Regulation of endospore formation in Bacillus subtilis

Spore formation in bacteria poses a number of biological problems of fundamental significance. Asymmetric cell division at the onset of sporulation is a powerful model for studying basic cell-cycle problems, including chromosome segregation and septum formation. Sporulation is one of the best understood examples of cellular development and differentiation. Fascinating problems posed by sporulation include the temporal and spatial control of gene expression, intercellular communication and various aspects of cell morphogenesis.     

Deletion of an rRNA gene set in Bacillus subtilis

One of the 10 rRNA gene sets found in a wild-type strain of Bacillus subtilis 168 was deleted, apparently by recombination between two tandemly repeated rRNA gene sets. The deletion strain grew as well as the wild-type strain under a variety of growth conditions and also showed no change in the sporulation efficiency.     

Processing of a membrane protein required for cell-to-cell signaling during endospore formation in Bacillus subtilis

Activation of the late prespore-specific RNA polymerase sigma factor sigma(G) during Bacillus subtilis sporulation coincides with completion of the engulfment process, when the prespore becomes a protoplast fully surrounded by the mother cell cytoplasm and separated from it by a double membrane system. Activation of sigma(G) also requires expression of spoIIIJ, coding for a membrane protein translocase of the YidC/Oxa1p/Alb3 family, and of the mother cell-specific spoIIIA operon. Here we present genetic and biochemical evidence indicating that SpoIIIAE, the product of one of the spoIIIA cistrons, and SpoIIIJ interact in the membrane, thereby linking the function of the spoIIIJ and spoIIIA loci in the activation of sigma(G). We also show that SpoIIIAE has a functional Sec-type signal peptide, which is cleaved during sporulation. Furthermore, mutations that reduce or eliminate processing of the SpoIIIAE signal peptide arrest sporulation following engulfment completion and prevent activation of sigma(G). SpoIIIJ-type proteins can function in cooperation with or independently of the Sec system. In one model, SpoIIIJ interacts with SpoIIIAE in the context of the Sec translocon to promote its correct localization and/or topology in the membrane, so that it can signal the activation of sigma(G) following engulfment completion.     

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.     

Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.     

Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.     

Deletion mutants of temperate Bacillus subtilis bacteriophage phi105.

Summary Six deletion mutants of temperate Bacillus subtilis phage 105 have been isolated on the basis of their increased resistance to chelating agents. The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes. All deletions are located in a region about 55–70% from one end of the DNA molecule, in the right half of the known genetic map of the phage. The segment 55–65% does not contain any genes essential for lytic growth or lysogenization. A gene(s) for immunity is located in a segment 65–70% from the left end.By electronmicroscopy of partially denatured 105 DNA two A-T rich regions have been localized in the right half of the molecule. One of these regions falls within the non-essential 55–65% DNA segment.     

Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis

The CotA laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.     

Coccus-shaped Bacillus subtilis cells are inhibited at stage 0 of sporulation

RodA and rodB mutations cause rod-shaped Bacillus subtilis cells to become coccus-shaped when the growth temperature is increased from 30 to 45 degrees C. At 30 degrees C four rod strains sporulated as well as the genetically closely related rod+ strains. In contrast, at 45 degrees C the sporulation frequencies of rod strains decreased approximately 10(2)- to 10(4)-fold, while those of rod+ strains remained either unchanged or decreased only slightly. Temperature shift experiments and ultrastructural data indicated that coccus-shaped cells were unable to form prespore septa and were, therefore, inhibited at stage 0 of sporulation.     

Autoregulation of alpha-amylase formation in Bacillus subtilis

The phenomenon of self-regulation of alpha-amylase formation in Bacillus subtilis is discovered. The dependence of regulatory effect upon the phase of culture development in shown.     

B-myb is required for inner cell mass formation at an early stage of development.

The myb gene family has three members, c-myb, A-myb, and B-myb, which have distinct expression patterns. Analyses of c-myb and A-myb mutant mice have indicated that c-myb and A-myb are important for hematopoiesis and spermatogenesis, respectively. However, there has been no evidence for a role for B-myb in development. To examine the role of B-myb in development, we generated B-myb-deficient mice by gene targeting. Although the heterozygous mutants were healthy, the homozygous mutants died at an early stage of development, around E4. 5-E6.5. In vitro culture of blastocyst indicated that B-myb is required for inner cell mass formation. Consistent with the important role of B-myb in early embryonic development, only B-myb among myb family members was expressed in embryonic stem cells. These results indicate that each of the three members of the myb gene family plays a distinct role during development.     

Characterization of LytH,a differentiation-associated peptidoglycan hydrolase of Bacillus subtilis involved in endospore cortex maturation

The cortex peptidoglycan from endospores of Bacillus subtilis is responsible for the maintenance of dormancy. LytH (YunA) has been identified as a novel sporulation-specific component with a role in cortex structure determination. The lytH gene was expressed only during sporulation, under the control of the mother cell-specific sigma factor sigma(K). Spores of a lytH mutant have slightly reduced heat resistance and altered staining when viewed by electron microscopy. Analysis of the peptidoglycan structure of lytH mutant spores shows the loss of muramic acid residues substituted with L-alanine and a corresponding increase in muramic acid residues substituted with tetrapeptide compared to those in the parent strain. In a lytH cwlD mutant, the lack of muramic acid residues substituted with L-alanine and delta-lactam leaves 97% of residues substituted with tetrapeptide. These results suggest that lytH encodes an L-Ala-D-Glu peptidase involved in production of single L-alanine side chains from tetrapeptides in the spore cortex. The lack of di- or tripeptides in a lytH mutant reveals the enzyme is an endopeptidase.