Biomedical subjects

P Setlow

Publications and source records attributed to P Setlow.

At least 181 records · Page 10Linked to original sources

PubMed1986

Determination of the chromosomal locations of four Bacillus subtilis genes which code for a family of small, acid-soluble spore proteins.

The chromosomal locations of four genes which code for small, acid-soluble spore proteins (SASP) in Bacillus subtilis have been determined. Although these four genes code for extremely homologous small, acid-soluble spore proteins (greater than 65% sequence identity), the genes are not clustered but are located at 70 degrees (adjacent to glyB [sspB gene]), 115 degrees (between metC and pyr cluster [sspD gene]), 180 degrees (between metB and kauA [sspC gene]), and 260 degrees (between ilvC and aroG [sspA gene]) on the B. subtilis genetic map.

Bacillus subtilis↗

PubMed1986

Cloning and nucleotide sequencing of genes for three small, acid-soluble proteins from Bacillus subtilis spores.

Three Bacillus subtilis genes (termed sspA, sspB, and sspD) which code for small, acid-soluble spore proteins (SASPs) have been cloned, and their complete nucleotide sequence has been determined. The amino acid sequences of the SASPs coded for by these genes are similar to each other and to those of the SASP-1 of B. subtilis (coded for by the sspC gene) and the SASP-A/C family of B. megaterium. The sspA and sspB genes are expressed only in sporulation, in parallel with each other and with the sspC gene. Two regions upstream of the postulated transcription start sites for the sspA and B genes have significant homology with the analogous regions of the sspC gene and the SASP-A/C gene family. Purification of two of the three major B, subtilis SASPs (alpha and beta) and determination of their amino-terminal sequences indicated that the sspA gene codes for SASP-alpha and that the sspB gene codes for SASP-beta. This was confirmed by the introduction of deletion mutations into the cloned sspA and sspB genes and transfer of these deletions into the B. subtilis chromosome with concomitant loss of the wild-type gene.

Amino Acid Sequence↗

PubMed1986

Cloning and nucleotide sequencing of genes for small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus".

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore germination and which reacted with antibodies raised against B. megaterium SASP. Genes coding for a B. stearothermophilus and a "T. thalpophilus" SASP as well as for two B. cereus SASP were cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus." This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than simply amino acid storage.

Amino Acid Sequence↗

PubMed1986

Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light.

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.

Bacillus subtilis↗

PubMed1986

Cloning and nucleotide sequence of the Bacillus megaterium gene coding for small, acid-soluble spore protein B.

The Bacillus megaterium gene coding for small, acid-soluble spore protein (SASP) B was cloned and its nucleotide sequence was determined. The amino acid sequence predicted from the DNA sequence was identical to that determined previously for SASP B, with the exception of the amino-terminal methionine predicted from the gene sequence which is presumably removed posttranslationally and an asparagine residue predicted at position 21 which was originally identified as an aspartate residue. The mRNA encoded by the SASP B gene is synthesized for only a discrete period midway in sporulation, in parallel with mRNAs coding for other SASPs. The small size of the SASP B mRNA (365 nucleotides) indicated that the mRNA is monocistronic. The SASP B gene itself hybridized strongly to only one band in Southern blots of restriction enzyme digests of B. megaterium DNA, suggesting that the SASP B gene is not a member of a highly conserved multigene family, as is the case for other SASP genes.

Amino Acid Sequence↗

PubMed1985

Genes for Bacillus megaterium small, acid-soluble spore proteins: nucleotide sequence of two genes and their expression during sporulation.

The complete nucleotide (nt) sequence of two Bacillus megaterium genes coding for small, acid-soluble spore proteins (SASP), termed C-1 and C-2, has been determined. The nt sequences of the genes are greater than 98% identical in the coding regions, greater than 90% identical in approx. 180 bp and approx. 50 bp of upstream and downstream flanking sequences, respectively, and exhibit features conserved in related B. megaterium SASP genes. Northern blot analyses showed that the SASP-C-1 and/or C-2 genes are transcribed during sporulation in parallel with the related SASP-C and C-3 genes. The promoter regions of the SASP-C-1 and C-2 genes were localized, based on the sizes of their mRNAs and the positions of transcription termination sequences. The SASP-C-1 and C-2 genes' promoter regions exhibit significant homology with those for the SASP-C and C-3 genes.

Bacillus megaterium↗

PubMed1985

Cloning of a small, acid-soluble spore protein gene from Bacillus subtilis and determination of its complete nucleotide sequence.

The first Bacillus subtilis small, acid-soluble spore protein (SASP) gene has been cloned by using previously cloned B. megaterium SASP genes as DNA-DNA hybridization probes. Determination of the DNA sequence of the B. subtilis SASP gene showed that it codes for a 72-residue protein (termed SASP-1) containing a single spore protease cleavage site as well as other sequences conserved in Bacillus megaterium SASPs A, C, C-1, C-2, and C-3. The B. subtilis SASP-1 genes's coding sequence is preceded by a potential Bacillus ribosome-binding site, and is followed by a sequence that could form a stem-and-loop structure characteristic of transcription termination sites. Upstream from the coding sequence there are no obvious homologies with other B. subtilis sporulation genes, but similarities with B. megaterium SASP genes are evident. SASP-1 mRNA (290 bases long) is absent from vegetative cells, but appears midway in sporulation and then disappears. The cloned SASP-1 gene hybridizes to three bands other than the SASP-1 gene itself in EcoRI or HindIII digests of B. subtilis DNA. Presumably these other bands represent SASP genes related to the SASP-1 gene, and we have been able to detect at least three such proteins in B. subtilis spores.

Bacillus megaterium↗

PubMed1984

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.

Amino Acid Sequence↗

PubMed1984

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.

Antigens, Bacterial↗

PubMed1984

Expression of Bacillus megaterium and Bacillus subtilis small acid-soluble spore protein genes during stationary-phase growth of asporogenous B. subtilis mutants.

The small acid-soluble spore proteins alpha and beta were not detected during stationary-phase growth of asporogenous Bacillus subtilis mutants blocked in stages 0, II, or III, but mutants blocked in stages IV or V accumulated nearly wild-type levels of these small acid-soluble spore proteins. Similar results were obtained when production of Bacillus megaterium C protein (also a small acid-soluble spore protein), as well as alpha and beta, were monitored in these mutants containing a recombinant plasmid carrying the B. megaterium C protein gene. The only exception was a spo0H mutant which synthesized a small amount of C protein, but no alpha or beta.

Bacillus megaterium↗

PubMed1984

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.

Bacillus megaterium↗

PubMed1984

Complete nucleotide sequence and start sites for transcription and translation of the Bacillus megaterium protein C gene.

The nucleotide sequence of the Bacillus megaterium protein C gene, encompassing the coding region and 341 base pairs of flanking regions, has been determined. The gene codes for a 72-residue protein whose predicted amino acid sequence is identical to that previously determined for protein C with the exception of an amino-terminal methionine predicted from the gene sequence, but not found in the mature protein. The translational initiation codon is preceded by an 11-base pair sequence highly complementary to the 3' terminus of B. megaterium 16S rRNA. Protection against S1 nuclease digestion by hybridization of a protein C gene fragment to RNA containing high levels of protein C mRNA localized the transcription initiation site 108 base pairs upstream from the translation start site. Upstream from the transcription initiation site there are no obvious homologies with conserved regions of promoters for previously described B. subtilis vegetative or sporulation genes.

Amino Acid Sequence↗

PubMed1983

Cloning of the gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium.

The structural gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium, has been cloned in Escherichia coli. Expression of the C-protein gene in E. coli requires an external transcription promoter and prevention of termination of transcription prior to transcription of all or part of the sequence coding for the C protein. The gene for the C protein is within a 5-kilobase DNA fragment, but this fragment does not code for either of the other two major low molecular weight spore proteins, suggesting that the structural genes for these proteins are not tightly linked.

Bacillus megaterium↗

PubMed1983

Enzymatic activity of precursors of Bacillus megaterium spore protease.

The protease that initiates rapid proteolysis during germination of Bacillus megaterium spores is synthesized during sporulation as a 46,000-molecular-weight polypeptide (P46) and is processed later in sporulation to a 41,000-molecular-weight polypeptide (P41), which is converted to a 40,000-molecular-weight polypeptide (P40) early in spore germination. P40 is known to be both tetrameric and enzymatically active. In this work, we show that P46 and P41 are both tetrameric, but that only P41 is enzymatically active. The identification of a zymogen form (P46) of this protease explains in part the regulation of the activity of this enzyme.

Bacillus megaterium↗

PubMed1983

Purification and characterization of a Bacillus megaterium disulfide reductase specific for disulfides containing pantethine 4',4"-diphosphate.

An NADH-linked disulfide reductase specific for disulfides containing pantethine 4',4"-diphosphate moieties was purified 23,000-fold to homogeneity from spores of Bacillus megaterium. The enzyme had a native molecular weight of 122,000 with two apparently identical subunits, contained one molecule of flavin adenine dinucleotide per subunit, and was inhibited by the vicinal dithiol reagent arsenite. The enzyme was active only on disulfides containing pantethine 4',4"-diphosphate moieties, including pantethine 4',4"-diphosphate, oxidized coenzyme A, and coenzyme A in disulfide linkage to acyl carrier protein. However, the Km values for pantethine 4',4"-diphosphate and oxidized coenzyme A were 0.65 and 7.4 mM, respectively. The enzyme was at a low level in log-phase cells but increased up to 10-fold early in the stationary phase and had a similar specific activity in both the mother cell and the forespore compartment; the enzyme activity fell only slowly during spore germination and outgrowth. The enzyme was not detected in several eucaryotic sources and was present in at most a low level in a number of gram-negative bacteria. Surprisingly, the specific activity of this enzyme varied more than 200-fold in extracts from different Bacillus species, with values in B. subtilis being 5- to 6-fold lower and values in B. cereus and B. sphaericus being 8- and 35-fold higher, respectively, than the maximum value in B. megaterium. However, the high specific activity in B. sphaericus did not represent more enzyme protein than in B. megaterium. The possible function of this newly discovered enzyme is discussed.

Bacillus↗

PubMed1983

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.

Bacillus megaterium↗

PubMed1983

Cyclic AMP is not detectable in Clostridium perfringens.

Cyclic AMP was not detected (less than 5 X 10(-9) M intracellular concentration) at any stage of growth or sporulation of two strains of Clostridium perfringens grown with or without methylxanthines. Only Bacillus and Lactobacillus, genera belonging to the same phylogenetic cluster, have previously exhibited undetectable levels of cyclic AMP.

Clostridium perfringens↗

PubMed1982

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.

Antigen-Antibody Complex↗