Biomedical subjects

R Losick

Publications and source records attributed to R Losick.

At least 19 recordsLinked to original sources

PubMed1992

Sporulation regulatory protein GerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes.

The mother-cell line of gene expression during sporulation in Bacillus subtilis is a hierarchical cascade consisting of at least four temporally controlled gene sets, the first three of which each contain a regulatory gene for the next gene set in the pathway. gerE, a member of the penultimate gene set, is a regulatory gene whose products is required for the transcriptional activation of genes (coat protein genes cotB and cotC) in the last gene set. The gerE product also influences the expression of other members of the penultimate gene set (coat protein genes cotA and cotD appear to be repressed and activated, respectively). We now report that the purified product of gerE (GerE) is a DNA-binding protein that adheres to the promoters for cotB and cotC. We also show that GerE stimulates cotB and cotC transcription in vitro by RNA polymerase containing the mother-cell sigma factor sigma K. These findings support the view that GerE is a positively acting, regulatory protein whose appearance at a late stage of development directly activates the transcription of genes in the last known temporal class of mother-cell-expressed genes. In addition, GerE stimulates cotD transcription and inhibits cotA transcription in vitro by sigma K RNA polymerase, as expected from in vivo studies, and, unexpectedly, profoundly inhibits in vitro transcription of the gene (sigK) that encodes sigma K. The effects of GerE on cotD and sigK transcription are just the opposite of the effects exerted by the earlier-appearing, mother-cell regulatory protein spoIIID, suggesting that the ordered appearance of first SpoIIID, then GerE, ensures proper flow of the regulatory cascade controlling gene expression in the mother cell.

Bacillus subtilis↗

PubMed1992

Crisscross regulation of cell-type-specific gene expression during development in B. subtilis.

Sporulation in Bacillus subtilis is a model for how cells of one type generate other differentiated cell types. During sporulation two cellular compartments arise that differ from each other and from the progenitor cell. Differential gene expression between the two is governed by the successive appearance of four transcription factors whose activities are coordinated in crisscross fashion between the two cells.

Bacillus subtilis↗

PubMed1992

Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens.

A key step in the biosynthesis of macrolide antibiotics is the assembly of a large macrocyclic lactone ring by a multienzyme protein complex called the polyketide synthase. In the species Streptomyces ambofaciens, the polyketide synthase for the assembly of the 16-membered ring of the macrolide antibiotic spiramycin is encoded by the biosynthetic gene srmG. Here we show that the accumulation of transcripts from the srmG promoter is governed by the regulatory gene srmR, whose predicted product, a 65 kDa polypeptide, is not significantly similar in its deduced amino acid sequence to that of previously reported proteins in the protein databases. The srmR gene product is also required for the accumulation of transcripts from srmX, an additional gene in the vicinity of srmR, but not for the accumulation of transcripts from srmR itself. Interestingly, mutations in srmR prevent the accumulation of transcripts from the spiramycin resistance gene srmB, but this is an indirect consequence of the failure of srmR mutants to produce spiramycin, which is an inducer of its own resistance gene. The possibility that srmR is the prototype for a new class of regulatory genes governing early events in the biosynthesis of macrolide antibiotics is discussed.

Amino Acid Sequence↗

PubMed1992

Characterization of bofA, a gene involved in intercompartmental regulation of pro-sigma K processing during sporulation in Bacillus subtilis.

Sporulating cells of the gram-positive bacterium Bacillus subtilis are partitioned into two cellular compartments called the mother cell and the forespore. Gene expression in the mother cell and the forespore is regulated differentially by the compartment-specific transcription factors sigma K and sigma G, respectively. Gene expression between the two compartments is also coordinated by a signal transduction pathway that couples the activation of sigma K (by processing of its inactive precursor pro-sigma K) in the mother cell to sigma G-directed gene expression in the forespore. To dissect the signal transduction pathway genetically, we previously isolated bypass of forespore mutations at loci called bofA and bofB that relieve the dependence of pro-sigma K processing on the action of sigma G. bofB mutations were previously shown to be allelic to the two-cistron sporulation operon spoIVF, which encodes the pro-sigma K-processing enzyme or its regulator. We now report that bofA mutations are located in a small open reading frame of 87 codons that encodes a putative integral membrane protein with three potential membrane-spanning domains. The possibility is discussed that BofA and the SpoIVF proteins form a heteromeric complex in the mother cell membrane that surrounds the forespore and that this complex mediates the intercompartmental coupling of pro-sigma K processing to events in the forespore.

Amino Acid Sequence↗

PubMed1992

Characterization of spoIVA, a sporulation gene involved in coat morphogenesis in Bacillus subtilis.

We report the cloning and characterization of the Bacillus subtilis sporulation locus spoIVA, mutations at which cause an unusual defect in spore formation in which the coat misassembles as swirls within the mother cell. We show that spoIVA is a single gene of 492 codons that is capable of encoding a polypeptide of 55 kDa. Transcription of spoIVA is induced at about the second hour of sporulation by the regulatory protein sigma E from two closely spaced promoters designated P1 and P2. Experiments in which the upstream promoter P1 was removed show that transcription of spoIVA from P2 is sufficient for efficient spore formation. Based on these and other findings, we infer that the spoIVA gene product is a morphogenetic protein; we discuss its role in the deposition of coat polypeptides around the developing forespore.

Amino Acid Sequence↗

PubMed1992

Identification of Bacillus subtilis genes for septum placement and shape determination.

The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells. We report the cloning of the wild-type allele of divIVB1 and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD. Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E. coli shape-determining genes mreB, mreC, and mreD. The B. subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodB1) that causes the production of aberrantly shaped cells under restrictive conditions. Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon. The possible involvement of min gene products in the switch from medial to polar placement of the septum during sporulation is discussed.

Amino Acid Sequence↗

PubMed1992

Characterization of the Bacillus subtilis sporulation gene spoVK.

The sporulation gene spoVK of Bacillus subtilis was cloned by use of the insertional mutation spoVK::Tn917 omega HU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.

Bacillus subtilis↗

PubMed1991

Compartmentalized expression of a gene under the control of sporulation transcription factor sigma E in Bacillus subtilis.

Immunoelectron microscopy was used to visualize the expression of a gene under the control of developmental transcription factor sigma E during spore formation in Bacillus subtilis. sigma E is generated by cleavage of an inactive proprotein (pro-sigma E) shortly after the formation of the sporulation septum, which partitions the sporangium into mother-cell and forespore compartments. Specific antibodies and gold-conjugated secondary antibodies were used to localize beta-galactosidase in thin sections of sporangia from cells bearing a lacZ transcriptional fusion to a gene (spoIID) under the direct control of sigma E. Transcription of spoIID was found to be induced shortly after the formation of the sporulation septum and was largely confined to the mother cell. Cell-type-specific transcription of genes under the control of sigma E could be responsible for establishing the mother-cell line of gene expression.

Bacillus subtilis↗

PubMed1991

Establishment of cell type by compartmentalized activation of a transcription factor.

Early in the process of spore formation in Bacillus subtilis a septum is formed that partitions the sporangium into daughter cells called the forespore and the mother cell. The daughter cells each have their own chromosome but follow dissimilar programs of gene expression. Differential gene expression in the forespore is now shown to be established by the compartmentalized activity of the transcription factor sigma F. The sigma F factor is produced prior to septation, but is active only in the forespore compartment of the post-septation sporangium. The sigma F factor is controlled by the products of sporulation operons spoIIA and spoIIE, which may be responsible for confining its activity to one of the daughter cells.

Bacillus subtilis↗

PubMed1991

Sporulation operon spoIVF and the characterization of mutations that uncouple mother-cell from forespore gene expression in Bacillus subtilis.

During the process of endospore formation in Bacillus subtilis the appearance of the mother-cell transcription factor sigma K by conversion from its inactive precursor pro-sigma K is coupled to events under the control of the forespore transcription factor sigma G. This intercompartmental coupling is believed to be mediated by the products of a sporulation locus called spoI V F because certain bypass-of-forespore (bof) mutations that map at the spoI V F locus relieve the dependence of pro-sigma K processing on the action of sigma G in the forespore. We now report that spoI V F is a two-cistron operon whose transcription is under the control of the sporulation transcription factor sigma E and whose products are likely to be integral membrane proteins. We show that the products of both the promoter-proximal (spoI V F A) and promoter-distal (spoI V F B) cistrons are required for spore formation at 37 degrees C, but that the spoI V F A gene product is dispensable at 30 degrees C. The bypass-of-forespore mutations are located at the extreme 3' end of the spoI V F A cistron, one such mutation causing a proline to serine substitution eight residues from the COOH terminus of SpoIVFA and another (a nonsense mutation) causing the absence of the terminal six amino acid residues of the protein. We also show that at a permissive temperature for spore formation spoI V F A null mutants exhibit a bypass-of-forespore phenotype. We hypothesize that SpoIVFA functions positively in stabilizing SpoIVFB, which we propose is thermolabile in the absence of the promoter-proximal gene product, and negatively in inhibiting the action of SpoIVFB. A model for intercompartmental coupling is presented in which SpoIVFB promotes pro-sigma K processing in response to a signal from the forespore that relieves or otherwise counteracts the inhibitory effect of SpoIVFA on SpoIVFB.

Amino Acid Sequence↗

PubMed1991

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor.

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.

Bacterial Proteins↗

PubMed1991

Differentiation and the establishment of cell type during sporulation in Bacillus subtilis.

Differentiation in Bacillus subtilis involves the formation of specialized cell types called the mother cell and the forespore. These differ from each other and from their parent in developmental fate. Establishment of the two cell types and their subsequent differentiation is governed by the compartmentalized action of six developmental transcription factors.

Bacillus subtilis↗

PubMed1991

Forespore-specific transcription of a gene in the signal transduction pathway that governs Pro-sigma K processing in Bacillus subtilis.

We present studies on the regulation of a developmental gene (spoIVB) whose product is required at a late stage of morphogenesis during the process of sporulation in Bacillus subtilis. Earlier work implicated the spoIVB gene product in a signal-transduction pathway that governs the conversion of pro-sigma K to the mature and active form of the mother cell sigma factor, sigma K, in response to a signal generated within the forespore chamber of the sporangium. We now show that (1) spoIVB is induced at the engulfment stage of sporulation, (2) this transcription is restricted to the forespore, and (3) spoIVB is under the direct control of the forespore sigma factor sigma G. The discovery that spoIVB is a forespore-expressed gene suggests that the spoIVB gene product, or a developmental event under its control, triggers the processing of pro-sigma K and thereby mediates the coupling of sigma K-directed gene expression in the mother cell to sigma G-directed gene expression in the forespore. We also show that spoIVB transcription is partially dependent on the action of the mother cell regulatory gene spoIIID, a finding that suggests that the transcription of certain forespore-expressed genes is influenced by events in the mother cell.

Bacillus subtilis↗

PubMed1991

Post-transcriptional control of a sporulation regulatory gene encoding transcription factor sigma H in Bacillus subtilis.

The transcriptional regulatory gene spoOH encodes an RNA polymerase sigma factor called sigma H that directs gene expression at an early stage of sporulation in the Gram-positive bacterium Bacillus subtilis. We now report that conditions that induce sporulation cause a rapid increase in the cellular concentration of sigma H. This increase could account for the stimulated transcription of certain sigma H-controlled genes at the onset of sporulation. Experiments in which the expression of spoOH was monitored by use of a spoOH-lacZ fusion and in which expression of spoOH was artificially manipulated by use of an isopropyl-beta-D-thiogalacto-side-inducible promoter indicate that sporulation-induced increases in the amount of sigma H are not controlled at the level of the transcription of its structural gene. Rather, we infer the existence of post-transcriptional control mechanisms that govern sigma H levels, and we present evidence suggesting that increases in the amount of sigma H at the start of sporulation are due to increased translation or stability of the spoOH mRNA and, to a lesser extent, decreased turnover of spoOH protein.

Adenosine↗

PubMed1991

Gene encoding two alkali-soluble components of the spore coat from Bacillus subtilis.

We report the cloning and characterization of a gene called cotF from Bacillus subtilis that encodes alkali-soluble polypeptides of 5 and 8 kDa that are components of the spore coat. The 5- and 8-kDa polypeptides are generated by proteolytic cleavage of the primary product of the cotF gene, which is 160 codons in length and is capable of encoding a polypeptide of 19 kDa. Amino acid sequence analysis indicates that the 5-kDa species is derived from the NH2-terminal portion of the primary gene product and that the 8-kDa species is derived from the COOH-terminal portion. A mutant bearing an in vitro-constructed cotF null mutation produced normal-looking spores that contained an apparently complete set of coat proteins except for the absence of the 5- and 8-kDa polypeptides. The map position of cotF is 349 degrees. Transcription of cotF commenced coincidently (during h 6 of sporulation) with genes known to be under the control of sporulation transcription factor sigma kappa.

Amino Acid Sequence↗

PubMed1990

A forespore checkpoint for mother cell gene expression during development in B. subtilis.

Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the bofA and/or bofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.

Bacillus subtilis↗

PubMed1990

Cascade regulation of spore coat gene expression in Bacillus subtilis.

Endospores of the Gram-positive bacterium Bacillus subtilis are encased in a tough protein shell known as the coat. The coat is composed of a dozen or more different structural proteins. We report the identification of and studies on the regulation of promoters governing the expression of coat protein (cot) genes designated B to E encoding polypeptides of 59, 12, 11 and 24 kDa, respectively. We show that transcription of genes B, C and D is governed by single promoters and that transcription of gene E is governed by tandem promoters designated P1 and P2. In extension of recent work on the transcription of cot gene A and the mother-cell regulatory genes gerE, sigK and spoIIID, we show that genes involved in coat formation are turned on in a regulatory cascade of at least four co-ordinately controlled gene sets. The cascade consists of: cotE as transcribed from its P1 promoter and spoIIID, which are turned on during hours three to four of sporulation; cotE as transcribed from its P2 promoter and sigK, which are turned on during hour five by the appearance of the product (a small DNA-binding protein) of spoIIID; cotA, cotD and gerE, which are turned on during hours five to six by the appearance of the product (sigma factor sigma K) of sigK; and cotB and cotC, which are turned on during hour seven by the appearance of the product (an inferred DNA-binding protein) of gerE. The cascade is hierarchical in that the first three gene sets each contain the regulatory gene that turns on the expression of the next gene set in the pathway. We also show that the level of expression of a member (cotC) of the terminal class of gene expression is strongly influenced by medium and that this effect directly or indirectly depends on the product of sporulation gene spoIV A.

Bacillus subtilis↗

PubMed1990

Two amino acids in an RNA polymerase sigma factor involved in the recognition of adjacent base pairs in the -10 region of a cognate promoter.

The recognition of promoter region -10 nucleotide sequences in prokaryotes is believed to be mediated by a segment of alpha-helix in a region of RNA polymerase sigma factors called 2.4. Earlier genetic studies implicated Thr-100 in region 2.4 of the Bacillus subtilis sigma factor sigma H in the recognition of the G.C base pair at position -13 in the -10 region (GAAT) of a cognate promoter. In confirmation of this assignment, we now show that a change-of-specificity mutant of sigma H in which Thr-100 was replaced with isoleucine suppresses a G.C----A.T nucleotide substitution at position -13 but not other "promoter down mutations" (causing impaired promoter activity) at positions -13, -12, and -11. We also show that a loss-of-contact mutant created by the replacement of Thr-100 with alanine (having a short side chain) enables sigma H to tolerate three different promoter down mutations at position -13 but not down mutations at other positions. Finally, we suggest the identification of an additional amino acid involved in base-pair recognition by the demonstration that the replacement of Arg-96 with alanine specifically suppresses an A.T----G.C promoter down mutation at position -12. The identification of amino acids that are four residues apart that are involved in the recognition of adjacent base pairs may fix the orientation of region 2.4 (its NH2 terminus being proximal to the promoter transcription start site) and is consistent with a model in which the recognition of promoter region -10 nucleotide sequences is mediated by an alpha-helix in which residues involved in base-pair contact are separated by one turn and clustered on one face of the helix.

Bacillus subtilis↗