Temporal regulation of the Bacillus subtilis early sporulation gene spo0F

The initiation of sporulation in Bacillus subtilis depends on seven genes of the spo0 class. One of these, spo0F, codes for a protein of 14,000 daltons. We studied the regulation of spo0F by using spo0F-lacZ translational fusions and also measured Spo0F protein levels by immunoassays. spo0F-lacZ and Spo0F levels increased as the cells entered the stationary phase, and this effect was repressed by glucose and glutamine. Decoyinine, which lowers GTP levels and allows sporulation in the presence of normally repressing levels of glucose, induced spo0F-lacZ expression and raised Spo0F levels. The expression of spo0F-lacZ was dependent on spo0A, -0B, -0E, -0F, and -0H genes, a spo0H deletion causing the strongest effect. In most respects, the spo0F gene was regulated in a manner similar to that of spoVG. However, the presence of an abrB mutation did not relieve the dependence of spo0F gene expression on spo0A, as it does with spoVG (P. Zuber and R. Losick, J. Bacteriol. 169:2223-2230, 1987).     

Cloning of an early sporulation gene in Bacillus subtilis.

A 0.8-megadalton BglII restriction fragment of Bacillus licheniformis cloned into the BglII site of plasmid pBD64 can complement spo0H mutations of Bacillus subtilis. The clone was isolated by selecting for the Spo+ phenotype and antibiotic resistance, using the helper system described by Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980). The insert is functional in both orientations and thus presumably has its own promoter. A deletion generated within the 0.8-megadalton insert by HindIII restriction and subsequent religation eliminates the ability of the cloned fragment to complement spo0H mutations. The cloned B. licheniformis deoxyribonucleic acid segment specifies the synthesis, in minicells, of a polypeptide of approximately 27,000 daltons. This protein is observed with both orientations, but not when the HindIII deletion is present in the cloned B. licheniformis chromosomal fragment. We have also demonstrated that ribonucleic acid complementary to the cloned B. licheniformis sporulation gene is transcribed in B. licheniformis both during vegetative growth and sporulation.     

The role of the sporulation gene spollIE in the regulation of prespore-specific gene expression in Bacillus subtilis

The spoIIIG gene encodes a sigma factor that determines prespore-specific gene expression during sporulation in Bacillus subtilis. Correct spatial and temporal expression of the spoIIIG gene depends on a number of other sporulation (spo) genes, but only one of these genes, spoIIIE, has a specific effect on spoIIIG expression and not on gene expression in the other differentiating cell, the mother cell. However, the spoIIIE gene is expressed predominantly before differentiation begins. Thus, its product must play an important role in sensing or determining the spatial localization of prespore-specific gene expression in this system.     

Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis

Summary Spore formation in the Gram-positive bacterium Bacillus subtilis is a last resort adaptive response to starvation. To initiate sporulation, the key regulator in this process, Spo0A, needs to be activated by the so-called phosphorelay. Within a sporulating culture of B. subtilis, some cells initiate this developmental program, while other cells do not. Therefore, initiation of sporulation appears to be a regulatory process with a bistable outcome. Using a single cell analytical approach, we show that the autostimulatory loop of spo0A is responsible for generating a bistable response resulting in phenotypic variation within the sporulating culture. It is demonstrated that the main function of RapA, a phosphorelay phosphatase, is to maintain the bistable sporulation gene expression. As rapA expression is quorum regulated, it follows that quorum sensing influences sporulation bistability. Deletion of spo0E, a phosphatase directly acting on Spo0A approximately P, resulted in abolishment of the bistable expression pattern. Artificial induction of a heterologous Rap phosphatase restored heterogeneity in a rapA or spo0E mutant. These results demonstrate that with external phosphatases, B. subtilis can use the phosphorelay as a tuner to modulate the bistable outcome of the sporulating culture. This shows that B. subtilis employs multiple pathways to maintain the bistable nature of a sporulating culture, stressing the physiological importance of this phenomenon.     

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.     

Cloning of sporulation gene spoIIG in Bacillus subtilis.

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.     

Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis.

We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation. Sporangia were doubly stained with propidium iodide to visualize the forespore and mother cell nucleoids and with fluorescein-conjugated antibodies to visualize the location of beta-galactosidase produced under the control of the sporulation RNA polymerase sigma factors sigma E and sigma F. In confirmation and extension of earlier reports, we found that expression of a lacZ fusion under the control of sigma E was confined to the mother cell compartment of sporangia at the septation (II) and engulfment (III) stages of morphogenesis. Conversely, sigma F-directed gene expression was confined to the forespore compartment of sporangia at postseptation stages of development. Little indication was found for sigma E- or sigma F-directed gene expression prior to septation or in both compartments of postseptation sporangia. Gene expression under the control of the forespore sigma factor sigma G also exhibited a high level of compartmentalization. A high proportion of sporangia exhibited fluorescence in our immunostaining protocol, which should be suitable for the subcellular localization of sporulation proteins for which specific antibodies are available.     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.