Activation Of σF Research Articles

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Noise in a phosphorelay drives stochastic entry into sporulation in Bacillus subtilis.

Entry into sporulation in Bacillus subtilis is governed by a phosphorelay in which phosphoryl groups from a histidine kinase are successively transferred via relay proteins to the response regulator Spo0A. Spo0A~P, in turn, sets in motion events that lead to asymmetric division and activation of the cell-specific transcription factor σF, a hallmark for entry into sporulation. Here, we have used a microfluidics-based platform to investigate the activation of Spo0A and σF in individual cells held under constant, sporulation-inducing conditions. The principal conclusions were that: (i) activation of σF occurs with an approximately constant probability after adaptation to conditions of nutrient limitation; (ii) activation of σF is tightly correlated with, and preceded by, Spo0A~P reaching a high threshold level; (iii) activation of Spo0A takes place abruptly just prior to asymmetric division; and (iv) the primary source of noise in the activation of Spo0A is the phosphorelay. We propose that cells exhibit a constant probability of attaining a high threshold level of Spo0A~P due to fluctuations in the flux of phosphoryl groups through the phosphorelay.

Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA.

Sporulation in the bacterium Bacillus subtilis is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σF, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σF. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σF is activated.

FtsA mutants of Bacillus subtilis impaired in sporulation.

Spore formation in Bacillus subtilis involves a switch in the site of cell division from the midcell to a polar position. Both medial division and polar division are mediated in part by the actin-like, cytokinetic protein FtsA. We report the isolation of an FtsA mutant (FtsA(D265G)) that is defective in sporulation but is apparently unimpaired in vegetative growth. Sporulating cells of the mutant reach the stage of asymmetric division but are partially blocked in the subsequent morphological process of engulfment. As judged by fluorescence microscopy and electron microscopy, the FtsA(D265G) mutant produces normal-looking medial septa but immature (abnormally thin) polar septa. The mutant was unimpaired in transcription under the control of Spo0A, the master regulator for entry into sporulation, but was defective in transcription under the control of sigmaF, a regulatory protein whose activation is known to depend on polar division. An amino acid substitution at a residue (Y264) adjacent to D265 also caused a defect in sporulation. D265 and Y264 are conserved among endospore-forming bacteria, raising the possibility that these residues are involved in a sporulation-specific protein interaction that facilitates maturation of the sporulation septum and the activation of sigmaF.

Mutational analysis of the early forespore/mother-cell signalling pathway in Bacillus subtilis.

Intercellular communication is a crucial phenomenon during spore development in Bacillus subtilis. It couples the establishment of a compartment-specific genetic program to the transcriptional activity of a sigma factor in the other compartment. It also keeps sigma factor activation in register with the morphological process. This study used directed mutagenesis to analyse the pathway that couples sigma E activation in the mother-cell to activation of sigma F in the forespore following asymmetric septation. Targets for mutagenesis in SpoIIGA (the receptor) were chosen based on the predicted topology of the protein when associated with the cell membrane. The results showed that a residue near the N terminus (D6), predicted to be exposed outside the cell, is required for receptor activity, whereas the major extracellular loop (between membrane domains IV and V) is dispensable for function. In contrast, mutations in SpoIIR (the signal) that partially blocked protein release (but not membrane translocation) had no effect on signal transduction. These results do not rule out the possibility that uncharacterized molecules intervene in the signalling pathway that establishes the mother-cell-specific developmental program during the early stage of sporulation.

Two-stage regulation of an anti-sigma factor determines developmental fate during bacterial endospore formation.

During endospore formation in Bacillus subtilis an asymmetric division produces two cells, forespore and mother cell, which follow different developmental paths. Commitment to the forespore-specific developmental path is controlled in part by the activation of the forespore-specific RNA polymerase sigma factor, sigma F. Activity of sigma F is inhibited in the mother cell by the anti-sigma factor SpoIIAB. In the forespore, sigma F directs transcription of the structural gene for sigma G. However, sigma G does not become active until after engulfment of the forespore is complete. This sigma G activity is dependent upon the products of the spoIIIA operon. We showed that sigma G is present but mostly inactive in a spoIIIA mutant. We also demonstrated that the anti-sigma factor SpoIIAB can bind to sigma G in vitro. Moreover, a mutant form of sigma G that binds SpoIIAB inefficiently in vitro was shown to function independently of SpoIIIA during sporulation. These and previously reported results support a model in which SpoIIAB functions as an inhibitor of sigma G activity during sporulation. Therefore, we propose that the anti-sigma factor SpoIIAB antagonizes both sigma F and sigma G activities, and that this antagonism is relieved in the forespore in two stages. In the first stage, which follows septation, a SpoIIAA-dependent mechanism partially relieves SpoIIAB inhibition of sigma F activity in the forespore. In the second stage, which follows forespore engulfment, a SpoIIIA-dependent process inactivates SpoIIAB in the forespore, resulting in the activation of sigma G.

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

An early step in sporulation of the bacterium Bacillus subtilis, is the formation of two compartments in the developing sporangium: the mother cell and the forespore. These compartments differ in their programs of gene expression and developmental fate. The establishment of cell type within this simple developmental program, is accomplished by the compartmentalization of sigma subunits of RNA polymerase. The localization of these sigma factors results in compartment-specific gene expression. Recent experiments have elucidated some of the early steps in the establishment of cell type. After septum formation, the activity of the sigma factor, sigma F, is confined to the forespore compartment. This, in turn, results in the localized expression of another developmental sigma factor, sigma G. The forespore localization of these two sigma factors, establishes the forespore line of gene expression. sigma F and sigma G also regulate mother cell events. sigma F activity in the forespore regulates the proteolytic processing of sigma E within the mother cell compartment. The localization sigma E activity leads to mother cell expression of another sigma factor, pro-sigma K. The proteolytic processing of pro-sigma K to mature sigma K is controlled by the forespore sigma factor, sigma G. Mature sigma K then directs the transcription of mother cell specific genes. Therefore, the initial localization of sigma F activity to the forespore compartment, orchestrates the establishment of cell type in both forespore and mother cell compartments.