Search PubMed⌕ Search

Biomedical subjects

R Losick

Publications and source records attributed to R Losick.

At least 145 records · Page 8Linked to original sources
PubMed1984

A promoter whose utilization is temporally regulated during sporulation in Bacillus subtilis.

The formation of endospores in the Gram-positive bacterium Bacillus subtilis proceeds according to a temporally ordered program of gene activation. To investigate timing mechanisms in sporulation gene expression, we have isolated and sequenced the promoter region for a B. subtilis gene known as 0.3 kb whose transcription is switched on at about stage III of development. The 5' terminus of the 0.3 kb mRNA was mapped by the S1 nuclease procedure to a position just upstream from its apparent ribosome binding site and initiation codon and just downstream from the transcription termination site for an adjacent gene. This information enabled us to construct a transcriptional fusion in which the 5' region of the 0.3 kb gene was joined to the lacZ gene of Escherichia coli. When introduced into cells of B. subtilis, the 0.3 kb-lacZ fusion caused the synthesis of a fusion-specified RNA that originated from within the 0.3 kb promoter region and extended into the adjacent E. coli DNA, and the induction of beta-galactosidase synthesis at the third to fourth hour of sporulation. Enzyme synthesis required the 0.3 kb promoter, since a deletion of the 5' region of the 0.3 kb gene in the transcription fusion eliminated the production of beta-galactosidase. Induction of the 0.3 kb-lacZ fusion was under developmental control, since the production of beta-galactosidase was blocked or substantially impaired by chromosomal mutations in the sporulation genes spoOB, spoIIA, spoIIE and spoIIIE, but not by a spoIIC mutation. We conclude that the 0.3 kb gene promoter is subject to a developmental clock, which delays its utilization until an intermediate stage of sporulation, and discuss models for how the timing of gene expression is regulated.

Bacillus subtilis↗
PubMed1984

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions.

A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cmr) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cmr B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl2-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRI or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.

Bacillus subtilis↗
PubMed1984

Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene.

A 1.3-kb restriction fragment carrying a cat gene derived from Staphylococcus aureus was inserted by ligation in both possible orientations into a HpaI restriction site located less than 300 bp from one end of Tn917. The resulting transposon derivatives were unimpaired in their ability to make and resolve transpositions into the chromosome of Bacillus subtilis and they displayed no detectable defect in expression of the inducible erm gene carried by the transposon. This demonstrates that the HpaI site itself, and perhaps the entire 250- to 300-bp region between the HpaI site and the nearest transposon terminal inverted repeat consists of nonessential DNA, and is there fore available to be modified or used as a cloning site with the expectation that the resulting transposon derivatives should be capable of normal transposition activity. To facilitate such manipulations, the HpaI site was "replaced" by a 24-bp DNA segment which contains a BamHI site flanked on either side by SmaI sites; these BamHI and SmaI sites are unique to the transposon. Several of the plasmid constructions undertaken in the course of this work illustrate ways in which homologous recombination may be used in conjunction with ligation in B. subtilis (and other bacteria, such as Streptococcus pneumoniae, which have similar mechanisms for DNA uptake during competence) to facilitate significantly the recovery of certain kinds of recombinant molecules.

Bacillus subtilis↗
PubMed1983

Deletion analysis of a complex promoter for a developmentally regulated gene from Bacillus subtilis.

SpoVG is a developmentally regulated gene from the spore-forming bacterium Bacillus subtilis. The transcription initiation region for spoVG consists of two overlapping promoters whose startpoints of RNA synthesis are ten base pairs apart (Moran et al., 1981a). These startpoints are separately utilized by two forms of RNA polymerase holoenzyme containing different species of B. subtilis sigma factor. We have constructed a series of deletion mutations that extend into the spoVG promoter region from the downstream and from the upstream directions. Transcription studies with these mutated promoters showed that the functional boundaries of the spoVG promoters extended from the region of the transcription startpoints into an upstream A + T-rich box, which was located 76 to 51 base pairs preceding the downstream startsite. We have unexpectedly discovered that propagation of the spoVG promoter region on a high copy number plasmid in B. subtilis interferes with the process of sporulation by impairing development at an early stage. This was not a general effect of promoter amplification, since the propagation on plasmids of two other strong Bacillus promoters had little or no effect on spore formation. Deletion analysis established that the region of spoVG causing sporulation inhibition closely correlated with DNA sequences required for efficient promoter utilization in vitro. We propose that amplification of spoVG titrates a sporulation-specific regulatory protein that binds at or near the region of transcription initiation.

Bacillus subtilis↗
PubMed1983

Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gene.

A developmentally regulated gene (spoVG) from the spore-forming bacterium Bacillus subtilis is expressed from two overlapping promoters, which direct transcription initiating from sites separated by 10 base pairs. Utilization of the upstream promoter is determined by an RNA polymerase sigma factor of molecular weight 37,000 (sigma 37). We report the isolation of a 32,000-molecular weight species of sigma factor (sigma 32), which exclusively dictates transcription initiation from the downstream promoter, and suggest a model for the way in which sigma-specific recognition sequences are intermeshed within the spoVG transcription initiation region.

Bacillus subtilis↗
PubMed1983

Use of a lacZ fusion to study the role of the spoO genes of Bacillus subtilis in developmental regulation.

A mutation in any one of eight spoO genes of Bacillus subtilis blocks the process of spore formation at its earliest stage. To investigate how the products of the spoO genes may be involved in developmental gene expression, we fused the lacZ gene of E. coli to spoVG, a sporulation gene whose induction at the onset of sporulation is under spoO control. In cells of Spo+ bacteria containing a single copy of the gene fusion, conditions leading to the onset of sporulation resulted in the induction of beta-galactosidase synthesis. This induction was moderately to severely impaired by mutations in any of seven spoO genes. Deletion and hybridization analysis demonstrated that this sporulation-induced enzyme synthesis was exclusively expressed from the two overlapping promoters, which comprise the spoVG transcription-initiation region, and that a small DNA segment (157 bp) containing the spoVG promoters was sufficient to cause spoO-dependent induction of the fused lacZ gene.

Bacillus subtilis↗
PubMed1983

Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917.

The Streptococcus faecalis transposon Tn917 was introduced into Bacillus subtilis by transformation of competent cells with the plasmid pAM alpha 1::Tn917 and was tested for transposition activity by selection for insertions into the temperate phage SP beta. Insertions were obtained at a frequency indicating relatively efficient movement of the element, and Southern hybridization analysis of a particular insertion confirmed it to be the result of a genuine transposition event. A restriction fragment from pAM alpha 1::Tn917 containing the transposon sequences was ligated into a temperature-sensitive plasmid (pBD95), and transpositions into the B. subtilis chromosome were selected by requiring the transposon drug resistance to be maintained at temperatures nonpermissive for plasmid replication. Insertions have been recovered at many chromosomal sites, including ones that produced auxotrophy of different kinds and ones that produced various different sporulation-defective phenotypes, indicating good prospects for the use of Tn917 as a tool for insertional mutagenesis in B. subtilis.

Bacillus subtilis↗
PubMed1982

Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis.

We have determined the nucleotide sequence of two Bacillus subtilis promoters (veg and tms) that are utilized by the principal form of B. subtilis RNA polymerase found in vegetative cells (sigma 55-RNA polymerase) and have compared our sequences to those of several previously reported Bacillus promoters. Hexanucleotide sequences centered approximately 35 (the "--35" region) and 10 (the "--10" region) base pairs upstream from the veg and tms transcription starting points (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed to Escherichia coli promoters. Conformity to the preferred --35 and --10 sequences may not be sufficient to promote efficient utilization by B. subtilis RNA polymerase, however, since three promoters (veg, tms and E. coli tac) that conform to these sequences and that are utilized efficiently by E. coli RNA polymerase were used with highly varied efficiencies by B. subtilis RNA polymerase. We have also analyzed mRNA sequences in DNA located downstream from eight B. subtilis chromosomal and phage promoters for nucleotide sequences that might signal the initiation of translation. In accordance with the rules of McLaughlin, Murray and Rabinowitz (1981), we observe mRNA nucleotide sequences with extensive complementarity to the 3' terminal region of B. subtilis 16S rRNA, followed by an initiation codon and an open reading frame.

Bacillus subtilis↗
PubMed1981

Nucleotide sequence of a Bacillus subtilis promoter recognized by Bacillus subtilis RNA polymerase containing sigma 37.

We report the nucleotide sequence of the promoter for a Bacillus subtilis gene (spoVC) whose transcription is controlled by a 37,000 dalton species of B. subtilis sigma factor known as sigma 37 but not by the principal- sigma factor of 55,000 daltons (sigma 55). Using S1 nuclease mapping we show that the startpoint for sigma 37-directed transcription of the spoVC gene in vitro corresponded closely to the 5' terminus of in vivo synthesized spoVC RNA. The binding site for sigma 37-containing RNA polymerase extended from 43 bp to 51 bp (positions -43 to -51) upstream from the transcription startpoint to 22 bp (position +22) downstream from the startpoint. The nucleotide sequence of the spoVC promoter differed significantly from promoters whose recognition is controlled by sigma 55 but was similar to other sigma 37- controlled promoters in regions known to be important in promoter recognition. Our results are consistent with the hypothesis (Lee and Pero, J. Mol. Biol., in press) that sigma factors work by contacting specific bases in both the -35 and -10 regions of promoters.

Bacillus subtilis↗
PubMed1981

A sporulation-induced sigma-like regulatory protein from B. subtilis.

We have isolated a sigma-like regulatory protein termed sigma 29 whose synthesis or association with Bacillus subtilis RNA polymerase was induced during spore formation. sigma 29 is a sporulation-specific component of RNA polymerase as it was absent in enzyme from an early-blocked sporulation mutant (SpoOA). We have demonstrated specific RNA synthesis by sigma 29-RNA polymerase using as a DNA template a cloned cluster of vegetative and sporulation genes from the purA-cysA region of the B. subtilis chromosome. The pattern of gene recognition by sigma 29-RNA polymerase was distinct from that observed for RNA polymerases containing sigma 55 or sigma 37, species of sigma factor that are present in vegetative cells of B. subtilis. A reconstitution experiment in which purified sigma 29 was added to core RNA polymerase demonstrates that sigma 29 was directly responsible for the altered transcriptional specificity of sporulation RNA polymerase. We propose that sigma 29 is a regulatory protein that controls developmental gene transcription at an early stage of spore formation.

Bacillus subtilis↗
PubMed1981

Promoter for a developmentally regulated gene in Bacillus subtilis.

We have determined the nucleotide sequence of the promoter for aB. subtilis gene (the 0.4 kb gene) whose transcription is under developmental control. Transcription of the 0.4 kb gene is turned on at the onset of sporulation; this RNA synthesis depends on the products of the B. subtilis regulatory genes (the spoO loci) that control the initiation of development. Recognition of the 0.4 kb gene promoter in vitro is dictated by novel species of B. subtilis RNA polymerase sigma factor known as sigma 37 and sigma 29 but not by the principal B. subtilis sigma factor sigma 55. Using S1 nuclease mapping, runoff transcription and dinucleotide priming, we have identified dual startpoints (separated by about 10 bp) for sigma37-directed transcription of the 0.4 kb gene These start-points correspond closely to the 5' termini of 0.4 kb RNA synthesized in vivo during the course of sporulation. Two forms of sigma37 containing RNA polymerase were distinguished that preferentially utilize either the upstream or the downstream startpoint in vitro. We investigated the requirements for sigma37-directed transcription by constructing in vitro deletion mutations that extend from the upstream direction into the 0.4 kb promoter region. One such deletion, which terminates 40 and 51 bp upstream from the transcription startpoints (thereby removing a highly AT-rich 26 bp sequence), completely eliminates transcription from the downstream startpoint but only partially inhibits transcription from the upstream startpoint. A second deletion that terminates at the upstream startpoint completely prevents transcription from both initiation sites. The implications of 0.4 kb gene promoter structure for developmentally regulated transcription of B, subtilis are discussed.

Bacillus subtilis↗
PubMed1981

Developmentally regulated transcription in a cloned segment of the Bacillus subtilis chromosome.

We describe a model system for studying developmentally regulated transcription during spore formation in Bacillus subtilis. This model system is a cloned cluster of genes known as 0.4 kb, ctc, and veg from the purA-cysA region of the B. subtilis chromosome. Each gene exhibited a distinct pattern of transcription in cells growing in glucose medium and in cells deprived of nutrients in sporulation medium. The 0.4 kb gene was transcribed at a low level in growing cells but was actively transcribed during nutrient deprivation in sporulation medium. This ribonucleic acid (RNA) synthesis was dependent upon the products of five B. subtilis genes that are involved in the initiation of spore formation:spo0A, spo0A, spo0E, spo0F, and spo0H. A mutation in any one of these regulatory genes severely restricted transcription of the 0.4 kb sequence. Transcription of the ctc gene was also turned on by nutrient deprivation, but this RNA synthesis was not impaired in spo0 mutants. Although not under spo0 control, the ctc gene probably corresponds to a locus, spoVC, whose product is required at a late stage of sporulation. Finally, the veg gene was actively transcribed both in growing cells and in nutrient-deprived cells. Like ctc RNA synthesis, transcription of the veg gene was not dependent upon the spo0 gene products. We propose that the spo0A, spo0B, spo0E, spo0F, and spo0H gene products are components of a pathway(s) that senses nutrient deprivation in B. subtilis and translates this environmental signal into the transcriptional activation of a subset of developmental genes.

Bacillus subtilis↗
PubMed1981

A cloned gene that is turned on at an intermediate stage of spore formation in Bacillus subtilis.

Cells of Bacillus subtilis synthesize a relatively long-lived ribonucleic acid (RNA) of about 300 bases during the course of spore formation. This transcript does not appear until an intermediate stage (III or IV) of development but is the predominant sporulation-specific transcript among RNAs of discrete size in late (stages IV to VI) developing cells. Appearance of the 300-base RNA is under sporulation control as this transcript could not be detected in cells of an early-blocked sporulation mutant (Spo0A). We have located the coding sequence for the 300-base RNA within a cloned chromosomal segment from the purA-cysA region that was previously shown to contain a cluster of genes that are actively transcribed during sporulation. The coding sequence for the 300-base RNA (designated as the 0.3 kb gene) mapped between a gene (veg) that was actively transcribed during growth and development and a gene (0.4 kb) that was turned on at the onset of sporulation. Although clustered within a small segment of the chromosome, the veg, 0.3 kb, and 0.4 kb transcription units exhibited, therefore, distinct patterns of temporally programmed gene expression. Models for the activation of the 0.3 kb gene at an intermediate stage of development are discussed.

Bacillus subtilis↗
PubMed1981

Identification of a new developmental locus in Bacillus subtilis by construction of a deletion mutation in a cloned gene under sporulation control.

We removed by recombinant deoxyribonucleic acid (DNA) techniques a small DNA segment from within a cloned gene (the 0.4 kb gene) in which transcription in under sporulation control in Bacillus subtilis. These deletion mutation was introduced into the B. subtilis chromosome by transformation with cloned DNA. Competent cells bearing a mutation (tms-26) that is closely linked to the 0.4 kb gene were transformed with linearized plasmid DNA containing the truncated 0.4 kb gene and the wild-type allele of the tms locus. Selection for Tms+ transformants yielded oligosporogenous mutants of unusually dark-brown colony pigmentation. This phenotype was caused by a mutation which mapped at or very near the site of the 0.4 kg gene deletion, whose presence and position in chromosomal DNA was confirmed by Southern hybridization analysis. Phase-contrast microscopy and electron microscopy showed that the mutation, which we designated as spoVG, impaired sporulation at about the fifth stage; bacteria harboring the spoVG mutation proceeded normally through stage IV of development but frequently lysed thereafter, apparently as a result of disintegration of an immature spore cortex. This identifies the 0.4 kb gene (or DNA in its immediate vicinity) as a new sporulation locus and shows that its product functions at a late stage in development.

Bacillus subtilis↗
PubMed1980

Novel RNA polymerase sigma factor from Bacillus subtilis.

A modified form of Bacillus subtilis RNA polymerase (RNA nucleotidyltransferase) has been isolated that exhibits distinctive transcriptional specificity. This modified enzyme transcribes two cloned genes from the purA-cysA region of the B. subtilis chromosome whose expression in vivo is associated with the process of sporulation. Neither of these genes is transcribed by the usual form of B. subtilis RNA polymerase holoenzyme containing a sigma factor of 55,000 daltons (sigma 55). The modified RNA polymerase lacks sigma 55 but contains a newly identified subunit of 37,000 daltons termed sigma 37. A reconstitution experiment in which sigma 37 was added to core RNA polymerase strongly suggests that sigma 37 is responsible for the transcriptional specificity of the modified RNA polymerase. Sigma 37 apparently acts at the level of promoter recognition; this transcriptional determinant enabled core RNA polymerase to form stable binary and ternary ("initiation") complexes with endonuclease restriction fragments containing promoters for the cloned B. subtilis genes.

Bacillus subtilis↗