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.

SummaryThe 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 genomewide 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 inhibited by Spo0A are genes encoding components of the DNA replication machinery and genes that govern flagellum biosynthesis and chemotaxis. Also included 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.

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.

The master regulator for entry into sporulation in Bacillus subtilis is the response regulator Spo0A, which directly governs the expression of about 121 genes. Using cells in which the synthesis of Spo0A was under the control of an inducible promoter or in which production of the regulatory protein was impaired by a promoter mutation, we found that sporulation required a high (threshold) level of Spo0A and that many genes in the regulon differentially responded to high and low doses of the regulator. We distinguished four categories of genes, as follows: (i) those that required a high level of Spo0A to be activated, (ii) those that required a high level of Spo0A to be repressed, (iii) those that were activated at a low level of the regulator, and (iv) those that were repressed at a low dose of the regulator. Genes that required a high dose of Spo0A to be activated were found to have low binding constants for the DNA-binding protein. Some genes that were turned on at a low dose of Spo0A either had a high binding constant for the regulatory protein or were activated by an indirect mechanism involving Spo0A-mediated relief of repression by the repressor protein AbrB. We propose that progressive increases in the level of Spo0A leads to an early phase of transcription in which genes that play auxiliary roles in development, such as cannibalism and biofilm formation, are turned on and a later phase in which genes that play a direct role in sporulation are activated.Entry into the developmental process of spore formation in Bacillus subtilis is governed by a master regulatory protein known as Spo0A (1, 21). Spo0A, which is a member of the response regulator family of DNA-binding proteins, is activated at the start of sporulation by a multicomponent phosphorelay consisting of at least three histidine autokinases, KinA, KinB, and KinC, and the phosphorelay proteins Spo0F and Spo0B (6). The kinases phosphorylate Spo0F. Spo0FϳP, in turn, transfers the phosphoryl group to Spo0B. Finally, Spo0BϳP transfers the phosphoryl group to, and thereby activates, Spo0A. Spo0A is additionally subject to control at the level of its synthesis by a positive feedback loop in which the regulatory protein indirectly stimulates the synthesis of the RNA polymerase sigma factor H , which, in turn, stimulates transcription of the gene for Spo0A as well as the genes for the phosphorelay components KinA and Spo0F (21). The level of phosphorylation of Spo0AϳP is also influenced by dedicated phosphatases that remove phosphoryl groups from Spo0FϳP (e.g., RapA) and from Spo0AϳP itself (Spo0E) (17,24,25). These regulatory mechanisms act in effect as a bistable switch in that under conditions that induce sporulation, only a portion of the cells in the population activate Spo0A, whereas the remainder of the cells do not (9, 16).Once activated by phosphorylation, the master regulator binds to a DNA sequence element known as the 0A box (21,23,33). In certain cases, such as the well-studied example of abrB (14,15,26,27,35,36,39), binding of Spo0AϳP to the 0...

Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase in Bacillus subtilis. Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase. To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotated B. subtilis open reading frames. In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H. This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes. In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H. Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.Bacterial sigma factors are positive regulators of gene expression that interact with core RNA polymerase and direct the initiation of transcription from defined promoter sequences (22, 25). The major sigma factor in most bacteria, sigma-A, is required for expression of many of the so-called housekeeping functions and the bulk of the RNA during growth. Many bacteria have multiple alternative sigma factors, which are responsible for directing transcription of specialized gene sets. Bacillus subtilis has at least 17 alternative sigma factors which are involved in a variety of processes, including certain stress responses, chemotaxis, and motility (25,30). One of the more dramatic examples of gene regulation by alternative sigma factors is the process of endospore formation (sporulation) in B. subtilis. The sporulation program of gene expression in B. subtilis is carried out under the direction of five alternative sigma factors whose activities are subject to spatial and temporal control (14, 51). Here we report the results of transcriptional profiling experiments aimed at identifying, on a genome-wide basis, genes under the control of one of these sigma factors, sigma-H.Sigma-H, the sigH (spo0H) gene product, directs the transcription of several genes that function in the transition from exponential growth to stationary phase, including the initiation of spore formation and entry into the state of genetic competence (1,7,12,20). Sigma-H is required at an early stage of sporulation and directly activates transcription of several sporulation genes including spo0A, spo0F, kinA, spo0M, spoVG, and spoVS and the spoIIA operon (2,4,28,41,42,46,54,59,61). Sigma-H also directs the transcription of several mem...

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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 fruitingbody-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.Formation of biofilms-surface-associated multicellular assemblages-is an important microbial survival strategy (4, 25). Relative to the rapid, continual, and extreme changes in environmental conditions that can characterize a planktonic existence, biofilms offer the constituent cells some shelter, enabling them to establish long-term relationships with each other and their immediate surroundings. Cells in different regions of a developing biofilm experience diverse environmental conditions, resulting in marked patterns of cellular differentiation (e.g., see references 5 and 16). Such spatiotemporal organization is particularly striking in biofilms formed by wild strains of the spore-forming bacterium Bacillus subtilis. Unlike strains that have been domesticated by decades of propagation in the laboratory (e.g., B. subtilis 168), which form thin and relatively undifferentiated biofilms, wild B. subtilis strains form elaborate multicellular communities that display conspicuous architectural features, such as fruiting-body-like aerial projections that extend from the surfaces of the biofilm. The tips of these fruiting bodies serve as preferential sites for spore formation (2).We are interested in discovering genes that are involved in biofilm and fruiting body formation. We have focused our efforts on a relatively undomesticated strain of B. subtilis, NCIB 3610 (hereafter referred to as "3610"), which forms robust and highly structured biofilms both in liquid and on solid medium (colonies). When inoculated into a standing culture of minimal medium, 3610 initially grows planktonically as motile, single cells. The ...

We describe a three-protein signal-transduction pathway that governs immunity to a protein toxin involved in cannibalism by the spore-forming bacterium Bacillus subtilis. Cells of B. subtilis enter the pathway to sporulate under conditions of nutrient limitation but delay becoming committed to spore formation by killing nonsporulating siblings and feeding on the dead cells. Killing is mediated by the exported toxic protein SdpC. We report that extracellular SdpC induces the synthesis of an immunity protein, SdpI, that protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor, SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SdpC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein.