Synthesis of spoIIA and spoVA mRNA in Bacillus subtilis

The expression of the spoIIA and spoVA sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoVA loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.     

Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis

A spoIIA::lacZ gene fusion has been used to investigate the dependence pattern of expression of the spoIIA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the hybrid gene, begins to appear about 30 to 60 min after the induction of sporulation. spoIIA expression is dependent upon the products of all of the known spoO loci but on none of the 'later' loci tested. The beta-galactosidase activity falls after 1.5 h in Spo+ cells and in late-blocked mutants, but continued accumulation of the enzyme occurs in certain stage II mutants. Kinetic experiments suggest that the fall in activity may be, in part, the result of regulation at the level of translation. Mutations in several loci, spo0J, spoIIIF and spoVIC, delay expression of the operon by 1-3 h. The significance of these results in terms of models for the control of gene expression during sporulation is discussed.     

The Products of the spoVA Operon Are Involved in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis

Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.     

Transcriptional control of synthesis of acid-soluble proteins in sporulating Bacillus subtilis

The major acid-soluble spore proteins (ASSPs) isolated from mature spores of Bacillus subtilis are designated alpha, beta, and gamma (about 60, 60, and 100 amino acids in length, respectively). Alpha and beta are very similar, and gamma is very similar to a less predominant ASSP called delta (about 115 amino acids). A minor and very basic ASSP called epsilon is the same size as alpha and beta but is unrelated antigenically. These and several minor ASSPs comprise at least three related families of sporulation-specific gene products. Expression of the alpha and beta genes, detectable as functional mRNA in vitro, coincides with the time of synthesis of all of the major ASSPs in vivo. This apparently coordinate expression is dependent on at least the spo0A, spoIIA, and spoIIIA loci, but not on the spoIVA or spoVA loci, consistent with the late stage of this expression (initiating at 3.5 h after the start of sporulation and peaking at 5 h after start of sporulation). A few minor ASSPs may be asynchronously expressed.     

Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis

The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the beta-galactosidase gene, lacZ, of Escherichia coli so that the expression of beta-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of beta-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID::lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spo0 lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make beta-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA----[spoIIG, spoIIE]----[spoIID, spoIIB, spoIIF].     

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.     

The spoIIIA locus is not a major determinant of prespore-specific gene expression during sporulation in Bacillus subtilis.

During sporulation in Bacillus subtilis, expression of several prespore-specific genes is strongly dependent on the spoIIIE and spoIIIG gene products. Previous reports have also indicated a requirement for the products of the spoIIIA locus. However, we have now systematically studied six different well-defined spoIIIA mutations and find that, relative to spoIIIE and spoIIIG mutations, they have only a minor effect on the expression of two different prespore-specific genes, spoVA and sspA. Moreover, we have shown that strain IS37, which has been used as a spoIIIA mutant in several previous studies, actually contains a lesion in the spo0A gene. We conclude that spoIIIA has a relatively minor or indirect role in the regulation of prespore-specific gene expression.     

Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.     

Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis

We have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations. The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000. Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA. A novel form of complementation analysis, based on derivatives of bacteriophage phi 105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations. The spoVA locus is the largest polycistronic sporulation operon yet characterized.     

Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two sister cells, the prespore and the mother cell. Many of the genes that regulate sporulation (spo genes) are thought to be expressed differentially. However, direct demonstration of differential gene expression, by fractionation of prespore and mother cell proteins, is possible only at a relatively late stage of development. H. De Lencastre and P. J. Piggot (J. Gen. Microbiol. 114:377-389, 1979) have described a genetic method for determining the cellular location of the requirement for spo gene expression. Here we describe a similar method based on the use of integrational plasmids that can insertionally inactivate any given spo gene. Loss of the integrated plasmid by homologous recombination leads to the restoration of spo gene function. If this occurs just before sporulation begins, the phenotypes of the progeny of heat-resistant spores should depend on whether the gene is required in the prespore or the mother cell. Thus, we show that for known prespore-specific genes, such as spoIIIG and spoVA, only phenotypically Spo+ progeny that have lost the integrated plasmid are produced. In contrast, for mother-cell-specific genes, such as spoIIIC and spoVJ, a substantial proportion of the progeny are asporogenous, having retained the integrated plasmid. On the basis of our results, the spoIID and spoIIIA genes, which are expressed soon after division, appear to be required only in the mother cell compartment.     

Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.     

Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae

Maltose transport and maltase activities were inactivated during sporulation of a MAL constitutive yeast strain harboring different MAL loci. Both activities were reduced to almost zero after 5 h of incubation in sporulation medium. The inactivation of maltase and maltose permease seems to be related to optimal sporulation conditions such as a suitable supply of oxygen and cell concentration in the sporulating cultures, and occurs in the fully derepressed conditions of incubation in the sporulation acetate medium. The inactivation of maltase and maltose permease under sporulation conditions in MAL constitutive strains suggests an alternative mechanism for the regulation of the MAL gene expression during the sporulation process.