Initiation Of Sporulation Research Articles

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.

Dimer Form of Phosphorylated Spo0A, a Transcriptional Regulator, Stimulates thespo0FTranscription at the Initiation of Sporulation inBacillus subtilis

The Spo0A protein ofBacillus subtilisis a transcriptional regulator that shows extensive homology to the regulator proteins in bacterial two-component regulatory systems. Phosphorylation of Spo0A is absolutely necessary for the initiation of sporulation. We now show that phospho-Spo0A is a dimer, binds specifically to thespo0Fpromoter region, and stimulates the transcription from the P2 promoter recognized by σH-RNA polymerase. Biochemical and biological analyses suggest that phospho-Spo0A interacts directly with the “0A-like box” sequence (TGTCGTA) located in thespo0Fpromoter region. Phosphorylation of Spo0A enhanced its affinity to the 0A-like box. Evidence is also presented that thespo0Fpromoter region contains a static bend having two sets of oligo(dA-dT) tracts. It was demonstrated that the bending region overlaps with the recognition site for the phospho-Spo0A.f2f2Present address: M. Asayama, Faculty of Agriculture, Ibaraki University, Ami, Ibaraki 300-03, Japan.

Possible role for the essential GTP-binding protein Obg in regulating the initiation of sporulation in Bacillus subtilis

We fused obg, encoding an essential GTP-binding protein in Bacillus subtilis, to the LacI-repressible, IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter Pspac. Depletion of Obg, following removal of IPTG, caused a defect in sporulation and in expression of sporulation genes that are activated by Spo0A approximately P. These defects were significantly relieved by a mutation in spo0A (rvtA11) that bypasses the normal phosphorylation pathway, indicating that Obg might normally be required, either directly or indirectly, to stimulate activity of the phosphorelay that activates Spo0A.

Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis.

Development of genetic competence in Bacillus subtilis is regulated by extracellular signaling molecules, including the ComX pheromone, a modified 9- or 10-amino-acid peptide. Here, we present characterization of a second extracellular competence stimulating factor (CSF). CSF appears to be, at least in part, a small peptide of between 520 and 720 daltons. Production of CSF requires several genes that are needed both for initiation of sporulation and development of competence (spo0H, spo0A, spo0B, and spo0F). Although both peptide factors regulate competence, two different sensing pathways mediate the response to the ComX pheromone and CSF. Analysis of double mutants indicated that ComX pheromone is on the same genetic pathway as the membrane-bound histidine protein kinase encoded by comP and that CSF is on the same genetic pathway as the oligopeptide permease encoded by spo0K. Furthermore, the cellular response to partly purified ComX pheromone requires the ComP histidine protein kinase, whereas the response to partly purified CSF requires the Spo0K oligopeptide permease. These two sensing pathways converge to activate competence genes. Both factors and their convergent sensing pathways are required for normal development of competence and might function to integrate different physiological signals.

Different roles for KinA, KinB, and KinC in the initiation of sporulation in Bacillus subtilis

Activation (phosphorylation) of the transcription factor encoded by spo0A is essential for the initiation of sporulation in Bacillus subtilis. At least three histidine protein kinases are involved in the phosphorylation of Spo0A. Under some growth conditions, KinA was the primary kinase, but under other conditions, KinB had the more critical role. KinC was required for the initial activation of Spo0A, even in the presence of KinA and KinB.

Isolation and characterization of kinC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinases KinA and KinB in Bacillus subtilis

Phosphorylation of the transcription factor encoded by spo0A is required for the initiation of sporulation in Bacillus subtilis. Production and accumulation of Spo0A-P is controlled by histidine protein kinases and the spo0 gene products. To identify additional genes that might be involved in the initiation of sporulation and production of Spo0A-P, we isolated genes which when present on a multicopy plasmid could suppress the sporulation defect of a spo0K mutant. kinC was one gene isolated in this way. A multicopy plasmid containing kinC completely or partially suppressed the sporulation defect caused by mutations in spo0K, kinA, spo0F, and spo0B, indicating that at least when overexpressed, KinC is capable of stimulating phosphorylation of Spo0A independently of the normal phosphorylation pathway. The predicted product of kinC is 428 amino acids long and is most similar to KinA and KinB, the histidine protein kinases involved in the initiation of sporulation. In otherwise wild-type strains, kinC null mutations caused little or no defect in sporulation under the conditions tested. However, in the absence of a functional phosphorelay (spo0F or spo0B), KinC appears to be the kinase responsible for phosphorylation of the sof-1 and rvtA11 forms of Spo0A.

Genetic Networks Controlling the Initiation of Sporulation and the Development of Genetic Competence in Bacillus subtilis

Genetic Networks Controlling the Initiation of Sporulation and the Development of Genetic Competence in Bacillus subtilis

Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant.

The spo0A gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spo0A mutant of B. subtilis. In this paper we describe the construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins. The spo0A gene of Bt was cloned and identified by its ability to transform a B. subtilis spo0A mutant to prototrophy. Its nucleotide sequence is homologous to the B. subtilis gene. The spo0A gene was replaced in the Bt genome with a disrupted copy to give an Spo- strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spo0A mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulated toxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spo0A mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.

Molecular genetics and the initiation of solventogenesis in Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052

A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spo0A plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii. These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5′ regulatory regions. This invites the speculation that they are under direct control of Spo0A, and additional data are now required to test this prediction.

Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis

The initiation of sporulation in B. subtilis is regulated by the Spo0A transcription factor, which is activated by phosphorylation to control developmental switching from the vegetative to the sporulation state. The level of phosphorylation of Spo0A is regulated by the phosphorelay, a signal transduction system based on the protein-histidine kinase-response regulator two-component paradigm. To initiate sporulation, the cell must recognize and interpret a large variety of environmental, metabolic, and cell cycle signals that influence the phosphorylation level of Spo0A. We describe here a family of protein-aspartate phosphatases with activity of Spo0F∼P, a response regulator component of the phosphorelay, that provide a mechanism for signal recognition and interpretation. These phosphatases function to drain the phosphorelay, lower Spo0A∼P levels, and prevent sporulation. The integration of diverse environmental signals that affect the initiation of sporulation likely occurs through the competition between opposing activities of protein kinases and protein phosphatases.

Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA‐binding domain

Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis. It is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-containing surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spoIVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria.

Spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis.

The spo0J gene of Bacillus subtilis is required for the initiation of sporulation. We show that the sporulation defect caused by null mutations in spo0J is suppressed by a null mutation in the gene located directly upstream from spo0J, soj (suppressor of spo0J). These results indicate that Soj inhibits the initiation of sporulation and that Spo0J antagonizes that inhibition. Further genetic experiments indicated that Soj ultimately affects sporulation by inhibiting the activation (phosphorylation) of the developmental transcription factor encoded by spo0A. In addition, the temperature-sensitive sporulation phenotype caused by the ftsA279 (spoIIN279) mutation was partly suppressed by the soj null mutation, indicating that FtsA might also affect the activity of Soj. Soj and Spo0J are known to be similar in sequence to a family of proteins involved in plasmid partitioning, including ParA and ParB of prophage P1, SopA and SopB of F, and IncC and KorB of RK2, spo0J was found to be required for normal chromosome partitioning as well as for sporulation. spo0J null mutants produced a significant proportion of anucleate cells during vegetative growth. The dual functions of Spo0J could provide a mechanism for regulating the initiation of sporulation in response to activity of the chromosome partition machinery.

Effect of glucose on sporulation and extracellular amylase production byClostridium perfringens type A in a defined medium

The effect of glucose and other sugars on sporulation and extracellular amylase production byClostridium perfringens NCTC 8679 type A in a defined medium was studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity, while disaccharides such as lactose, maltose, and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10mm except for fructose (25mm). Levels of heat-resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in this strain ofC. perfringens.

Subunit composition and domain structure of the Spo0A sporulation transcription factor of Bacillus subtilis

The Spo0A transcription factor is responsible for the initiation of sporulation and is active in transcription only after phosphorylation by a specific signal transduction pathway, the phosphorelay. The effect of phosphorylation on the physical properties of Spo0A was determined. Spo0A and Spo0A approximately P both behaved as monomers during Sephacryl chromatography and gel electrophoresis, suggesting that phosphorylation did not modify the oligomerization state of the protein. Trypsin digested Spo0A at a single cleavage site between residues 142 and 143 within a hinge connecting two tightly folded domains. The amino domain retains ability to be phosphorylated by the phosphorelay. The carboxyl domain is active as a DNA-binding protein and retains the sequence specificity of the intact molecule for 0A boxes on the abrB promoter as revealed by footprinting studies. The carboxyl domain stimulated in vitro transcription from the spoIIG promoter 5-fold greater than an equal amount of Spo0A and about half as well as equivalent amounts of Spo0A approximately P. Thus, the unphosphorylated amino domain inhibits the transcription stimulation activity of the carboxyl domain. We suggest that phosphorylation activates transcription regulation functions of Spo0A by modifying the spatial relationships of the amino and carboxyl domains.

Activation of spo0A transcription by sigma H is necessary for sporulation but not for competence in Bacillus subtilis.

spo0A and spo0H are needed for the initiation of sporulation and for the development of genetic competence in Bacillus subtilis. Transcription of spo0A initiates from two promoters, Pv and Ps. Pv is active during vegetative growth and is recognized by RNA polymerase containing sigma A. Expression from Ps increases during sporulation and depends on sigma H, the spo0H gene product. A deletion mutation, spo0A delta Ps, that removes the promoter controlled by sigma H blocked sporulation but had no detectable effect on competence. These results indicate that expression of spo0A from Ps is necessary for sporulation and that the requirement for spo0H in competence development is not due to its role in expression of spo0A.

Sigma factors, asymmetry, and the determination of cell fate in Bacillus subtilis.

Soon after the initiation of sporulation, Bacillus subtilis divides asymmetrically to produce sister cells that have very different developmental fates. Recently, it has been proposed that the differential gene expression which begins soon after this division is due to cell-specific activation of the transcription factors sigma F and sigma E in the prespore and the mother cell, respectively. We describe the use of a method for the localization of gene expression in individual sporulating cells that lends strong support to the cell-specific localization of sigma F and sigma E activities. The dependence of sigma E activity on integrity of the gene encoding sigma F has led to the suggestion that activation of sigma F in the prespore leads to a directional signal that triggers activation of sigma E only in the mother cell. Here we show that sigma E actually specifies the fate of the mother cell; in the absence of sigma E, two prespore-like cells are made. The appearance of sigma F activity at both poles of a sigma E-deficient mutant supports the idea that sigma F normally remains latent in the mother cell and that its activation depends on some morphological or physiological feature of the prespore. We present a model for the generation of asymmetry and the establishment of cell fate in B. subtilis.

A developmental checkpoint couples the initiation of sporulation to DNA replication in Bacillus subtilis.

Spore formation in Bacillus subtilis requires the generation of two distinct cell types, each with an active chromosome that becomes committed to a defined program of gene expression. Here we show that a developmental checkpoint couples the initiation of sporulation, and the subsequent formation of these two cell types, to DNA replication early during development. Inhibiting the initiation of chromosomal replication prevents the onset of sporulation and inhibits expression of several genes that are normally induced early during development. This defect in gene expression is due to inhibition of the multi-component phosphorylation pathway needed to activate the developmental transcription factor encoded by spo0A. The target affected by inhibiting the initiation of replication is neither Spo0A nor the major kinase, KinA, needed for production of Spo0A approximately P. Rather, the target appears to be one of the proteins that transfers phosphate from the kinase to the Spo0A transcription factor. The signal that couples activity of the phosphorelay to the initiation of DNA replication is different from the previously described DNA damage signal that inhibits the phosphorelay during SOS induction in a recA-dependent response. Thus, DNA replication as well as DNA damage signals control production of Spo0A approximately P and initiation of sporulation.

Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation

Early during endospore formation in the bacterium Bacillus subtilis, two distinct cell types are formed. The initiation of this developmental pathway requires several physiological conditions (e.g., nutrient deprivation) and is controlled by the Spo0A transcription factor. We have found that in a culture of sporulating cells, there are two subpopulations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early developmental gene expression remains uninduced. We measured the expression of developmental (spo) genes in single cells of B. subtilis by using spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained with a dye that fluoresces upon hydrolysis by beta-galactosidase, and the fluorescence in individual cells was measured with a flow cytometer. For Spo+ cells, we found that the proportion of the population expressing early developmental genes correlates well with the fraction of the population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0A transcription factor cause a decrease in the size of the subpopulation expressing early developmental genes that are directly activated by Spo0A approximately P. Again, the size of the subpopulation correlates well with the fraction of cells that produce spores. These results indicate that a threshold level of activated Spo0A (Spo0A approximately P) or of a component of the phosphorylation pathway must accumulate to induce sporulation gene expression and that most of the cells that are able to induce the expression of early genes that are directly activated by Spo0A approximately P go on to produce mature spores.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the phosphorelay and the initiation of sporulation in Bacillus subtilis.

The initiation of sporulation of bacteria is a complex cellular event controlled by an extensive network of regulatory proteins that serve to ensure that a cell embarks on this differentiation process only when appropriate conditions are met. The major signal-transduction pathway for the initiation of sporulation is the phosphorelay, which responds to environmental, cell cycle, and metabolic signals, and phosphorylates the Spo0A transcription factor activating its function. Signal input into the phosphorelay occurs through activation of kinases to phosphorylate a secondary-messenger protein, Spo0F. Spo0F-P serves as a substrate for phosphoprotein phosphotransferase, Spo0B, which phosphorylates Spo0A. The pathway is regulated by transcriptional control of its component proteins and by regulating phosphate flux through the pathway. This is accomplished by several regulatory proteins, and by activated Spo0A, which regulates transcription of genes for its own synthesis. Spo0A-P indirectly controls the transcription of numerous genes by regulating the level of other transcription regulators and directly activates the transcription of several regulatory proteins and sigma factors required for progression to the second stage of sporulation. Although the pathway and regulatory proteins have been identified, the signals and effectors for these regulators remain a mystery.

Characterization of a new sporulation factor in Bacillus subtilis.

We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase. It is required in small amounts and is resistant to various proteolytic agents. Several spo mutants were tested for the ability to produce functional sporulation factor. All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells. Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation.