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R Losick

Publications and source records attributed to R Losick.

At least 127 records · Page 7Linked to original sources
PubMed1988

Cloned gene encoding the delta subunit of Bacillus subtilis RNA polymerase.

Core RNA polymerase and several forms of RNA polymerase holoenzyme from Bacillus subtilis are found in association with a 21,500-kDa polypeptide called delta (delta). We have cloned the structural gene (rpoE) for delta by using a hybridization probe a synthetic oligodeoxynucleotide that was designed on the basis of a partial NH2-terminal amino acid (aa) sequence of purified delta protein. The rpoE gene was found to encode a 173-aa polypeptide of a predicted Mr of 20,400. Genetic and physical mapping experiments placed rpoE at 325 degrees on the B. subtilis chromosome and established the gene order rpoE-ctrA-spo0F. The delta subunit is known to enhance the specificity of transcription in vitro by bacterial and phage SP01-modified forms of polymerase, but replacement of rpoE by an in vitro-constructed deletion-mutated gene was found not to impair viability, sporulation, or the growth of phage SP01.

Amino Acid Sequence↗
PubMed1988

Visualizing gene expression in time and space in the filamentous bacterium Streptomyces coelicolor.

Streptomycetes are prokaryotic microorganisms that exhibit a complex, mycelial fungus-like cycle of morphological differentiation. Development involves at least two spatially separated types of cells: the branching hyphae of the substrate mycelium, which penetrate the stratum upon which the colony feeds, and the upwardly protruding hyphae of the aerial mycelium, which undergo metamorphosis into spores. The luciferase-encoding luxA and luxB operon of the luminescent marine bacterium Vibrio harveyi was used as a promoter probe to visualize gene expression in differentiating colonies of Streptomyces coelicolor. Promoters for developmental genes of several kinds gave distinctive temporal and spatial patterns of light emission.

DNA, Bacterial↗
PubMed1988

Identification of the promoter for a spore coat protein gene in Bacillus subtilis and studies on the regulation of its induction at a late stage of sporulation.

The cotA (pig) gene of Bacillus subtilis encodes a 65,000 Mr protein that is a component of the spore coat and is responsible for the brown pigment characteristic of colonies in which cells are undergoing sporulation. To study developmental regulation of the cotA gene, we identified its promoter and studied its transcription in a large number of mutants blocked at various stages of sporulation and germination. Deletion analysis showed that induction and efficient transcription of cotA required DNA sequences extending no more than 55 base pairs (bp) upstream (and no more than 130 bp downstream) from the 5' terminus of cotA mRNA. Transcription from the cotA promoter was found to be switched on at approximately the time (4 to 5 h after the onset of sporulation) of spore coat synthesis and deposition. Strikingly, this transcription was substantially inhibited in almost all asporogenous mutants blocked prior to the developmental stage (V) of spore coat formation. cotA transcription was also impaired in several stage V mutants but not in other stage V mutants or in mutants blocked in germination. The germination mutant gerE caused a several-fold overexpression of cotA. The dependence of cotA expression on so many genes required at early to intermediate stages of sporulation suggests that transcription of this spore coat gene is somehow coupled (directly or through several intervening steps) to a morphological or physiological feature(s) of the developing sporangium.

Amino Acid Sequence↗
PubMed1988

Extracellular control of spore formation in Bacillus subtilis.

Spore formation in the Gram-positive bacterium Bacillus subtilis has been classically viewed as an example of unicellular differentiation that occurs in response to nutritional starvation. We present evidence that B. subtilis produces an extracellular factor(s) that is required, in addition to starvation conditions, for efficient sporulation. This factor is secreted and accumulates in a cell density-dependent fashion such that cells at a low density sporulate poorly under conditions in which cells at a high density sporulate efficiently. Conditioned medium (sterile filtrate) from cells grown to a high density contains this extracellular differentiation factor (EDF-A) and stimulates spore formation of cells at low density under normal starvation conditions. EDF-A is heat-resistant, protease-sensitive, and dialyzable, indicating that it is at least in part an oligopeptide. Production of EDF-A is reduced or eliminated in spoOA and spoOB mutants, which are defective in many processes associated with the end of vegetative growth. Mutations in abrB, which suppress many of the pleiotropic phenotypes of spoOA mutants, restore production of EDF-A.

Bacillus subtilis↗
PubMed1988

Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore.

Endospores of Bacillus subtilis are encased in a two-layer protein shell known as the coat, which consists of a lammellar-like inner layer and an electron-dense outer layer. We report the cloning of the structural gene (designated cotE) for an alkali-soluble coat protein of 24 kD and show that the cotE gene product is a morphogenic protein required in the assembly of the outer coat. The nucleotide sequence of cotE reveals an open reading frame capable of encoding a 181-residue-long polypeptide of 21 kD. A cotE mutant was created by replacing the chromosomal gene, which was located at 145 degrees on the chromosome, with an in vitro constructed, deletion-mutated gene. The resulting cotE mutant formed normal-looking (optically refractile) spores that were heat resistant but were sensitive to lysozyme and somewhat impaired in germination. Ultrastructural analysis indicated that the mutant spores lacked the electron-dense outer layer of the coat but retained a normal-looking inner coat. The mutant spores were pleiotropically deficient in several coat proteins, including the product of cotE and the products of previously cloned cot genes A-C. Based on experiments in which expression of the cotA and cotC genes was found to be unimpaired in cotE mutant cells, we infer that the cotE gene product is involved in the assembly of the products of cotA-cotC, and certain other proteins into the electron-dense outer layer of the coat.

Amino Acid Sequence↗
PubMed1988

Suppression of ctc promoter mutations in Bacillus subtilis.

Transcription from the Bacillus subtilis ctc promoter is induced as cells enter stationary phase under conditions in which the enzymes of the tricarboxylic acid cycle are repressed. This transcription requires the presence of a secondary form of RNA polymerase, E sigma B, that is found in exponentially growing cells and in early-stationary-phase cells. Starting with a defective ctc promoter that had either a base substitution at position -15 or a base substitution at position -36, we were able to identify four independent second-site mutations within these mutated promoters that suppressed the effect of the original mutations and thereby restored function to the ctc promoter. Three of these mutated promoters had an additional base substitution(s) at positions -5, -9, or both -5 and -9 that enhanced their utilization in vivo by E sigma B, whereas one of the promoters had a single-base-pair deletion in the -15 region that placed it under a completely different form of regulation than that of the wild-type ctc promoter. In addition to mutations in the ctc promoter region, we also isolated three classes of mutants that exhibited increased ctc expression. The effects of the mutations in these strains were not allele specific, since they increased expression from both mutant and wild-type ctc promoters. One class of mutants which affected expression from the ctc promoter carried mutations that blocked the activity of the tricarboxylic acid cycle. A second class of mutations mapped near cysA and was unable to sporulate. Three-factor transformation crosses and complementation analysis indicated that one of these mutations was an allele of spo0H. The third class of mutations is closely linked to dal and may define a regulatory gene for sigB, the sigma B structural gene.

Acetyltransferases↗
PubMed1988

Promoter determining the timing and spatial localization of transcription of a cloned Streptomyces coelicolor gene encoding a spore-associated polypeptide.

Streptomyces coelicolor is a filamentous, gram-positive bacterium that exhibits a complex cycle of morphological differentiation involving the formation of an aerial mycelium of multinucleoid hyphae which undergo septation to form long chains of spores. We report the identification of two proteins of 13 and 3 kilodaltons, designated SapA and SapB, respectively, that are produced during formation of the aerial mycelium and are found in assocation with purified, mature spores. We cloned the structural gene (sapA) for one of these spore-associated proteins. Nucleotide sequence analysis suggests that the 13-kilodalton polypeptide is derived from a larger pre- or preproprotein containing a leader sequence of 37 amino acids. Nuclease protection-hybridization analysis and experiments using the Vibrio harveyi, luciferase-encoding luxAB operon as a gene tag demonstrated that expression of sapA is controlled from a promoter contained within a region of less than 110 base pairs in length, whose transcription start site is located approximately 50 base pairs upstream from the initiation codon for the sapA open reading frame. Transcription of sapA was induced at the time of appearance of the aerial mycelium, and the level of sapA transcripts was significantly reduced in certain mutants blocked in aerial mycelium (bld) and or spore (whi) formation. As further evidence of the association of sapA transcription with morphological differentiation, experiments in which we monitored sapA transcription topographically by use of a sapA-luxAB operon fusion demonstrated a close spatial correlation between colony regions undergoing aerial mycelium formation and zones of sapA-promoted light emission.

Amino Acid Sequence↗
PubMed1988

The promoter for a sporulation gene in the spoIVC locus of Bacillus subtilis and its use in studies of temporal and spatial control of gene expression.

We have identified the transcription start site and regulatory region governing the expression of a sporulation gene in the spoIVC locus of Bacillus subtilis. Efficient expression and developmental regulation of this gene was controlled from a promoter region that extended no more than 110 base pairs upstream and no more than 4 base pairs downstream from the start site of transcription, on which basis we infer that spoIVC is regulated at the level of transcription initiation. Using a transcriptional fusion of the spoIVC gene to the lacZ gene of Escherichia coli, we found that spoIVC expression was turned on at the third to fourth hour of sporulation (at about the developmental stage [IV] that its products are required in spore formation) and that this transcription was largely restricted to the mother cell chamber of the sporangium. Mutations in many different spo genes (causing blocks at stages 0 to V) were found to influence (negatively and positively) the level of spoIVC expression. Our results distinguish the mode of spoIVC regulation from that of previously studied sporulation genes and indicate that it is representative of a new regulon of mother cell-specific gene expression.

Bacillus subtilis↗
PubMed1987

Genes encoding spore coat polypeptides from Bacillus subtilis.

Endospores of the Gram-positive bacterium Bacillus subtilis are encased in a tough protein shell, known as the coat, that consists of a dozen or more different polypeptides. We have cloned structural genes designated cotA, cotB, cotC and cotD that encode spore coat proteins of Mr 65,000, 59,000, 12,000 and 11,000, respectively. These genes were cloned by using as hybridization probes synthetic oligonucleotides that were designed on the basis of partial NH2-terminal sequence determinations of the purified coat proteins. To determine the location of the cot genes on the chromosome and to study their function genetically, we tagged each gene by insertion of a chloramphenicol-resistance determinant (cat) within its coding sequence. We then replaced each wild-type cot gene in the chromosome with the corresponding, insertionally inactivated gene. Genetic mapping experiments showed that cotA, cotB, cotC and cotD were located at 52 degrees, 290 degrees, 168 degrees and 200 degrees, respectively, on the B. subtilis chromosome. None of the cot::cat insertion mutants were Spo-, but spores of the cotD mutant were found to germinate somewhat more slowly than did wild-type spores, and the cotA mutant was found to be blocked in the appearance of the brown pigment characteristic of colonies of wild-type sporulating cells. Physical and genetic experiments established that cotA was identical to a previously identified gene called pig, known to be responsible for sporulation-associated pigment production. Spores from all four insertion mutants exhibited the wild-type pattern of coat polypeptides, except for the absence in each instance of the corresponding product of the cot gene that had been insertionally inactivated.

Amino Acid Sequence↗
PubMed1987

Genetic analysis of Bacillus subtilis spo mutations generated by Tn917-mediated insertional mutagenesis.

Mutations that cause sporulation defects (spo mutations) often identify developmentally regulated transcription units or genes whose products are required for the expression of sporulation-specific regulons. We report here the isolation, genetic analysis and phenotypic characterization of spo mutations produced by insertional mutagenesis with transposon Tn917, a form of mutagenesis that facilitates genetic and physical manipulation of mutated genes in many ways. Twenty-four insertional spo mutations were studied in detail. On the basis of transformation-mediated and transduction-mediated linkage analysis and a range of phenotypic tests, these mutations were assigned to 20 distinct loci, at least 9 of which are different from the 40 previously described spo loci. The insertional mutations caused blocks at a variety of different stages of sporulation, and therefore probably identify genes active at different times during sporulation. In addition to increasing substantially the total of known spo loci, we anticipate that this collection will include representatives of many of the temporally regulated sets of genes that comprise the overall program of sporulation-specific gene activation in Bacillus subtilis. Given the kinds of manipulations that are possible with genes disrupted by Tn917 insertions, this should significantly facilitate efforts to understand the regulation of these gene sets.

Bacillus subtilis↗
PubMed1987

Effects of plasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subtilis.

Transcription of the sporulation gene spoVG of Bacillus subtilis is induced at the onset of spore formation and depends on the products of the regulatory genes spoOA, spoOB, and spoOH. We describe two effects of propagating the promoter region of spoVG on a multicopy plasmid replicon in B. subtilis cells. One effect is that transcription from the plasmid-borne spoVG promoter is altered with respect to the time of its induction and the dependence on spoO gene products. An example of this effect is that plasmid propagation was observed to relieve substantially the inhibitory effect of a mutation in spoOH, the spoO gene upon which spoVG promoter activity is most strongly dependent. We present results which suggest that propagation on a plasmid replicon causes an alteration in the conformation of spoVG promoter DNA which somehow compensates for the defective spoOH gene product. Plasmid propagation did not, however, entirely eliminate the requirement for the spoOH gene product; little or no spoVG-directed RNA synthesis was observed in cells bearing a putative spoOH deletion mutation, a finding which indicates that SpoOH protein plays an indispensable role in spoVG promoter utilization. Another effect of propagating the promoter region of spoVG on a multicopy plasmid is to inhibit sporulation. S1 nuclease mapping experiments suggest that amplification of spoVG on a multicopy plasmid causes the titration of a transcription factor or minor form of RNA polymerase holoenzyme required for utilization of one of the two overlapping promoters which comprise the spoVG transcription initiation region.

Bacillus subtilis↗
PubMed1987

Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis.

Tyrocidine is a cyclic decapeptide antibiotic which is produced and secreted by stationary-phase cells of the sporeforming bacterium Bacillus brevis. We identified the promoter for the B. brevis structural gene (tycA) for tyrocidine synthetase I, the enzyme catalyzing the first step in tyrocidine biosynthesis, and studied its regulation in cells of B. brevis and Bacillus subtilis. Transcription from the tycA promoter was induced at the end of the exponential phase of the growth cycle in B. brevis cells growing in sporulation medium. To study the regulation of tycA in B. subtilis, we constructed a derivative of the B. subtilis bacteriophage SP beta containing a transcriptional fusion of the tycA promoter to the lacZ gene of Escherichia coli and introduced the tycA-lacZ operon fusion by means of specialized transduction into sporulation mutants known to be blocked in sporulation-associated antibiotic production. Our principal finding was that tycA-directed lacZ expression was impaired in the stage-0 mutants with mutations spo0A, spo0B, and spo0E but not in spo0C, spo0F, spo0H, or spo+ bacteria. The dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation, which caused tycA-lacZ to be transcribed constitutively at all stages of growth. A simple model is proposed for the mechanism of tycA induction based on the Spo0A-dependent inactivation of Ab-B protein, which is proposed to be a negative regulator of tycA transcription.

Bacillus↗
PubMed1987

Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis.

Transcription of the Bacillus subtilis gene spoVG is induced at the onset of sporulation and is dependent on the products of the stage-0 regulatory genes spo0A, spo0B, and spo0H. We show here that the dependence of spoVG transcription on Spo0A and Spo0B (but not Spo0H) can be bypassed by a mutation at abrB, a previously identified locus at which mutations that suppress some of the phenotypes of spo0A are often located, or by a cis-acting mutation within the spoVG promoter. To explain the epistatis of abrB to spo0A and spo0B mutations, we propose that AbrB acts, directly or indirectly, to block transcription of spoVG and that Spo0A and Spo0B cause inactivation of the abrB gene product(s). Spo0A-Spo0B-dependent inactivation of AbrB could be a general explanation for the pleiotropic effects of spo0A and spo0B mutations on B. subtilis gene expression.

Bacillus subtilis↗
PubMed1987

Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis.

sigma B (sigma 37) is a secondary species of RNA polymerase sigma factor found in the gram-positive bacterium Bacillus subtilis. To study the function of sigma B genetically, we sought mutations that block the expression of a gene (ctc) known to be transcribed by sigma B-containing RNA polymerase in vitro. One such mutation, called crl, was found to map in or near the structural gene (sigB) for sigma B. To determine directly whether mutations in sigB would prevent transcription of ctc, we replaced sigB in the B. subtilis chromosome with insertion and deletion mutations that disrupted the sigma B coding sequence. Like crl, these in vitro-constructed mutations blocked expression of ctc, but had little or no effect on viability, sporulation, expression of the sporulation gene spoVG, or production of sporulation-associated alkaline protease. Using fusions of ctc to the reporter genes xylE and lacZ, we also identified mutations that enhanced ctc expression. One such mutation, called socB, was found to be located in an open reading frame immediately downstream of sigB.

Bacillus subtilis↗
PubMed1986

Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis.

The ctc gene of Bacillus subtilis is transcribed in vitro by the minor RNA polymerase holoenzyme forms, E sigma 37 and E sigma 32. To study the expression and regulation of ctc in vivo, we constructed operon and translational fusions of the ctc promoter region to the lacZ gene of Escherichia coli. Our results indicate that ctc is regulated at the transcriptional level, and that this RNA synthesis is maximally induced at the end of the exponential phase of growth under nutritional conditions which inhibit the activity of the tricarboxylic acid cycle. Analysis of in vitro-constructed deletion mutations extending into the ctc promoter region demonstrated that the region required for this regulation is no greater than 53 base-pairs in length. We also compared the expression of ctc to that of another B. subtilis gene, which is transcribed by E sigma 37 and E sigma 32 in vitro, the sporulation gene spoVG. Although the ctc and spoVG promoter regions are recognized by the same forms of RNA polymerase in vitro, our results show that they differ strikingly in the nutritional and genetic requirements for their expression in vivo.

Bacillus subtilis↗
PubMed1986

Gene encoding the sigma 37 species of RNA polymerase sigma factor from Bacillus subtilis.

sigma 37 is a minor species of RNA polymerase sigma factor found in the Gram-positive bacterium Bacillus subtilis. sigma 37 governs the transcription in vitro of genes that are turned on at an early stage in spore formation, as well as other genes that are switched on at the end of the exponential phase of growth but that are not under sporulation control. To study the role of sigma 37 in B. subtilis gene expression, we have cloned the gene for this minor species of sigma factor in Escherichia coli by using as a hybridization probe a synthetic oligonucleotide that was designed on the basis of the NH2-terminal amino acid sequence of sigma 37 protein. We determined the nucleotide sequence of the entire sigma 37 gene, which was found to encode a 262-amino acid residue polypeptide of 29.9 kDa. The predicted amino acid sequence of sigma 37 showed significant homology to that of other sigma proteins in a region that has been proposed to be the site of binding of these factors to core RNA polymerase. Genetic mapping experiments placed the gene for sigma 37, herein designated sigB, at 40 degrees on the genetic map of Piggot and Hoch [Piggot, P. & Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179]. An insertion mutation was constructed in sigB and found not to impair growth or sporulation.

Amino Acid Sequence↗