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R Losick

Publications and source records attributed to R Losick.

At least 19 recordsLinked to original sources
PubMed2004

SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2).

The sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365,023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma-butyrolactone signalling system, members of the actinomycete-specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA-containing genes is part of a large bldA-dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB-deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.

Amino Acid Sequence↗
PubMed2001

Differential gene expression governed by chromosomal spatial asymmetry.

The activity of the transcription factor sigmaF is confined to one (the forespore) of two cells created by asymmetric division during sporulation in B. subtilis. We show that sigmaF activation is partly governed by the position of the gene for the unstable anti-sigmaF factor SpoIIAB. Because cytokinesis precedes chromosome segregation, most of the chromosome is translocated into the forespore after division. We hypothesize that because spoIIAB enters the forespore late, SpoIIAB lost to proteolysis is temporarily not replenished. Thus, chromosome asymmetry would be translated into the asymmetric distribution of SpoIIAB. Supporting this idea, transposition of spoIIAB to sites present in the forespore at the time of division impaired sporulation when a second pathway that participates in sigmaF activation was disabled.

Bacillus subtilis↗
PubMed2001

Fruiting body formation by Bacillus subtilis.

Spore formation by the bacterium Bacillus subtilis has long been studied as a model for cellular differentiation, but predominantly as a single cell. When analyzed within the context of highly structured, surface-associated communities (biofilms), spore formation was discovered to have heretofore unsuspected spatial organization. Initially, motile cells differentiated into aligned chains of attached cells that eventually produced aerial structures, or fruiting bodies, that served as preferential sites for sporulation. Fruiting body formation depended on regulatory genes required early in sporulation and on genes evidently needed for exopolysaccharide and surfactin production. The formation of aerial structures was robust in natural isolates but not in laboratory strains, an indication that multicellularity has been lost during domestication of B. subtilis. Other microbial differentiation processes long thought to involve only single cells could display the spatial organization characteristic of multicellular organisms when studied with recent natural isolates.

Bacillus subtilis↗
PubMed2001

Self-reinforcing activation of a cell-specific transcription factor by proteolysis of an anti-sigma factor in B. subtilis.

The transcription factor sigma(F), which is activated in a cell-specific manner during sporulation in B. subtilis, is initially held in an inactive complex by the anti-sigma factor SpoIIAB. The anti-anti-sigma factor SpoIIAA reacts with SpoIIAB.sigma(F) to induce the release of free sigma(F) and free SpoIIAB. We now report that free SpoIIAB is subject to proteolysis and that it is protected from degradation by sigma(F) in the SpoIIAB.sigma(F) complex and by SpoIIAA in an alternative complex. Proteolysis requires residues located near the extreme C terminus of SpoIIAB and is dependent upon the ClpCP protease. The reaction of SpoIIAA with SpoIIAB.sigma(F) and the resulting degradation of newly released SpoIIAB could set up a self-reinforcing cycle that locks on the activation of sigma(F).

Amino Acid Sequence↗
PubMed2001

Morphological coupling in development: lessons from prokaryotes.

At certain junctures in development, gene transcription is coupled to the completion of landmark morphological events. We refer to this dependence on morphogenesis for gene expression as "morphological coupling." Three examples of morphological coupling in prokaryotes are reviewed in which the activation of a transcription factor is tied to the assembly of a critically important structure in development.

Animals↗
PubMed2001

A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis.

We present evidence for a three-protein inhibitor of polar division that locks in asymmetry after the formation of a polar septum during sporulation in Bacillus subtilis. Asymmetric division involves the formation of cytokinetic Z-rings near both poles of the developing cell. Next, a septum is formed at one of the two polar Z-rings, thereby generating a small, forespore cell and a mother cell. Gene expression under the control of the mother-cell transcription factor sigmaE is needed to block cytokinesis at the pole distal to the newly formed septum. We report that this block in polar cytokinesis is mediated partly by sigmaE-directed transcription of spoIID, spoIIM and spoIIP, sporulation genes that were known to be involved in the subsequent process of forespore engulfment. We find that a spoIID, spoIIM and spoIIP triple mutant substantially mimicked the bipolar division phenotype of a sigmaE mutant and that cells engineered to produce SpoIID, SpoIIM and SpoIIP prematurely were inhibited in septum formation at both poles. Consistent with the hypothesis that SpoIID, SpoIIM and SpoIIP function at both poles of the sporangium, a GFP--SpoIIM fusion localized to the membrane that surrounds the engulfed forespore and to the potential division site at the distal pole.

Bacillus subtilis↗
PubMed2001

Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis.

Entry into sporulation in Bacillus subtilis is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell. Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell. Replication origin regions were visualized by the use of a cassette of tandem copies of lacO that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI. The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented. On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore. Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (smc) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division. A small proportion ( approximately 1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region. Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ. A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.

Bacillus subtilis↗
PubMed2001

Cytological evidence for association of the ends of the linear chromosome in Streptomyces coelicolor.

The chromosome of the filamentous bacterium Streptomyces coelicolor is linear, but the genetic map is circular. We present cytological evidence based on the use of fluorescence in situ hybridization showing that the ends of the chromosome frequently colocalize, in agreement with the idea that the ends are held together, effectively forming a circular chromosome. These observations provide a possible explanation for how a linear bacterial chromosome can exhibit a circular genetic map.

Base Sequence↗
PubMed2001

RNA polymerase sigma factor that blocks morphological differentiation by Streptomyces coelicolor.

The filamentous bacterium Streptomyces coelicolor undergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation. Genes required for the initiation of aerial mycelium formation have been termed bld (bald), describing the smooth, undifferentiated colonies of mutant strains. By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a bld mutant harboring an insertion in a previously uncharacterized gene, SCE59.12c, renamed here rsuA. The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin. The rsuA gene encodes an apparent anti-sigma factor and is located immediately downstream of SCE59.13c, renamed here sigU, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors. The absence of rsuA in a strain that contained sigU caused a block in development, and the overexpression of sigU in an otherwise wild-type strain caused a delay in aerial mycelium formation. However, a strain in which both rsuA and sigU had been deleted was able to undergo morphological differentiation normally. We conclude that the rsuA-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded by sigU. We also conclude that the sigU-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage.

Amino Acid Sequence↗
PubMed2000

Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation.

The filamentous soil bacterium Streptomyces coelicolor undergoes a complex cycle of morphological differentiation involving the formation of an aerial mycelium and the production of pigmented antibiotics. We have developed a procedure for generating insertional mutants of S. coelicolor based on in vitro transposition of a plasmid library of cloned S. coelicolor DNAs. The insertionally mutated library was introduced into S. coelicolor, and transposon insertions were recovered at widely scattered locations around the chromosome. Many of the insertions revealed previously uncharacterized genes, and several caused novel mutant phenotypes, such as altered pigment production, enhanced antibiotic sensitivity, delayed or impaired formation of aerial hyphae, and a block in spore formation. The sporulation mutant harbored an insertion in one of three adjacent genes that are apparently unique to Streptomyces but are each represented by at least 20 paralogs at dispersed locations in the chromosome. Individual members of the three families often are found grouped together in a characteristic arrangement, suggesting that they have a common function.

Amino Acid Sequence↗
PubMed2000

The transcriptional profile of early to middle sporulation in Bacillus subtilis.

Spore formation by Bacillus subtilis is governed by global changes in gene transcription. We used nylon-substrate DNA arrays representing approximately 96% of the predicted open reading frames in the B. subtilis chromosome to compare the pattern of transcripts from wild-type cells with the pattern from cells mutant for the sporulation transcription factors Spo0A or final sigma(F). We found 520 genes whose transcript levels were at least 3-fold dependent on Spo0A but not on final sigma(F), and an additional 66 genes whose transcript levels were dependent upon both regulatory proteins. Two strategies were used to help assign genes to the direct control of a particular developmental regulatory protein. In one approach, we analyzed the effects on global gene expression of artificially producing a constitutively active form of Spo0A during growth. In a second approach, Hidden Markov models were used to identify promoters likely to be activated by Spo0A, final sigma(F), or a third sporulation transcription factor, final sigma(E). In addition to detecting known sporulation genes, we identified many genes of unknown function whose patterns of expression and regulation suggest that they could be involved in sporulation. Disruption of two such newly identified genes, yabP and yabQ, blocked sporulation at a late stage.

Amino Acid Sequence↗
PubMed1999

A family of membrane-embedded metalloproteases involved in regulated proteolysis of membrane-associated transcription factors.

We present evidence that the sporulation protein SpoIVFB of Bacillus subtilis is a member of a newly recognized family of metalloproteases that have catalytic centers adjacent to or within the membrane. SpoIVFB is required for converting the membrane-associated precursor protein, pro-sigma(K), to the mature and active transcription factor sigma(K) by proteolytic removal of an N-terminal extension of 20 amino acids. SpoIVFB and other family members share the conserved sequence HEXXH, a hallmark of metalloproteases, as well as a second conserved motif NPDG, which is unique to the family. Both motifs, which are expected to form the catalytic center of the protease, overlap hydrophobic segments that are predicted to be separate transmembrane domains. The only other characterized member of this family of membrane-embedded metalloproteases is the mammalian Site-2 protease (S2P), which is required for the intramembrane cleavage of the eukaryotic transcription factor sterol regulatory element binding protein (SREBP). We report that amino acid substitutions in the two conserved motifs of SpoIVFB impair pro-sigma(K) processing and sigma(K)-directed gene expression during sporulation. These results and those from a similar analysis of S2P support the interpretation that both proteins are founding members of a family of metalloproteases involved in the activation of membrane-associated transcription factors. Thus, the pathways that govern the activation of the prokaryotic transcription factor pro-sigma(K) and the mammalian transcription factor SREBP not only are analogous but also use processing enzymes with strikingly homologous features.

Amino Acid Motifs↗
PubMed1999

Septation, dephosphorylation, and the activation of sigmaF during sporulation in Bacillus subtilis.

Cell-specific activation of transcription factor sigmaF during sporulation in Bacillus subtilis requires the formation of the polar septum and the activity of a serine phosphatase (SpoIIE) located in the septum. The SpoIIE phosphatase indirectly activates sigmaF by dephosphorylating a protein (SpoIIAA-P) in the pathway that controls the activity of the transcription factor. By use of a SpoIIE-GFP fusion protein in time-course and time-lapse experiments and by direct visualization of septa in living cells, we show that SpoIIE is present in the predivisional sporangium, where it often localizes near both cell poles in structures known as E-rings. We also present evidence consistent with the view that SpoIIE is present in both progeny cells after polar division. These findings are incompatible with a model for the control of sigmaF activity in which the phosphatase is simply sequestered to one cell. Instead, we conclude that the function of SpoIIE is subject to regulation, and we present evidence that this occurs in two stages. The first stage, which involves the phosphatase function of SpoIIE, depends on the cell division protein FtsZ and could correspond to the FtsZ-dependent assembly of SpoIIE into E-rings. The second stage occurs after the dephosphorylation of SpoIIAA-P and is dependent on the later-acting, cell-division protein DivIC. Evidence based on the use of modified and mutant forms of the phosphatase protein indicates that SpoIIE blocks the capacity of unphosphorylated SpoIIAA to activate sigmaF until formation of the polar septum is completed.

Bacillus subtilis↗
PubMed1999

Extracellular complementation and the identification of additional genes involved in aerial mycelium formation in Streptomyces coelicolor.

Morphogenesis in the bacterium Streptomyces coelicolor involves the formation of a lawn of hair-like aerial hyphae on the colony surface that stands up in the air and differentiates into chains of spores. bld mutants are defective in the formation of this aerial mycelium and grow as smooth, hairless colonies. When certain pairs of bld mutants are grown close to one another on rich sporulation medium, they exhibit extracellular complementation such that one mutant restores aerial mycelium formation to the other. The extracellular complementation relationships of most of the previously isolated bld mutants placed them in a hierarchy of extracellular complementation groups. We have screened for further bld mutants with precautions intended to maximize the discovery of additional genes. Most of the 50 newly isolated mutant strains occupy one of three of the previously described positions in the hierarchy, behaving like bldK, bldC, or bldD mutants. We show that the mutations in some of the strains that behave like bldK are bldK alleles but that others fall in a cluster at a position on the chromosome distinct from that of any known bld gene. We name this locus bldL. By introducing cloned genes into the strains that exhibit bldC or bldD-like extracellular complementation phenotypes, we show that most of these strains are likely to contain mutations in genes other than bldC or bldD. These results indicate that the genetic control of aerial mycelium formation is more complex than previously recognized and support the idea that a high proportion of bld genes are directly or indirectly involved in the production of substances that are exchanged between cells during morphological differentiation.

Cell Differentiation↗