Biomedical subjects

Richard Losick

Publications and source records attributed to Richard Losick.

At least 37 records · Page 2Linked to original sources

PubMed2004

The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis.

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: sigma(E), sigma(K), GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The sigma(E) factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the sigma(E) regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of sigma(K). Next, sigma(K) activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by sigma(K) while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation.

Amino Acid Motifs↗

PubMed2004

A threshold mechanism governing activation of the developmental regulatory protein sigma F in Bacillus subtilis.

The RNA polymerase sigma factor sigma(F) is a developmental regulatory protein that is activated in a cell-specific manner following the formation of the polar septum during the process of spore formation in the bacterium Bacillus subtilis. Activation of sigma(F) depends on the membrane-bound phosphatase SpoIIE, which localizes to the septum, and on the formation of the polar septum itself. SpoIIE is responsible for dephosphorylating and thereby activating the phosphoprotein SpoIIAA, which, in turn, triggers the release of sigma(F) from the anti-sigma(F) factor SpoIIAB. Paradoxically, however, the presence of unphosphorylated SpoIIAA is insufficient to cause sigma(F) activation as SpoIIAA reaches substantial levels in mutants blocked in polar septation. We now describe mutants of SpoIIE, SpoIIAA, and SpoIIAB that break the dependence of sigma(F) activation on polar division. Analysis of these mutants indicates that unphosphorylated SpoIIAA must reach a threshold concentration in order to trigger the release of sigma(F) from SpoIIAB. Evidence is presented that this threshold is created by the action of SpoIIAB, which can form an alternative, long lived complex with SpoIIAA. We propose that formation of the SpoIIAA-SpoIIAB complex serves as a sink that traps SpoIIAA in an inactive state and that only when unphosphorylated SpoIIAA is in excess to the sink does activation of sigma(F) take place.

Amino Acid Sequence↗

PubMed2004

Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility.

Undomesticated strains of Bacillus subtilis, but not laboratory strains, exhibit robust swarming motility on solid surfaces. The failure of laboratory strains to swarm is caused by a mutation in a gene (sfp) needed for surfactin synthesis and a mutation(s) in an additional unknown gene(s). Insertional mutagenesis of the undomesticated 3610 strain with the transposon mini-Tn10 was carried out to discover genes needed for swarming but not swimming motility. Four such newly identified swarming genes are reported, three of which (swrA, swrB, and efp) had not been previously characterized and one of which (swrC) was known to play a role in resistance to the antibacterial effect of surfactin. Laboratory strains were found to harbour a frameshift mutation in the swrA gene. When corrected for the swrA mutation, as well as the mutation in sfp, laboratory strains regained the capacity to swarm and did so as robustly as the wild strain. The swrA mutation was an insertion of an A:T base pair in a homopolymeric stretch of eight A:T base pairs, and readily reverted to the wild type. These findings suggest that the swrA insertion and its reversion take place by slipped-strand mispairing during DNA replication and that swarming motility is subject to phase variation.

Amino Acid Sequence↗

PubMed2004

Novel genes that influence development in Streptomyces coelicolor.

Filamentous soil bacteria of the genus Streptomyces carry out complex developmental cycles that result in sporulation and production of numerous secondary metabolites with pharmaceutically important activities. To further characterize the molecular basis of these developmental events, we screened for mutants of Streptomyces coelicolor that exhibit aberrant morphological differentiation and/or secondary metabolite production. On the basis of this screening analysis and the subsequent complementation analysis of the mutants obtained we assigned developmental roles to a gene involved in methionine biosynthesis (metH) and two previously uncharacterized genes (SCO6938 and SCO2525) and we reidentified two previously described developmental genes (bldA and bldM). In contrast to most previously studied genes involved in development, the genes newly identified in the present study all appear to encode biosynthetic enzymes instead of regulatory proteins. The MetH methionine synthase appears to be required for conversion of aerial hyphae into chains of spores, SCO6938 is a probable acyl coenzyme A dehydrogenase that contributes to the proper timing of aerial mycelium formation and antibiotic production, and SCO2525 is a putative methyltransferase that influences various aspects of colony growth and development.

Acyl-CoA Dehydrogenase↗

PubMed2004

Genes involved in formation of structured multicellular communities by Bacillus subtilis.

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.

Bacillus subtilis↗

PubMed2004

Insulation of the sigmaF regulatory system in Bacillus subtilis.

The transcription factors sigmaF and sigmaB are related RNA polymerase sigma factors that govern dissimilar networks of adaptation to stress conditions in Bacillus subtilis. The two factors are controlled by closely related regulatory pathways, involving protein kinases and phosphatases. We report that insulation of the sigmaF pathway from the sigmaB pathway involves the integrated action of both the cognate kinase and the cognate phosphatase.

Bacillus subtilis↗

PubMed2004

Dynamic patterns of subcellular protein localization during spore coat morphogenesis in Bacillus subtilis.

Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins. Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP). YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium. YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore. Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore. Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore. Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis. We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization.

Bacillus subtilis↗

PubMed2004

Unmasking novel sporulation genes in Bacillus subtilis.

The Bacillus subtilis transcription factor sigma(E) directs the expression of a regulon of 262 genes, but null mutations in only a small fraction of these genes severely impair sporulation. We have previously reported that mutations in seven sigma(E)-controlled genes cause a mild (2- to 10-fold) defect in sporulation. In this study, we found that pairwise combinations of some of these seven mutations led to strong synthetic sporulation phenotypes, especially those involving the ytrHI operon and ybaN. Double mutants of ybaN and ytrH and of ybaN and ytrI had >10,000-fold lower sporulation efficiencies than the wild type. Thin-section electron microscopy revealed a block in cortex formation for the ybaN ytrH double mutant and coat defects for the ybaN single and ybaN ytrI double mutants. Sporulating cells of a ybaN ytrI double mutant and of a ybaN ytrHI triple mutant exhibited a pronounced loss of dipicolinic acid (DPA) between hours 8 and 24 of sporulation, in contrast to the constant levels seen for the wild type. An analysis of the spore cortex peptidoglycans of the ybaN ytrI and ybaN ytrHI mutants showed striking decreases in the levels of total muramic acid by hour 24 of sporulation. These data, along with the loss of DPA in the mutants, suggest that the developing spores were unstable and that the cortex underwent degradation late in sporulation. The existence of otherwise hidden sporulation pathways indicates that functional redundancy may mask the role of hitherto unrecognized sporulation genes.

Bacillus subtilis↗

PubMed2003

Assembly of the SpoIIIE DNA translocase depends on chromosome trapping in Bacillus subtilis.

Sporulation in Bacillus subtilis is an attractive system in which to study the translocation of a chromosome across a membrane. Sporulating cells contain two sister chromosomes that are condensed in an elongated axial filament with the origins of replication anchored at opposite poles of the sporangium. The subsequent formation of a septum near one pole divides the sporangium unequally into a forespore (the smaller compartment) and a mother cell. The septum forms around the filament, trapping the origin-proximal region of one chromosome in the forespore. As a consequence, the trapped chromosome transverses the septum with the remainder being left in the mother cell. Next, SpoIIIE assembles at the middle of the septum to create a translocase that pumps the origin-distal, two-thirds of the chromosome into the forespore. Here, we address the question of how the DNA translocase assembles and how it localizes to the septal midpoint. We present evidence that DNA transversing the septum is an anchor that nucleates the formation of the DNA translocase. We propose that DNA anchoring is responsible for the assembly of other SpoIIIE-like DNA translocases, such as those that remove trapped chromosomes from the division septum of cells undergoing binary fission.

Bacillus subtilis↗

PubMed2003

Cannibalism by sporulating bacteria.

Spore formation by the bacterium Bacillus subtilis is an elaborate developmental process that is triggered by nutrient limitation. Here we report that cells that have entered the pathway to sporulate produce and export a killing factor and a signaling protein that act cooperatively to block sister cells from sporulating and to cause them to lyse. The sporulating cells feed on the nutrients thereby released, which allows them to keep growing rather than to complete morphogenesis. We propose that sporulation is a stress-response pathway of last resort and that B. subtilis delays a commitment to spore formation by cannibalizing its siblings.

Adenosine Triphosphate↗

PubMed2003

The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division.

Gene transcription at the onset of sporulation in Bacillus subtilis is governed by Spo0A, a member of the response regulator family of transcription factors. Spo0A is traditionally viewed as the master regulator for entry into development. We now report that Spo0A continues to function after the initiation phase of sporulation and that it becomes a cell-specific transcription factor when the sporangium is divided into a mother cell and forespore. We observed that (1) Spo0A and Spo0A-directed gene transcription reached high levels in the mother cell; (2) an activated form of Spo0A impaired sporulation when produced in the forespore but not when produced in the mother cell; and (3) an inhibitor of Spo0A called Spo0A-N impaired sporulation and Spo0A-directed transcription when produced in the mother cell but not when produced in the forespore. Spo0A-N, which corresponds to the NH(2)-terminal domain of Spo0A, was shown to compete with the full-length response regulator for phosphorylation by the phosphorelay protein Spo0B. We propose that Spo0A is the earliest-acting transcription factor in the mother-cell line of gene expression and that in terms of abundance and transcriptional activity Spo0A may function predominantly as a cell-specific regulatory protein.

Bacillus subtilis↗

PubMed2003

The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis.

We report the identification and characterization on a genome-wide basis of genes under the control of the developmental transcription factor sigma(E) in Bacillus subtilis. The sigma(E) factor governs gene expression in the larger of the two cellular compartments (the mother cell) created by polar division during the developmental process of sporulation. Using transcriptional profiling and bioinformatics we show that 253 genes (organized in 157 operons) appear to be controlled by sigma(E). Among these, 181 genes (organized in 121 operons) had not been previously described as members of this regulon. Promoters for many of the newly identified genes were located by transcription start site mapping. To assess the role of these genes in sporulation, we created null mutations in 98 of the newly identified genes and operons. Of the resulting mutants, 12 (in prkA, ybaN, yhbH, ykvV, ylbJ, ypjB, yqfC, yqfD, ytrH, ytrI, ytvI and yunB) exhibited defects in spore formation. In addition, subcellular localization studies were carried out using in-frame fusions of several of the genes to the coding sequence for GFP. A majority of the fusion proteins localized either to the membrane surrounding the developing spore or to specific layers of the spore coat, although some fusions showed a uniform distribution in the mother cell cytoplasm. Finally, we used comparative genomics to determine that 46 of the sigma(E)-controlled genes in B.subtilis were present in all of the Gram-positive endospore-forming bacteria whose genome has been sequenced, but absent from the genome of the closely related but not endospore-forming bacterium Listeria monocytogenes, thereby defining a core of conserved sporulation genes of probable common ancestral origin. Our findings set the stage for a comprehensive understanding of the contribution of a cell-specific transcription factor to development and morphogenesis.

Bacillus subtilis↗

PubMed2003

Evidence in support of a docking model for the release of the transcription factor sigma F from the antisigma factor SpoIIAB in Bacillus subtilis.

Cell-specific activation of the transcription factor sigmaF during the process of sporulation in Bacillus subtilis is governed by an antisigma factor SpoIIAB and an anti-antisigma factor SpoIIAA. SpoIIAB, which exists as a dimer, binds to sigmaF in a complex of stoichiometry sigmaF.SpoIIAB2. Escape from the complex is mediated by SpoIIAA, which reacts with the complex to cause the release of free sigmaF. Previous evidence indicated that Arg-20 in SpoIIAB is a contact site for both sigmaF and SpoIIAA and that contact with sigmaF is mediated by Arg-20 on only one of the two subunits in the sigmaF.SpoIIAB2 complex. Here we report the construction of heterodimers of SpoIIAB in which one subunit is wild type and the other subunit is a mutant for Arg-20. We show that the dissociation constant for the binding of sigmaF to the heterodimer was similar to that for the wild type, a finding consistent with the idea that sigmaF contacts Arg-20 on only one of the two subunits. Although SpoIIAA was highly effective in causing the release of sigmaF from the wild type homodimer, the anti-antisigma factor had little effect on the release of sigmaF from the heterodimer. This finding is consistent with a model in which SpoIIAA docks on the sigmaF.SpoIIAB2 complex, making contact with the subunit in which Arg-20 is not in contact with sigmaF. SpoIIAB is both an anti-sigmaF factor and a protein kinase that phosphorylates and thereby inactivates SpoIIAA. We show that SpoIIAA effectively displaces sigmaF from a complex of sigmaF with a mutant (SpoIIABR105A) that is impaired in the kinase function of SpoIIAB. This result shows that SpoIIAA-mediated displacement of sigmaF from SpoIIAB does not require concomitant phosphorylation of SpoIIAA.

Bacillus subtilis↗

PubMed2003

Swarming motility in undomesticated Bacillus subtilis.

Swarming motility was identified and characterized in an undomesticated strain of Bacillus subtilis. Rapid surface migration was preceded by a cell density-dependent lag period, which could be eliminated if actively swarming cells were used as the inoculum. The leading edge of the swarm was characterized by multicellular rafts of highly flagellated cells. Flagellum biosynthesis and surfactant production were required for swarming. Swarming was not found in any of several standard laboratory strains. Laboratory strains are characteristically unable to produce surfactant, but such a strain remained unable to swarm even when surfactant was provided by extracellular complementation. We conclude that robust swarming is a feature of undomesticated B. subtilis and that this behaviour has been lost or attenuated in laboratory strains through the accumulation of multiple genetic defects.

Bacillus subtilis↗

PubMed2003

The Spo0A regulon of Bacillus subtilis.

The master regulator for entry into sporulation in Bacillus subtilis is the DNA-binding protein Spo0A, which has been found to influence, directly or indirectly, the expression of over 500 genes during the early stages of development. To search on a genome-wide basis for genes under the direct control of Spo0A, we used chromatin immunoprecipitation in combination with gene microarray analysis to identify regions of the chromosome at which an activated form of Spo0A binds in vivo. This information in combination with transcriptional profiling using gene microarrays, gel electrophoretic mobility shift assays, using the DNA-binding domain of Spo0A, and bioinformatics enabled us to assign 103 genes to the Spo0A regulon in addition to 18 previously known members. Thus, in total, 121 genes, which are organized as 30 single-gene units and 24 operons, are likely to be under the direct control of Spo0A. Forty of these genes are under the positive control of Spo0A, and 81 are under its negative control. Among newly identified members of the regulon with transcription that was stimulated by Spo0A are genes for metabolic enzymes and genes for efflux pumps. Among members with transcription that was in-hibited by Spo0A are genes encoding components of the DNA replication machinery and genes that govern flagellum biosynthesis and chemotaxis. Also in-cluded in the regulon are many (25) genes with products that are direct or indirect regulators of gene transcription. Spo0A is a master regulator for sporulation, but many of its effects on the global pattern of gene transcription are likely to be mediated indirectly by regulatory genes under its control.

Bacillus subtilis↗

PubMed2003

Unique degradation signal for ClpCP in Bacillus subtilis.

Regulation of the cell-specific transcription factor sigma(F) in the spore-forming bacterium Bacillus subtilis involves the antisigma factor SpoIIAB. Contributing to the activation of sigma(F) is the degradation of SpoIIAB in a manner that depends on the protease ClpCP. Here we show that the three residues (LCN) located at the extreme C terminus of SpoIIAB are both necessary and sufficient for this degradation. We also report that the use of the LCN extension as a degradation signal for ClpCP is unique to SpoIIAB.

Adenosine Triphosphatases↗

PubMed2003

A second PDZ-containing serine protease contributes to activation of the sporulation transcription factor sigmaK in Bacillus subtilis.

Gene expression late during the process of sporulation in Bacillus subtilis is governed by a multistep, signal transduction pathway involving the transcription factor sigma(K), which is derived by regulated proteolysis from the inactive proprotein pro-sigma(K). Processing of pro-sigma(K) is triggered by a signaling protein known as SpoIVB, a serine protease that contains a region with similarity to the PDZ family of protein-protein interaction domains. Here we report the discovery of a second PDZ-containing serine protease called CtpB that contributes to the activation of the pro-sigma(K) processing pathway. CtpB is a sporulation-specific, carboxyl-terminal processing protease and shares several features with SpoIVB. We propose that CtpB acts to fine-tune the regulation of pro-sigma(K) processing, and we discuss possible models by which CtpB influences the sigma(K) activation pathway.

Bacillus subtilis↗

PubMed2003

Subcellular localization of a small sporulation protein in Bacillus subtilis.

SpoVM is an unusually small (26-residue-long) protein that is produced in the mother cell chamber of the sporangium during the process of sporulation in Bacillus subtilis. We investigated the subcellular localization of SpoVM, which is believed to be an amphipathic alpha-helix, by using a fusion of the sporulation protein to the green fluorescence protein (GFP). We found that SpoVM-GFP is recruited to the polar septum shortly after the sporangium undergoes asymmetric division and that the fusion protein localizes to the mother cell membrane that surrounds the forespore during the subsequent process of engulfment. We identified a patch of three residues near the N terminus of the proposed alpha-helix that is needed both for proper subcellular localization and for SpoVM function. We also identified a patch of residues on the opposite face of the helix and residues near both ends of the protein that are needed for SpoVM function but not for subcellular localization. Subcellular localization of SpoVM-GFP was found to require an unknown gene(s) under the control of the mother cell transcription factor sigmaE. We propose that the N-terminal patch binds to an unknown anchoring protein that is produced under the control of sigmaE and that other residues important in SpoVM function to recruit an unknown sporulation protein(s) to the mother cell membrane that surrounds the forespore. Our results provide evidence that SpoVM function depends on proper subcellular localization.

Amino Acid Sequence↗