Biomedical subjects

P Setlow

Publications and source records attributed to P Setlow.

At least 109 records · Page 6Linked to original sources

PubMed1994

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation.

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.

Bacillus megaterium↗

PubMed1993

Manganese(II) activation of 3-phosphoglycerate mutase of Bacillus megaterium: pH-sensitive interconversion of active and inactive forms.

The effects of manganese(II) ions and of pH were studied on 3-p-glycerate mutase purified from Bacillus megaterium. Mn2+ ions converted the enzyme within a few minutes from a catalytically inactive form to one that was catalytically active even after Mn2+ had been removed. The enzyme reverted over 60-90 min to the inactive form, from which further activation-deactivation cycles could be elicited. The slow, temperature-dependent, activation, and deactivation is suggestive of change in protein conformation. No other metal ion was found that activated more than 4% as much as Mn2+. Activation by Mn2+ was strongly pH-dependent in the physiological pH range, consistent with displacement of 2 H+. Together with the pH dependence of the catalytic activity itself, the system displayed pronounced pH sensitivity in the pH range 6.5-8.0. The findings suggest that pH changes, documented for forming and germinating spores of B. megaterium, can account for much of the mutase control associated with accumulation and later utilization of 3-phosphoglycerate depots.

Bacillus megaterium↗

PubMed1993

Binding of small, acid-soluble spore proteins to DNA plays a significant role in the resistance of Bacillus subtilis spores to hydrogen peroxide.

Dormant spores of Bacillus subtilis which lack the majority of the alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha- beta- spores) that coat the DNA in wild-type spores are significantly more sensitive to hydrogen peroxide than are wild-type spores. Hydrogen peroxide treatment of alpha- beta- spores causes DNA strand breaks more readily than does comparable treatment of wild-type spores, and alpha- beta- spores, but not wild-type spores, which survive hydrogen peroxide treatment have acquired a significant number of mutations. The hydrogen peroxide resistance of wild-type spores appears to be acquired in at least two incremental steps during sporulation. The first increment is acquired at about the time of alpha/beta-type SASP synthesis, and the second increment is acquired approximately 2 h later, at about the time of dipicolinic acid accumulation. During sporulation of the alpha- beta- strain, only the second increment of hydrogen peroxide resistance is acquired. In contrast, sporulation mutants which accumulate alpha/beta-type SASP but progress no further in sporulation acquire only the first increment of hydrogen peroxide resistance. These findings strongly suggest that binding of alpha/beta-type SASP to DNA provides one increment of spore hydrogen peroxide resistance. Indeed, binding of alpha/beta-type SASP to DNA in vitro provides strong protection against cleavage of DNA by hydrogen peroxide.

Bacillus subtilis↗

PubMed1993

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase.

Penicillin-binding protein 4* (PBP 4*) was purified from Bacillus subtilis, its N-terminal sequence was determined, and the coding gene, termed pbpE, was cloned and sequenced. The predicted amino acid sequence of PBP 4* exhibited similarity to those of other penicillin-recognizing enzymes. Downstream of pbpE there was a second gene, termed orf2, which exhibited sequence similarity with aspartate racemase. The two genes were found to constitute an operon adjacent to and divergently transcribed from the sacB gene at 296 degrees on the chromosomal map. A weak beta-lactamase activity was associated with PBP 4*, but no enzymatic activity was found for the product of orf2. Mutation of pbpE, orf2, or both genes resulted in no observable effect on growth, sporulation, spore heat resistance, or spore germination. A translational pbpE-lacZ fusion was weakly expressed during vegetative growth and was significantly induced at the onset of sporulation. This induction depended on the activity of the spo0A product in relieving repression by the abrB repressor. A single transcription start site which was apparently dependent on E sigma A was detected upstream of pbpE.

Amino Acid Isomerases↗

PubMed1993

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpF gene, which codes for a putative class A high-molecular-weight penicillin-binding protein.

The partial nucleotide sequence of a gene encoding a Bacillus subtilis homolog to the Escherichia coli ponA gene, encoding penicillin-binding protein 1A, was previously reported. The remaining part of this gene, termed pbpF, was isolated, and its nucleotide sequence was completed. Deletion of this gene did not alter the profile of B. subtilis penicillin-binding proteins observed after gel electrophoresis and resulted in no observable phenotype. A transcriptional pbpF-lacZ fusion was weakly expressed during vegetative growth. Expression diminished during the first hours of sporulation but was slightly induced in the forespore compartment during late sporulation. This sporulation expression was dependent on spoIIIG, which encodes the forespore-specific transcription factor sigma G. A single transcription start site which was apparently directly dependent on E sigma A was detected in vegetative cells.

Amino Acid Sequence↗

PubMed1993

Cloning and nucleotide sequence of a plasmid-carried gene coding for a minor small, acid-soluble protein from Bacillus megaterium spores.

The gene (termed sspG) coding for a small, acid-soluble protein (SASP) from spores of Bacillus megaterium QMB1551, termed SASP-G, has been cloned, and its nucleotide sequence has been determined. SASP-G is a 42-residue protein containing 2 tryptophan and 11 lysine residues, including a hexalysine sequence, and is not homologous to any previously described SASP. The sspG gene appears to be an additional member of the sigma G regulon. No gene homologous to sspG is present in B. cereus T or B. subtilis 168. The reason for the absence of sspG from other Bacillus species appears to be that in B. megaterium, sspG is present only on a 111-kb plasmid; this plasmid is not present in B. cereus T or B. subtilis 168. The sspG gene is the first forespore-expressed gene found to be on a plasmid.

Amino Acid Sequence↗

PubMed1993

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.

Bacillus subtilis↗

PubMed1993

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis.

A number of Bacillus subtilis genes involved in NAD biosynthesis have been cloned and sequenced. One of the genes encodes a polypeptide homologous to Escherichia coli L-aspartate oxidase, and its mutation resulted in a nicotinic acid (Nic)-dependent phenotype; this gene was termed nadB. A second open reading frame (orf2) was found downstream of nadB, and an insertional plasmid separating orf2 and nadB also gave a Nic-dependent phenotype. This result suggests that orf2 may also be involved in NAD biosynthesis and that nadB and orf2 are in the same operon. Upstream of nadB was a third gene, transcribed in the opposite direction to that of nadB-orf2. The amino acid sequence derived from the third gene was quite similar to those derived from nifS genes of various nitrogen-fixing bacteria; therefore, the third gene was termed nifS. As with nadB and orf2, mutations in nifS also resulted in a Nic-dependent phenotype. The promoter regions of nadB and nifS overlapped each other and both contained -10 and -35 sequences which resemble those of E sigma A-type promoters. Transcription from both the nifS and nadB promoters, as well as expression of a nadB-lacZ fusion, was repressed by Nic. However, nadB transcription and nadB-lacZ expression were decreased, at most, only slightly by a deletion in nifS. The possible role of the nifS gene product in NAD biosynthesis is discussed.

Amino Acid Oxidoreductases↗

PubMed1993

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase.

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.

Amino Acid Sequence↗

PubMed1993

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

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.

Amino Acid Sequence↗

PubMed1993

The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked.

Determination by amino acid analyses of the percentage of diaminopimelic acid in the spore cortex of Bacillus megaterium and Bacillus subtilis which is involved in interpeptide cross-links gave values of 31 to 37%. This finding supports the idea that the cortex volume could undergo significant changes in response to changes in pH or ionic strength and could thus play an active role in reducing the water content of the spore protoplast during sporulation.

Amino Acids↗

PubMed1993

Levels of small molecules in dormant spores of Sporosarcina species and comparison with levels in spores of Bacillus and Clostridium species.

Dormant spores of Sporosarcina halophila and Sporosarcina ureae contained no detectable ATP, significant levels of ADP, even higher levels of AMP, and a large pool of 3-phosphoglyceric acid, similar to what is found in dormant spores of Bacillus and Clostridium species. Sporosarcina halophila and S. ureae spores also contained significant pools of free amino acids, in particular glutamic acid, as in the case with spores of Bacillus but not Clostridium species. Levels of monovalent and divalent inorganic cations were comparable in spores of Sporosarcina, Clostridium, and Bacillus species, and cation levels in spores of the slight halophile S. halophila were similar to those in S. ureae spores. These data suggest that levels of small molecules are generally similar in spores of all Gram-positive organisms, and further suggest that these levels reflect fundamental and conserved features of the sporulation process and dormant spores in these organisms. The data are also consistent with the proposed close evolutionary relationship between Bacillus and Sporosarcina species.

Adenine Nucleotides↗

PubMed1992

Synthesis and characterization of a 29-amino acid residue DNA-binding peptide derived from alpha/beta-type small, acid-soluble spore proteins (SASP) of bacteria.

A 29-amino acid residue peptide (SASP-peptide) derived from the sequence of the putative DNA-contacting portion at the carboxyl terminus of an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis has been synthesized by automated solid-phase methods and tested for its ability to interact with DNA. Circular dichroism (CD) spectroscopy reveals an interaction between this SASP-peptide and DNA, both by an increase in alpha-helix content of the peptide (which alone has a mostly random conformation) and by enhancement of the 275-nm CD band of the DNA. In contrast to results with intact alpha/beta-type SASP, however, the peptide does not induce a B----A conformational transition in the DNA. The SASP-peptide also binds to poly(dG).poly(dC) and protects this polynucleotide against DNase I digestion and UV light-induced cytosine dimer formation, parallel to findings made previously with native alpha/beta-type SASP. The results confirm the hypothesis that the carboxyl-terminal region of the alpha/beta-type SASP directly contacts DNA and possesses some, but not all, of the functional characteristics of the intact molecule.

Amino Acid Sequence↗

PubMed1992

DNA in dormant spores of Bacillus species is in an A-like conformation.

The DNA in dormant spores of Bacillus species is associated with alpha/beta-type small, acid-soluble proteins (SASP), which are double-stranded DNA-binding proteins whose amino acid sequence has been highly conserved in evolution. In vitro these proteins bind most strongly to DNA which readily adopts an A-like conformation, as binding of alpha/beta-type SASP causes DNA to assume an A-like conformation. As predicted by this conformational change in DNA, binding of alpha/beta-type SASP to relaxed but covalently closed plasmid DNA results in the introduction of a large number of negative supercoils. Associated with the conformational change in DNA brought about by alpha/beta-type SASP binding is a change in its photochemistry such that ultraviolet irradiation does not generate pyrimidine dimers, but rather a thyminyl-thymine adduct termed spore photoproduct (SP). The latter two properties of DNA complexed with alpha/beta-type SASP in vitro are similar to those of DNA in dormant spores of Bacillus species in which: (i) plasmid DNA has a much higher number of negative supercoils than plasmid in growing cells; and (ii) ultraviolet irradiation produces SP and no pyrimidine dimers, while only pyrimidine dimers are formed in growing cells. During sporulation these changes in the properties of spore DNA take place in parallel with synthesis of alpha/beta-type SASP, and the magnitude of the changes is greatly reduced in mutants that make low amounts of these proteins. A straightforward interpretation of these data is that DNA in dormant spores of Bacillus species is in an A-like conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Identification of Bacillus subtilis genes for septum placement and shape determination.

The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells. We report the cloning of the wild-type allele of divIVB1 and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD. Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E. coli shape-determining genes mreB, mreC, and mreD. The B. subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodB1) that causes the production of aberrantly shaped cells under restrictive conditions. Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon. The possible involvement of min gene products in the switch from medial to polar placement of the septum during sporulation is discussed.

Amino Acid Sequence↗

PubMed1992

Properties of purified sporlets produced by spoII mutants of Bacillus subtilis.

A number of abortively disporic spoII mutants of Bacillus subtilis released their forespore compartments (termed stage II sporlets) after mother cell lysis during sporulation in nutrient exhaustion or resuspension media. Stage II sporlets were viable and contained levels of ATP and a number of enzymes similar to those in cells 2 to 3 h after sporulation. However, stage II sporlets carried out essentially no macromolecular synthesis, a result suggesting that they were in a quiescent state. The nucleoid of these quiescent stage II sporlets was significantly condensed relative to that in the original vegetative cells, as was previously found to take place 1 to 2 h after initiation of sporulation (B. Setlow, N. Magill, P. Febbroriello, L. Nakhimousky, D. E. Koppel, and P. Setlow, J. Bacteriol. 173:6270-6278, 1991). Stage II sporlets may be a useful model system for analysis of forespore properties early in stage II of sporulation.

Bacillus subtilis↗

PubMed1992

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.

Adenosine Triphosphate↗

PubMed1992

Interaction between DNA and alpha/beta-type small, acid-soluble spore proteins: a new class of DNA-binding protein.

DNA in spores of Bacillus and Clostridium species is associated with small, acid-soluble proteins (SASP) of the alpha/beta type; the presence of these proteins is a major factor in causing spore resistance to UV light, alpha/beta-type SASP did not bind to single-stranded DNA, single- or double-stranded RNA, or DNA-RNA hybrids in vitro. However, these proteins bound a variety of double-stranded DNAs and conferred protection against DNase cleavage. The binding of alpha/beta-type SASP to DNA saturated at a protein/DNA ratio (wt/wt) of 4:1 to 5:1, which is approximately 1 SASP per 4 bp. alpha/beta-type SASP-DNA interaction did not require divalent cations, was independent of pH between 6 and 8, and, for some SASP-DNA pairs, was relatively insensitive to salt up to 0.3 M. The relative affinity of alpha/beta-type SASP for different DNAs was poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than plasmid pUC19 greater than poly(dA-dT).poly(dA-dT), with poly(dA).poly(dT) giving no detectable binding. This order in alpha/beta-type SASP-DNA affinities parallels the facility with which the DNAs adopt an A-like conformation, the conformation in alpha/beta-type SASP-DNA complexes. An oligo(dG).oligo(dC) of 12 bp was bound by alpha/beta-type SASP. While a 26-bp oligo(dG).oligo(dC) bound more tightly than the 12-mer, there was no significant increase in affinity for alpha/beta-type SASP with further increase in size of oligo(dG).oligo(dC). In contrast, binding of alpha/beta-type SASP to oligo(dA-dT).oligo(dA-dT) was minimal up to at least a 70-mer, and binding to poly(dA-dT).poly(dA-dT) was very cooperative. In addition to blocking DNase digestion, binding of alpha/beta-type SASP to DNA blocked (i) cleavage of the DNA backbone by hydroxyl radicals and orthophenanthroline-Cu2+, (ii) DNA cleavage by restriction enzymes, in particular those with specificity for GC-rich sequences; and (iii) in vitro transcription of some but not all genes. However, methylation of dG residues by dimethyl sulfate was not affected by alpha/beta-type SASP binding.

Bacterial Proteins↗