Biomedical subjects

R Losick

Publications and source records attributed to R Losick.

At least 91 records · Page 5Linked to original sources

PubMed1993

SpoIIAB is an anti-sigma factor that binds to and inhibits transcription by regulatory protein sigma F from Bacillus subtilis.

The sigma F factor is a regulatory protein that is responsible for directing gene expression in the forespore compartment of developing cells of the spore-forming soil bacterium Bacillus subtilis. The sigma F factor is encoded by the promoter-distal member of sporulation operon spoIIA, which consists of cistrons called spoIIAA, spoIIAB, and spoIIAC. Genetic evidence indicates that the activity of sigma F is negatively regulated by the product (SpoIIAB) of the spoIIAB cistron. We now report that SpoIIAB is capable of binding to sigma F and inhibiting its capacity to direct transcription by core RNA polymerase from the promoter for a forespore-expressed gene. SpoIIAB is an anti-sigma factor that may be directly involved in the compartmentalization of sigma F-directed gene expression.

Bacillus subtilis↗

PubMed1993

Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces coelicolor.

The formation of an aerial mycelium by the filamentous bacterium Streptomyces coelicolor is determined in part by a small morphogenetic protein called SapB. A collection of representative bald (bld) mutants, which are blocked in aerial mycelium formation, are all defective in the production of this protein and regain the capacity to undergo morphological differentiation when SapB is supplied exogenously. We now report that most of the bld mutants are rescued for SapB production and aerial mycelium formation when grown near certain other bld mutants. Extracellular complementation experiments of this kind indicate that morphological differentiation is governed by a hierarchical cascade of at least four kinds of intercellular signals. At least one such signal is present in conditioned medium. It is resistant to boiling and protease treatment, and it remains effective even when diluted up to eightfold in fresh medium.

Adult↗

PubMed1993

An unusually small gene required for sporulation by Bacillus subtilis.

We report the cloning and characterization of an unusually small gene called spoVM whose product is required for normal formation of the cortex and coat during sporulation in Bacillus subtilis. The spoVM gene is adjacent to, and in convergent orientation with, the B. subtilis homologue to the Escherichia coli gene for ribosomal protein L28. The spoVM open reading frame is only 26 codons in length and is capable of encoding a polypeptide of 3 kDa. The short length of spoVM was verified by means of complementation experiments with wild-type and deletion-mutated copies of the open reading frame and by engineering the synthesis of the spoVM gene product in E. coli. Transcription of spoVM was induced during the second hour of sporulation (approximately stage II) by the appearance of the sporulation RNA polymerase sigma factor, sigma E. Efficient transcription of spoVM additionally required the action of the sporulation DNA-binding protein SpoIIID. Because spoVM was not strongly required for the transcription of several genes expressed at late times in development, its protein product is likely to play a morphogenetic rather than a regulatory role in sporulation.

Amino Acid Sequence↗

PubMed1993

Sporulation gene spoIIB from Bacillus subtilis.

We have cloned and characterized the sporulation gene spoIIB from Bacillus subtilis. In extension of previous nucleotide sequence analysis, our results show that the order of genes in the vicinity of spoIIB is valS folC comC spoIIB orfA orfB mreB mreC mreD minC minD spoIVFA spoIVFB L20 orfX L24 spoOB obg pheB pheA. All 20 genes have the same orientation; the direction of transcription is from valS to pheA. We show that spoIIB is a 332-codon-long open reading frame whose transcription is under sporulation control. The deduced amino acid sequence of the spoIIB gene product, a 36-kDa polypeptide, is highly charged and contains a stretch of uncharged amino acids that could correspond to a transmembrane segment. Surprisingly, mutations in spoIIB, including an in vitro-constructed null mutation, cause only a mild impairment of spore formation in certain otherwise wild-type bacteria. However, when combined with mutations in another sporulation gene, spoVG, mutations in spoIIB cause a severe block in spore formation at the stage (stage II) of septum formation. (As with spoIIB mutations, mutations in spoVG cause little impairment in sporulation on their own.) The nature of the spoIIB spoVG mutant phenotype is discussed in terms of the events involved in the maturation of the sporulation septum and in the activation of sporulation transcription factors sigma F and sigma E.

Amino Acid Sequence↗

PubMed1993

Multilevel regulation of the sporulation transcription factor sigma K in Bacillus subtilis.

Gene expression in the mother-cell compartment of the Bacillus subtilis sporangium is governed in part by the sporulation transcription factor sigma K. The production of sigma K is controlled at three levels: by a chromosomal rearrangement that generates the sigma K-coding sequence (sigK), by compartment-specific transcription of sigK, and by conversion of the inactive pro-protein product of sigK (pro-sigma K) to sigma K. To investigate the function of these multiple levels of regulation, we constructed a set of strains that bypass the chromosomal rearrangement, pro-protein processing, or both levels of control. Here we show that one of the functions of the chromosomal rearrangement and pro-protein processing is to prevent inappropriate production of sigma K under nonsporulation conditions. In the absence of both of these levels of control, a low level of sigma K-directed gene expression is observed during stationary phase after growth in rich medium. The appearance of sigma K under these conditions is probably due to a low level of sigma K-directed transcription from the sigK promoter in a positive feedback loop. We also report the construction of a strain that produces high levels of sigma K during growth. Using this strain, we demonstrate that the production of sigma K during growth is sufficient to induce a cascade of gene expression that closely mimics late events in the mother-cell line of gene expression.

Bacillus subtilis↗

PubMed1993

Cloning and characterization of a gene required for assembly of the Bacillus subtilis spore coat.

During endospore formation in Bacillus subtilis, approximately a dozen proteins are synthesized and assembled around the prespore to form a protective coat. Little is known about the assembly process, but several of the genes encoding these coat proteins are expressed in the mother cell compartment, where the proteins accumulate on the outer side of the developing endospore. Transcription of these genes is directed by the mother cell-specific sigma factor, sigma K, during the later stages of endospore development. sigma E may direct expression of the genes that encode proteins that function in the earliest stages of coat assembly. By screening for sigma E-dependent promoters, we cloned a gene, designated spoVID, required for assembly of a normal spore coat. Expression of spoVID was initiated at about the second hour of sporulation and continued throughout development from a sigma E-dependent promoter. The spoVID gene was located on the B. subtilis chromosome just downstream of the previously characterized hemAXCDBL operon and is predicted to encode an extremely acidic protein with 575 residues. Insertion mutants of spoVID produced refractile spores that were resistant to heat and to chloroform but were sensitive to lysozyme. Electron microscopic examination of sporulating spoVID mutant cells revealed normal morphological development up to about the third hour of sporulation. However, during the later stages of development the coat proteins assembled into aberrant structures that occurred freely in the mother cell cytoplasm and that consisted of reiterations of the single inner and outer layers that normally make up the spore coat.

Amino Acid Sequence↗

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↗