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Biomedical subjects

P Setlow

Publications and source records attributed to P Setlow.

At least 163 records · Page 9Linked to original sources
PubMed1989

The expression of a highly expressed Bacillus subtilis gene is not reduced by introduction of multiple codons normally not present in such genes.

Four serine or threonine codons were introduced into a highly expressed Bacillus subtilis gene. The introduced codons were ones either common in highly expressed B. subtilis genes, or never used in such genes. Strikingly, the level and rate of expression of the modified genes containing either type of extra codons was identical. This suggests that in B. subtilis codon usage patterns may play little or no role in effecting the level of gene expression.

Bacillus subtilis↗
PubMed1989

Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.

During sporulation of Bacillus subtilis, two identical genomes segregate in two compartments, the forespore and mother cell. These genomes are expressed differentially, with some genes such as sspE turned on only in the forespore. In vitro transcription of sspE was obtained only with RNA polymerase extracted from sporulating cells. Fractionation of factors associated with this enzyme and reconstitution with core RNA polymerase from vegetative cells generated an enzyme accurately transcribing sspE in vitro and led to purification of a polypeptide with the amino-terminal sequence of the spoIIIG product. Inactivation of spoIIIG abolished expression of sspE and five other forespore-specific genes, whereas synthesis of the spoIIIG product in vegetative cells rapidly turned these genes on. Therefore, spoIIIG encodes a sigma-factor, sigma G, which controls the expression of multiple genes in the forespore compartment.

Amino Acids↗
PubMed1989

Evidence for an additional temporal class of gene expression in the forespore compartment of sporulating Bacillus subtilis.

We present evidence indicating that the previously studied, sporulation-induced gene 0.3 kb, which encodes a stable RNA present at late developmental stages, is transcribed in the forespore chamber of sporulating cells of Bacillus subtilis. Compartmentalized gene expression was demonstrated on the basis of subcellular fractionation experiments in which severalfold-higher levels of 0.3 kb-directed beta-galactosidase specific activity were observed in forespore extracts than in extracts from the mother cell and dependence studies in which 0.3 kb transcription was found to be blocked in mutants bearing mutations in spoIIIA, spoIIIE, and spoIIIG, genes which are known to govern forespore gene expression. Also, 0.3 kb transcription could be switched on during growth in cells in which transcription of the forespore regulatory gene spoIIIG was engineered to be activated in response to the lac inducer IPTG (isopropyl-beta-D-thiogalactopyranoside). Although it is transcribed in the forespore, 0.3 kb is switched on at a later developmental stage than other previously studied forespore-expressed genes, and hence it appears to be representative of an additional temporal class of compartmentalized gene expression.

Bacillus subtilis↗
PubMed1989

Promoter specificity of sigma G-containing RNA polymerase from sporulating cells of Bacillus subtilis: identification of a group of forespore-specific promoters.

During sporulation in Bacillus subtilis, expression of the genes sspA, sspB, sspC, sspD, and sspE, which encode a family of small, acid-soluble spore proteins, as well as of the spoVA and gdh operons is transcriptionally activated at stage III of sporulation only in the forespore compartment. Transcription of these genes is mediated by RNA polymerase containing sigma G (E sigma G), the product of the sigG gene, which is itself expressed at stage III in the developing forespore. We have determined the 5' ends of transcripts generated both in vivo and in vitro by the action of E sigma G on various genes of B. subtilis and other bacilli. The 5' ends of the in vivo and in vitro mRNAs were found to coincide and were therefore considered to define the transcription initiation sites for the genes examined. We identified highly homologous DNA sequences centered at 35 and 10 base pairs preceding the transcriptional start sites of the genes examined. Consequently, we propose that these sequences define a class of promoters recognized only by E sigma G which allow transcription of genes expressed uniquely at stage III in the developing forespore.

Bacillus subtilis↗
PubMed1988

Levels of mRNAs which code for small, acid-soluble spore proteins and their LacZ gene fusions in sporulating cells of Bacillus subtilis.

The levels of mRNAs from genes (sspA, B and E) which code for major small, acid-soluble, spore proteins of Bacillus subtilis have been determined, as well as the levels of mRNAs from ssp-lacZ gene fusions. Increasing the gene dosage of ssp-lacZ fusions resulted in parallel increases in both the ssp-lacZ mRNA level and the rate of b-galactosidase accumulation. Similarly, an 11-fold increase in sspE gene dosage gave a comparable increase in sspE mRNA, but at most a 1.5-fold increase in the amount of sspE gene product accumulated. In contrast, an 11-fold increase in the dosage of the sspA or B genes had no significant effect on the level of total sspA plus sspB mRNA, but did alter the ratios of these mRNAs as well as the amount of their gene products, to reflect the altered ratio of the two genes. These results suggest that intact ssp genes, but not ssp-lacZ gene fusions, are subject to feedback regulation of gene expression, with this regulation of the sspA and B genes effected by modulation of mRNA levels, while the feedback regulation of the sspE gene is at the post-transcriptional level.

Bacillus subtilis↗
PubMed1988

Decreased UV light resistance of spores of Bacillus subtilis strains deficient in pyrimidine dimer repair and small, acid-soluble spore proteins.

Loss of small, acid-soluble spore protein alpha reduced spore UV resistance 30- to 50-fold in Bacillus subtilis strains deficient in pyrimidine dimer repair, but gave only a 5- to 8-fold reduction in UV resistance in repair-proficient strains. However, both repair-proficient and -deficient spores lacking this protein had identical heat and gamma-radiation resistance.

Bacillus subtilis↗
PubMed1988

Regulation of expression of genes coding for small, acid-soluble proteins of Bacillus subtilis spores: studies using lacZ gene fusions.

We constructed in-frame translational fusions of the Escherichia coli lacZ gene with four genes (sspA, sspB, sspD, and sspE) which code for small, acid-soluble spore proteins of Bacillus subtilis, and integrated these fusions into the chromosomes of various B. subtilis strains. With single copies of the fusions in wild-type B. subtilis, beta-galactosidase was synthesized only during sporulation, with the amounts accumulated being sspB much greater than sspE greater than or equal to sspA greater than or equal to sspD. Greater than 97% of the beta-galactosidase was found in the developing forespore, and the great majority was incorporated into mature spores. Less than 2% of the maximum amount of beta-galactosidase was made when these fusions were introduced into B. subtilis strains blocked in stages 0 and II of sporulation, as well as in some stage III mutants. Other stage III mutants, as well as stage IV and V mutants, had no effect on beta-galactosidase synthesis. Increasing the copy number of the sspA-, sspD-, or sspE-lacZ fusions (up to 17-fold for sspE-lacZ) in wild-type B. subtilis resulted in a parallel increase in the amount of beta-galactosidase accumulated (again only in sporulation and with greater than 95% in the developing forespore), with no significant effect on wild-type small, acid-soluble spore protein production. Similarly, the absence of one or more wild-type ssp genes or the presence of multiple copies of wild-type ssp genes had no effect on the expression of the lacZ fusions tested. These data indicate that these ssp-lacZ fusions escape the autoregulation seen for the intact sspA and sspB genes. Strikingly, the kinetics of beta-galactosidase synthesis were identical for all four ssp-lacZ fusions and paralleled those of glucose dehydrogenase synthesis. Similarly, all asporogenous mutants tested had identical effects on both glucose dehydrogenase and ssp-lacZ fusion expression.

Bacillus subtilis↗
PubMed1988

Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease.

Four genes (ssp genes) coding for small, acid-soluble spore proteins of Bacillus megaterium and the gene for the protease that cleaves them during germination were cloned in the integratable plasmid pJH101. Each plasmid was integrated into the B. megaterium chromosome by a Campbell-type mechanism, allowing mapping of all five genes. The gene for the small, acid-soluble spore protein-specific protease (gpr) mapped near rib, and the sspA gene mapped between argA and hisA. The three other genes of the spp gene family (sspB, -D, and -F) all mapped near metC/D, with the order: sspF-sspD-metC/D-hemA-argO-sspB. While neither gpr nor sspF has been mapped in B. subtilis, the positions of the sspA, -B, and -D loci are similar in B. megaterium and B. subtilis, suggesting that the members of this multigene family have not recently undergone significant movement on the chromosome. It appears that more gene rearrangement has occurred in the flanking genes than has occurred in the ssp family of genes producing the small, acid-soluble spore proteins.

Bacillus megaterium↗
PubMed1988

Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis.

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.

Bacillus subtilis↗
PubMed1988

Properties of spores of Bacillus subtilis strains which lack the major small, acid-soluble protein.

Bacillus subtilis strains containing a deletion in the gene coding for the major small, acid-soluble, spore protein (SASP-gamma) grew and sporulated, and their spores initiated germination normally, but outgrowth of SASP-gamma- spores was significantly slower than that of wild-type spores. The absence of SASP-gamma had no effect on spore protoplast density or spore resistance to heat or radiation. Consequently, SASP-gamma has a different function in spores than do the other major small, acid-soluble proteins.

Bacillus subtilis↗
PubMed1987

Thymine-containing dimers as well as spore photoproducts are found in ultraviolet-irradiated Bacillus subtilis spores that lack small acid-soluble proteins.

Dormant spores of a Bacillus subtilis mutant that lacks two major small, acid-soluble spore proteins are very sensitive to UV irradiation, which in spores generates about half the amount of thymine-containing dimers formed by comparable irradiation of vegetative cells. Irradiation of mutant spores also produces spore photoproducts, but again only about one-half the amount formed in comparably irradiated wild-type spores. These findings suggest that the high UV sensitivity of the mutant spores is due to the production of pyrimidine dimers, which are not found in UV-irradiated wild-type spores, and that the high level of small, acid-soluble proteins found in wild-type spores is directly involved in spore UV resistance by facilitating a conformational change in spore DNA, preventing pyrimidine dimer formation.

Bacillus subtilis↗
PubMed1987

Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis.

The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP-gamma is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs. The SASP-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 340 nucleotides long. The results of hybridization of an sspE gene probe to Southern blots of B. subtilis DNA suggested that there is only a single gene coding for the SASP-gamma type of protein in B. subtilis. This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B. subtilis chromosome, with concomitant loss of the wild-type gene. This sspE deletion strain sporulated well, but lacked the SASP-gamma type of protein.

Amino Acid Sequence↗
PubMed1987

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

The nucleotide sequences of the single genes coding for the B-type small, acid-soluble spore proteins (SASP) of Bacillus cereus, B. stearothermophilus, and "Thermoactinomyces thalpophilus" were determined, and the amino acid sequences of all B-type SASP were compared. While this type of SASP showed significant sequence conservation around the two spore protease cleavage sites, alignment of these sequences required the introduction of gaps, and even then only 19 of the residues were conserved exactly in all five proteins. However, all five B-type SASP did contain a large (27 to 35-residue), rather well-conserved amino acid sequence repeat, and four of the five proteins had well-conserved regions of 14 to 17 amino acids which appeared three times.

Amino Acid Sequence↗
PubMed1987

Different small, acid-soluble proteins of the alpha/beta type have interchangeable roles in the heat and UV radiation resistance of Bacillus subtilis spores.

Spores of Bacillus subtilis strains which carry deletion mutations in one gene (sspA) or two genes (sspA and sspB) which code for major alpha/beta-type small, acid-soluble spore proteins (SASP) are known to be much more sensitive to heat and UV radiation than wild-type spores. This heat- and UV-sensitive phenotype was cured completely or in part by introduction into these mutant strains of one or more copies of the sspA or sspB genes themselves; multiple copies of the B. subtilis sspD gene, which codes for a minor alpha/beta-type SASP; or multiple copies of the SASP-C gene, which codes for a major alpha/beta-type SASP of Bacillus megaterium. These findings suggest that alpha/beta-type SASP play interchangeable roles in the heat and UV radiation resistance of bacterial spores.

Bacillus subtilis↗
PubMed1986

Genes for Bacillus megaterium small, acid-soluble spore proteins: cloning and nucleotide sequence of three additional genes from this multigene family.

Three genes coding for small, acid-soluble spore proteins (SASP) were cloned from Bacillus megaterium, using previously cloned B. megaterium SASP genes (SASP-C and -C-3) as DNA-DNA hybridization probes. One gene (SASP-A) codes for the A protein, a previously identified major SASP. The other two (termed genes for SASP-C-4 and -C-5) are extremely similar in much of their nucleotide sequence to the previously cloned B. megaterium SASP-C-2 gene. The proteins coded for by all these SASP genes had extensive sequence homology with each other and with those coded for by the B. megaterium SASP-C, -C-1, -C-2, and -C-3 genes. Their coding sequences are preceded by strong ribosome-binding sites and are followed by regions of dyad symmetry which presumably are transcription stop sites. The SASP-A, -C-4, and -C-5 genes are expressed in parallel during sporulation, and their transcription start points were localized by the size of the mRNAs produced. The sequences localized 10 and 35 base pairs upstream from the transcription start points show significant homology with the analogous regions of the SASP-C, -C-1, -C-2, and -C-3 genes. The identification of seven closely related SASP genes in B. megaterium indicates that the SASP are the products of a very extensive multigene family.

Amino Acid Sequence↗