Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation

We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate. The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 mM L-glutamate, and D-methionine was used instead of L-methionine. When in this way conditions of partial amino acid deprivation were achieved, a partial "stringent response" occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation. In the corresponding relaxed (relA) mutants, the changes of guanine nucleotides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.     

Nitrofurantoin prompts the stringent response in Bacillus subtilis

Nitrofurantoin causes the stringent response in Bacillus subtilis. After exposure of a stringent strain to this drug, the intracellular concentrations of guanosine 3'-diphosphate 5'-diphosphate (ppGpp), guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and ATP increased, while that of GTP decreased. In a relaxed strain no accumulation of ppGpp or pppGpp was observed, but both GTP and ATP declined after the addition of nitrofurantoin. Protein synthesis was equally sensitive to nitrofurantoin in both the stringent and relaxed strains, but the drug inhibited RNA accumulation only in the stringent strain, not in the relaxed strain. Nitrofurantoin also caused the accumulation of ppGpp in Escherichia coli and Serratia marcescens.     

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.     

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.     

Induction of stringent response by streptomycin in Bacillus subtilis cells

A stringent response was induced in Bacillus subtilis vegetative cells by streptomycin. This was confirmed as follows: In B. subtilis stringent cells (BR16S), stable RNA synthesis was repressed, and pppGpp and ppGpp were transiently synthesized in the presence of required amino acids and streptomycin. However, these phenomena were not observed in the isogenic relaxed strain (BR16R) under the same conditions. On the other hand, tetracyclines did not induce the response, and, moreover, the stringent response to streptomycin upon pretreatment of the stringent cells with the antibiotics was released.     

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.     

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.     

Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression.

The Bacillus subtilis gsiA operon was induced rapidly, but transiently, as cells entered the stationary phase in nutrient broth medium. A mutation at the gsiC locus caused sporulation to be defective and expression of gsiA to be elevated and prolonged. The sporulation defect in this strain was apparently due to persistent expression of gsiA, since a gsiA null mutation restored sporulation to wild-type levels. Detailed mapping experiments revealed that the gsiC82 mutation lies within the kinA gene, which encodes the histidine protein kinase member of a two-component regulatory system. Since mutations in this gene caused a substantial blockage in expression of spoIIA, spoIIG, and spoIID genes, it seems that accumulation of a product of the gsiA operon interferes with sporulation by blocking the completion of stage II. It apparently does so by inhibiting or counteracting the activity of KinA.     

Evidence that Bacillus subtilis sporulation induced by the stringent response is caused by the decrease in GTP or GDP.

Partial amino acid deprivation of Bacillus subtilis, which evokes the stringent response, initiates sporulation not because the highly phosphorylated guanine nucleotides guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) increase but because GTP decreases. This was shown with a mutant (Myc) partially resistant to mycophenolate, an inhibitor of IMP dehydrogenase. Upon amino acid deprivation, the Myc mutant (62032) showed the usual increase in ppGpp and pppGpp but a reduced decrease in GTP, and only few cells sporulated. Extensive sporulation was restored by the addition of mycophenolate or decoyinine, and inhibitor of GMP synthetase, which caused a further decrease in GTP.     

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.     

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.     

Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion.

Bacillus subtilis strains carrying div-341 or sacU mutations, or both, have been characterized to reveal the roles of both genes in the initiation of sporulation, as well as in cell division and exoenzyme secretion. Both mutations were closely linked by transformation and caused the pleiotropic effects on sporulation and sporulation-associated events. Some sacU mutations (sacUh) resulted in hyperproduction of exoenzymes, reduced autolysis, and an ability to sporulate in the presence of excess nutrients. The div-341 mutation, on the other hand, resulted in filamentous growth at a higher temperature (45 degrees C) and showed spo0 properties at an intermediate permissive temperature (37 degrees C) in the usual sporulation medium. However, the div-341 strain sporulated better than wild-type strain at 37 degrees C in the presence of excess nutrients. Exoenzyme production and autolysis were reduced at 37 degrees C in the div-341 strain. A double mutant with sacUh32 and div-341 showed the complex phenotypes. It showed the sacUh32 property of autolysis and exoenyzme secretion. It showed the sacUh32 property of sporulation at 30 degrees C and the div-341 property at 37 degrees C. Slow growth and defective spore outgrowth of the div-341 strain at 37 degrees C were not observed in the double-mutant strain. Based on pleiotropic phenotypes and close linkages of both mutations, we discuss the relationship between the sacU and div-341 genes and their roles in sporulation, exoenzyme secretion, and cell division.