline phosphatase.

Amino Acid Sequence↗
PubMed2000

Lack of a significant role for the PerR regulator in Bacillus subtilis spore resistance.

Bacillus subtilis cells lacking the PerR repressor which regulates transcription of genes encoding oxidative stress protective proteins grew at 30-50% the rate of wild-type cells, and perR cultures accumulated rapidly growing suppressor mutants lacking the catalase whose expression is regulated by PerR. However, perR spores which retained the perR regulated catalase were obtained on plates. These perR spores had levels of oxidative stress protective proteins from 7- to 50-fold higher than those in wild-type spores, but perR spore resistance to heat, hydrogen peroxide and t-butyl hydroperoxide was essentially identical to that of wild-type spores, indicating that elevated levels of proteins that protect growing cells from oxidizing agents play no role in dormant spore resistance to these compounds. However, germinated perR spores were much more resistant to alkyl hydroperoxides than were wild-type spores.

Bacillus subtilis↗
PubMed2000

Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation.

The DNA in the core of spores of Bacillus species is saturated with a group of small, acid-soluble proteins (SASP) that protect DNA from a variety of harsh treatments and play a major role in spore resistance and long-term spore survival. During spore germination, SASPs are rapidly degraded to amino acids and this degradation is initiated by a sequence-specific protease called germination protease (GPR), which exhibits no obvious mechanistic or amino acid sequence similarity to any known class of proteases. GPR is synthesized during sporulation as an inactive tetrameric zymogen termed P(46), which later autoprocesses to a smaller form termed P(41), which is active only during spore germination. Here, we report the crystal structure of P(46) from Bacillus megaterium at 3.0 A resolution and the fact that P(46) monomer adopts a novel fold. The asymmetric unit contains two P(46) monomers and the functional tetramer is a dimer of dimers, with an approximately 9 A channel in the center of the tetramer. Analysis of the P(46) structure and site-directed mutagenesis studies have provided some insight into the mechanism of zymogen activation as well as the zymogen's lack of activity and the inactivity of P(41) in the mature spore.

Amino Acid Sequence↗
PubMed2000

Analysis of the regulation and function of five genes encoding small, acid-soluble spore proteins of Bacillus subtilis.

Four genes [sspI, sspK, sspM, and sspO (originally called cotK)] encoding minor small, acid-soluble proteins (SASP) unique to spores of Bacillus subtilis are expressed only in the forespore compartment of sporulating cells of this organism. The sspI, sspK and sspM genes are monocistronic, while sspO is the first gene in a likely operon with sspP (originally called cotL), which also encodes a putative very small protein. Transcription of these genes is primarily, if not exclusively, by RNA polymerase with the forespore-specific sigma factor, sigma(G). Sequences centered 10 and 35nt upstream of the 5'-ends of sspI, sspK, sspM and sspO also show homology to the -10 and -35 sequences recognized by sigma(G). Mutations deleting these genes cause the loss of the appropriate SASP from spores, and the sspK, sspM and sspO (and likely sspP) mutations had no discernable effect on sporulation, spore properties or spore germination. Loss of sspI also had no effect on sporulation, spore properties or spore germination, but DeltasspI spores had a significant defect in spore outgrowth.

Bacillus subtilis↗