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R Losick

Publications and source records attributed to R Losick.

At least 73 records · Page 4Linked to original sources
PubMed1995

Localization of protein implicated in establishment of cell type to sites of asymmetric division.

Asymmetric division in Bacillus subtilis generates progeny cells with dissimilar fates. SpoIIE, a membrane protein required for the establishment of cell type, was shown to localize near sites of potential polar division. SpoIIE initially localizes in a bipolar pattern, coalescing at marks in the cell envelope at which asymmetric division can take place. Then, during division, SpoIIE becomes restricted to the polar septum and is lost from the distal pole. Thus, when division is complete, SpoIIE sits at the boundary between the progeny from which it dictates cell fate by the activation of a cell-specific transcription factor.

Bacillus subtilis↗
PubMed1995

Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division.

Cell fate is determined by cell-specific activation of transcription factor sigma F after asymmetric division during sporulation by Bacillus subtilis. The activity of sigma F is governed by SpoIIAA, SpoIIAB, and SpoIIE, a membrane protein localized at the polar septum. SpoIIAB binds to and inhibits sigma F, and SpoIIAA inhibits SpoIIAB, which prevents SpoIIAB from binding to sigma F. SpoIIAB is also a serine kinase that inactivates SpoIIAA. Here, it is demonstrated that SpoIIE dephosphorylates SpoIIAA-P and overcomes SpoIIAB-mediated inhibition of sigma F. The finding that SpoIIE is a serine phosphatase links asymmetric division to the pathway governing cell-specific gene transcription.

Adenosine Triphosphate↗
PubMed1995

Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis.

We present biochemical evidence for an intercellular signal transduction pathway in B. subtilis. This pathway governs the conversion of the proprotein pro-sigma E to mature transcription factor sigma E. Proteolytic processing is mediated by the membrane protein SpollGA and is triggered by the inferred extracellular signal protein SpollR. A factor in conditioned medium from B. subtilis cells engineered to produce SpollR during growth triggered processing in protoplasts of B. subtilis cells that had been engineered to produce SpollGA and pro-sigma E. The factor was also detected in, and partially purified from, extracts of SpollR-producing cells of E. coli. We speculate that SpollGA is both a receptor and a protease and the SpollR interacts with SpollGA on the outside of the cytoplasmic membrane, activating the intracellular protease domain of SpollGA.

Amino Acid Sequence↗
PubMed1995

Visualization of the subcellular location of sporulation proteins in Bacillus subtilis using immunofluorescence microscopy.

We describe the application of immunofluorescence microscopy to visualization of the subcellular localization of proteins involved in coat morphogenesis and chromosome packaging during the process of sporulation in Bacillus subtilis. In confirmation and extension of previous findings, we show that SpolVA, which is responsible for guiding coat formation to the surface of the outer membrane that surrounds the developing spore, assembles into a shell that is located close to or on the surface of this enveloping membrane. CotE, which is responsible for the formation of the outer layer of the coat, assembles into a second shell of apparently larger diameter. Assembly of SpolVA could be detected as early as the morphological stage of polar septation and closely followed the enveloping membrane of the mother cell during the stage of engulfment, thereby providing a sensitive and diagnostic marker for this phagocytic-like process. Surprisingly, the chromosome of the developing spore and the small, acid-soluble proteins, known as alpha/beta-type SASPs, that are known to coat the spore chromosome, were found to co-localize to a doughnut-like ring of approximately 1 micrometer in diameter. The use of a double mutant lacking the alpha/beta-type SASP demonstrated that these high abundance, DNA-binding proteins are responsible for packaging the chromosome of the developing spore into this unusual structure. We conclude that sporulation in B. subtilis is a fertile system for addressing cell biological problems in a bacterium and that immunofluorescence microscopy provides a sensitive method for visualizing protein subcellular localization at high resolution.

Bacillus subtilis↗
PubMed1995

Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis.

We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation. Sporangia were doubly stained with propidium iodide to visualize the forespore and mother cell nucleoids and with fluorescein-conjugated antibodies to visualize the location of beta-galactosidase produced under the control of the sporulation RNA polymerase sigma factors sigma E and sigma F. In confirmation and extension of earlier reports, we found that expression of a lacZ fusion under the control of sigma E was confined to the mother cell compartment of sporangia at the septation (II) and engulfment (III) stages of morphogenesis. Conversely, sigma F-directed gene expression was confined to the forespore compartment of sporangia at postseptation stages of development. Little indication was found for sigma E- or sigma F-directed gene expression prior to septation or in both compartments of postseptation sporangia. Gene expression under the control of the forespore sigma factor sigma G also exhibited a high level of compartmentalization. A high proportion of sporangia exhibited fluorescence in our immunostaining protocol, which should be suitable for the subcellular localization of sporulation proteins for which specific antibodies are available.

Bacillus subtilis↗
PubMed1995

Identification and characterization of sporulation gene spoVS from Bacillus subtilis.

We report the identification and characterization of an additional sporulation gene from Bacillus subtilis called spoVS, which is induced early in sporulation under the control of sigma H. We show that spoVS is an 86-codon-long open reading frame and is capable of encoding a protein of 8,796 Da which exhibits little similarity to other proteins in the databases. Null mutations in spoVS have two contrasting phenotypes. In otherwise wild-type cells they block sporulation at stage V, impairing the development of heat resistance and coat assembly. However, the presence of a spoVS mutation in a spoIIB spoVG double mutant (which is blocked at the stage [II] of polar septation) acts as a partial suppressor, allowing sporulation to advance to a late stage. The implications of the contrasting phenotypes are discussed in the context of the formation and maturation of the polar septum.

Amino Acid Sequence↗
PubMed1995

An additional GerE-controlled gene encoding an abundant spore coat protein from Bacillus subtilis.

We describe the identification and characterization of a gene, herein designated cotG, encoding an abundant coat protein from the spores of Bacillus subtilis. The cotG open reading frame is 195 codons in length and is capable of encoding a polypeptide of 24 kDa that contains nine tandem copies of the 13-amino-acid long, approximately repeated sequence H/Y-K-K-S-Y-R/C-S/T-H/Y-K-K-S-R-S. cotG is located at 300 degrees on the genetic map close to another coat protein gene, cotB. The cotG and cotB genes are in divergent orientation and are separated by 1.3 kb. Like the promoter for cotB, the cotG promoter is induced at a late stage of sporulation under the control of the RNA polymerase sigma factor sigma K and the DNA-binding protein GerE. The -10 and -35 nucleotide sequences of the cotG promoter resemble those of other promoters recognized by sigma K-containing RNA polymerase, and centered 70 bp upstream of the apparent start site is a sequence that matches the consensus binding site for GerE. Spore coat proteins from a newly constructed cotG null mutant lack not only CotG but also CotB, a finding that suggests that CotG may be a morphogenetic protein that is required for the incorporation of CotB into the coat.

Amino Acid Sequence↗
PubMed1995

Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis.

We report the use of the green fluorescent protein (GFP) of Aequorea victoria to visualize cell-specific gene expression and protein subcellular localization during sporulation in Bacillus subtilis. Sporangia bearing the gene (gfp) for the green fluorescent protein fused to genes under the control of the sporulation transcription factor sigma F exhibited a forespore-specific pattern of fluorescence. Forespore-specific fluorescence could be detected with fusions to promoters that are utilized with low (csfB) and high (sspE-2G) efficiency by sigma F-containing RNA polymerase. Conversely, a mother cell-specific pattern of fluorescence was observed in sporangia bearing a transcriptional fusion of gfp to a spore coat protein gene (cotE) under the control of sigma E and an in-frame fusion to a regulatory gene (gerE) under the control of sigma K. An in-frame fusion of gfp to cotE demonstrated that GFP can also be used to visualize protein subcellular localization. In sporangia producing the CotE-GFP fusion protein, fluorescence was found to localize around the developing spore, and this localization was dependent upon SpoIVA, a morphogenetic protein known to determine proper localization of CotE.

Bacillus subtilis↗
PubMed1995

Adjacent and divergently oriented operons under the control of the sporulation regulatory protein GerE in Bacillus subtilis.

The DNA-binding protein GerE is the latest-acting regulatory protein in the mother cell line of gene expression during sporulation in Bacillus subtilis. GerE directs the transcription of several genes that encode structural components of the protein coat that encases the mature spore. We report on the identification and characterization of a cluster of additional genes whose transcription is dependent on GerE. These genes, which are located in the replication terminus region of the chromosome (181 degrees on the genetic map), are arranged in adjacent and divergently oriented operons called cgeAB and cgeCDE, which consist of two and at least three genes, respectively. CgeD, the product of the second member of the cgeCDE operon, is strikingly similar to the product of a B. subtilis gene (ipa-63d) of unknown function and is similar at its amino terminus to certain glycosyl transferases involved in polysaccharide biosynthesis. Strains with mutations in the cgeAB and cgeCDE operons produce spores with altered surface properties, on which basis we propose that proteins encoded by these operons influence maturation of the outermost layer of the spore, perhaps by glycosylation of coat proteins at the spore surface.

Amino Acid Sequence↗
PubMed1994

An adenosine nucleotide switch controlling the activity of a cell type-specific transcription factor in B. subtilis.

The sigma F factor establishes cell type-specific gene transcription during sporulation in B. subtilis. sigma F is negatively regulated by SpollAB, which forms complexes with sigma F or SpollAA. ATP and its nonhydrolyzable analogs stimulate the formation of the SpollAB.sigma F complex, whereas ADP stimulates the formation of the SpollAB.SpollAA complex. Which protein SpollAB associates with is determined by the concentrations of the two nucleotides, on which basis we propose a partner-switching model for the regulation of sigma F: [formula: see text] Consistent with this model, SpollAA reverses SpollAB-mediated inhibition of sigma F-directed transcription in a manner that depends on ADP. Cell-specific activation of sigma F could be due to an alteration in adenosine nucleotide levels in one cell of the sporangium.

Adenosine Diphosphate↗
PubMed1994

Establishment of cell type specific gene transcription during sporulation in Bacillus subtilis.

Asymmetric cell division during the process of sporulation in the bacterium Bacillus subtilis generates dissimilar progeny that exhibit distinct programs of gene transcription. Recent work reveals a partner switching mechanism that governs the activity of the sporulation regulatory protein sigma F and that may be responsible for the establishment of cell type specific gene transcription.

Adenine Nucleotides↗
PubMed1994

Subcellular localization of proteins involved in the assembly of the spore coat of Bacillus subtilis.

Spores of the bacterium Bacillus subtilis are encased in a two-layered protein shell, which consists of an electron-translucent, lamellar inner coat, and an electron-dense outer coat. The coat protein CotE is both a structural component of the coat and a morphogenetic protein that is required for the assembly of the outer coat. We now show that CotE is located in the outer coat of the mature spore and that at an intermediate stage of sporulation, when the developing spore (the forespore) is present as a free protoplast within the sporangium, CotE is localized in a ring that surrounds the forespore but is separated from it by a small gap. We propose that the ring is the site of assembly of the outer coat and that the gap is the site of formation of the inner coat. Assembly of the ring depends on the sporulation protein SpoIVA, which sits close to or on the surface of the outer membrane that encircles the forespore. We propose that SpoIVA creates a basement layer around the forespore on which coat assembly takes place. The subcellular localization and assembly of CotE and other coat proteins are therefore determined by the capacity of SpoIVA to recognize and adhere to a specific surface within the sporangium, the outer membrane of the forespore.

Bacillus subtilis↗
PubMed1994

Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ.

A homologue of the bacterial cell division gene ftsZ was cloned from the filamentous bacterium Streptomyces coelicolor. The gene was located on the physical map of the chromosome at about '11 o'clock' (in the vicinity of glkA, hisA and trpB). Surprisingly, a null mutant in which the 399-codon ftsZ open reading frame was largely deleted was viable, even though the mutant was blocked in septum formation. This indicates that cell division may not be essential for the growth and viability of S. coelicolor. The ftsZ mutant was able to produce aerial hyphae but was unable to produce spores, a finding consistent with the idea that ftsZ is required in order for aerial hyphae to undergo septation into the uninucleoid cells that differentiate into spores.

Amino Acid Sequence↗
PubMed1994

Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G.

The Bacillus subtilis RNA polymerase sigma factor sigma G is a cell-type-specific regulatory protein that governs the transcription of genes that are expressed at an intermediate to late stage of sporulation in the forespore compartment of the sporangium. Here we report the identification of a mutation (lon-1) that causes inappropriate transcription of genes under the control of sigma G under nutritional and genetic conditions in which sporulation is prevented. The mutation is located at 245 degrees on the genetic map and lies within a newly identified open reading frame that is predicted to encode a homolog to Lon protease. Inappropriate transcription of sigma G-controlled genes in the lon-1 mutant is not prevented by mutations in genes that are normally required for the appearance of sigma G during sporulation but is prevented by a mutation in the structural gene (spoIIIG) for sigma G itself. In light of previous work showing that spoIIIG is subject to positive autoregulation, we propose that Lon protease is responsible (possibly by causing degradation of sigma G) for preventing sigma G-directed transcription of spoIIIG and hence the accumulation of sigma G in cells that are not undergoing sporulation. An integrated physical and genetic map is presented that encompasses 36 kb of uninterrupted DNA sequence from the lon pheA region of the chromosome, corresponding to 245 degrees to 239 degrees on the genetic map.

ATP-Dependent Proteases↗
PubMed1994

Characterization of a cell division gene from Bacillus subtilis that is required for vegetative and sporulation septum formation.

We report the cloning and characterization of a cell division gene, herein designated divIC, from the gram-positive, spore-forming bacterium Bacillus subtilis. This gene was previously identified on the basis of a temperature-sensitive mutation, div-355, that blocks septum formation at restrictive temperatures. We show that the divIC gene is a 125-codon open reading frame that is capable of encoding a protein of 14.7 kDa and that div-355 is a 5-bp duplication near the 3' end of the open reading frame. We also show that divIC is an essential gene by use of an in vitro-constructed null mutation. In confirmation and extension of earlier results, we show that divIC is necessary for both vegetative and sporulation septum formation, and we demonstrate that it is required for the activation of genes expressed under the control of the sporulation transcription factors sigma F and sigma E. The divIC gene is located 1.3 kb upstream of the coding sequence for the sporulation gene spoIIE. Between divIC and spoIIE is a 128-codon open reading frame whose predicted product contains a region of similarity to the RNA-binding domains of polynucleotide phosphorylase and ribosomal protein S1 from Escherichia coli and two putative tRNA genes for methionyl-tRNA and glutamyl-tRNA, the gene order being divIC orf128 tRNA(Met) tRNA(Glu) spoIIE.

Amino Acid Sequence↗