Cannibalism: a social behavior in sporulating Bacillus subtilis

A social behavior named cannibalism has been described during the early stages of sporulation of the Gram-positive Bacillus subtilis. This phenomenon is based on the heterogeneity of sporulating populations, constituted by at least two cell types: (1) sporulating cells, in which the master regulator of sporulation Spo0A is active, and (2) nonsporulating cells, in which Spo0A is inactive. Sporulating cells produce two toxins that act cooperatively to kill the nonsporulating sister cells. The nutrients released by the dead cells into the starved medium are used for growth by the sporulating cells that are not yet fully committed to sporulate, and as a result, sporulation is arrested. This review outlines the molecular mechanisms of the killing and immunity to the toxins, the regulation of their production and other examples of killing of siblings in microorganisms. The biological significance of this behavior is discussed.     

Binding of Radioactive Benzylpenicillin to Asporogenous Mutants of Bacillus subtilis During Postexponential Growth

The specific penicillin binding capacity of a postexponential culture of Staphylococcus aureus remains constant, but that of a sporulating Bacillus subtilis culture fluctuates dramatically. An initial decrease in binding capacity during presporulation events is followed by two distinct intervals of enhanced specific binding capacity during the postlogarithmic growth of a sporulating B. subtilis culture. The first peak of enhanced binding occurs during septation, when enzymes for germ cell wall formation are present; and the second peak coincides with cortical biosynthesis. The specific postlogarithmic binding capacities of a number of Spo(-) mutants of B. subtilis were examined to ascertain if specific asporogenous mutations altered the binding pattern observed with the wild-type organism. Four distinct postexponential binding patterns were recognized: (i) a low, constant binding capacity resembling the binding pattern of S. aureus, (ii) a decrease in binding capacity with no subsequent significant peaks, (iii) a decrease in binding capacity followed by a single peak corresponding to the first peak seen with the wild type, (iv) a pattern similar to the wild type. The fourth pattern was observed in a mutant blocked during stage III of sporogenesis which produced forespores that never became refractile. Mutations blocking either one or both periods of enhanced postlogarithmic binding were interspersed throughout a linkage group of spore genes next to lys-2 on the B. subtilis chomosome.     

Formation of competent Bacillus subtilis cells.

The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth. After formation, competent cells were no longer sensitive to the above agents. Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence. A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium. A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation. Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation. Competent cells were formed even in media which supported sporulation. The development of competence was also studied with spo0 mutants at 10 different loci. Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J. These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients. It is suggested that common steps are involved both in forespore septation and in competent cell formation.     

Netropsin stimulates the formation of an extracellular proteinase and suppresses protein turnover in sporulating Bacillus megaterium

Abstract Netropsin stimulated the rate of synthesis of an extracellular metalloproteinase in Bacillus megaterium incubated in a sporulation medium. The antibiotic delayed but did not suppress the decrease in the ability to synthesize the proteinase occurring at later sporulation stages. Netropsin also stimulated the synthesis of the proteinase when added to a growing culture; it inhibited the increase of protein turnover which was switched on between the 2nd and 3rd hour in the sporulating population. No refractile spores were developed during 6 h at 35°C in the antibiotic-treated culture. In the control 60% of sporulating cells were observed under similar conditions.     

Appearance of a New Species of Ribonucleic Acid During Sporulation in Saccharomyces cerevisiae

In the course of study on ribonucleic acid (RNA) metabolism during sporulation in Saccharomyces cerevisiae, a new species of RNA (20S) was observed in sporulating cells by polyacrylamide gel electrophoresis. The relative content of this RNA to total RNA increased linearly early in sporulation. Labeled adenine was preferentially incorporated into 20S RNA during the early stages of sporulation. The correlation between the physiological and genetic control of sporulation and the synthesis of 20S RNA are discussed.     

Lipid Synthesis During Sporulation of Saccharomyces cerevisiae

Lipid synthesis was studied in both sporulating (diploid) and nonsporulating (haploid) cells of Saccharomyces cerevisiae. Two phases of lipid synthesis occur in diploid cells transferred to sporulation medium. Phase I, which occurs during the first 12 h of exposure to sporulation medium, was also observed in the haploid strains. Phase II, occurring from the 20th to the 25th h, coincided with the appearance of mature asci and was observed only in the diploid cells. The majority of phospholipid synthesis took place during period I, whereas neutral lipid synthesis occurred during both periods. Phospholipid synthesis was virtually identical in both type and quantity in the sporulating and nonsporulating strains.     

Teichoic acid and lipid metabolism during sporulation of Bacillus megaterium KM.

The biochemistry of teichoic acid and lipid metabolism has been studied during sporulation of Bacillus megaterium KM. Measurements of cell-wall and membrane teichoic acid have shown that net synthesis of these polymers ceases at the onset of sporulation. Pulse-labelling studies show that the period of asymmetric septation and forespore engulfment is marked by an initiation of turnover of membrane teichoic acid but not of wall teichoic acid. This is reflected in the presence of inner-membrane teichoic acid and the virtual absence of wall teichoic acid in dormant spores. The total amount of lipid phosphorus in the sporulating cell increases by 70% as a result of asymmetric septation and subsequent engulfment of the forespore. The phosphorus requirement for this synthesis is derived from a pool formed during exponential growth, which is not exchangeable with extracellular Pi during sporulation. These results suggest that during sporulation a proportion of the glycerol 3-phosphate produced by preferential degradation of membrane teichoic acid formed during exponential growth is used for phospholipid synthesis during sporulation.     

Visualization of Penicillin-Binding Proteins during Sporulation of Streptomyces griseus

We used fluorescein-tagged β-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.     

End3p-mediated endocytosis is required for spore wall formation in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, vesicles transported to the vicinity of spindle pole bodies are fused to each other to generate bilayered prospore membranes (PSMs). PSMs encapsulate the haploid nuclei that arise from the meiotic divisions and serve as platforms for spore wall deposition. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3Delta, sporulated poorly and the spores produced lost resistance to ether vapor, suggesting that END3-mediated endocytosis is important for sporulation. End3p-GFP localized to cell and spore peripheries in vegetative and sporulating cells and colocalized with actin structures. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3Delta. Analysis of meiosis in end3Delta mutants revealed that the meiotic divisions occurred with wild-type kinetics. Furthermore, PSMs were assembled normally. However, the levels of proteins required for spore wall synthesis and components of the spore wall layers at spores were reduced, indicating that end3Delta mutants are defective in spore wall synthesis. Thus, END3-mediated endocytosis is important for spore wall formation. Additionally, cytological analyses suggest that trafficking between the plasma membrane and PSMs is important earlier during sporulation.     

Penicillin Binding Components in Bacillius subtilis During Sporulation

The penicillin binding components of sporulating Bacillus subtilis 3610 were compared with those of the vegetative organism. No qualitative difference in binding components was observed. The total binding, however, varied with the phase of sporulation.     

Two active forms of valyl-tRNA synthetase from Bacillus subtilis: alteration related to the early stages of sporulation

Two enzymatically active forms, E-I and E-II, of valyl-tRNA synthetase [EC 6.1.1.9] from cells at various stages in the life cycle of Bacillus subtilis 168 LTT, germinated cells, vegetative cells (t-0.5), sporulating cells (t0, t1, t3, and t4), forespores and mature spores, were analyzed by hydroxyapatite column chromatography. The E-II activity was detected in the main fraction of valyl-tRNA synthetase during the life cycle of B. subtilis 168 LTT. The high activity of E-II at t0 decreased rapidly in the stationary and sporulating phases. On the other hand, the E-I activity increased in the early sporulating stage and was about twofold higher at t3 than at t0. After t3, this activity also decreased rapidly and was not detected in forespores and mature spores. The relative amount of E-I at t0 was 3.4% of the total valyl-tRNA synthetase activity eluted from the hydroxyapatite column, 12.9% at t1 and 29.2% at t3, but it was less than 10% at t4 and in germinated cells. The alteration in E-I and E-II activities was also observed in cells of B. subtilis NIG 1121 (spo+), W23 and 168W, but not in any asporogenous mutant strain studied. These results show that the alteration in the valyl-tRNA synthetase activity appears only during the early stages of sporulation and is closely related to the sporulation of B. subtilis.     

Effects of antibiotics on synthesis and persistence of sigma E in sporulating Bacillus subtilis.

A potential regulatory link between the activation of a sporulation-specific sigma factor (sigma E) and forespore septum formation was investigated by treating Bacillus subtilis with inhibitors of protein or peptidoglycan synthesis and monitoring the consequences of these treatments on sigma E activation and septation. Western blot (immunoblot) and electron microscopic analyses revealed that both the formation of sigma E and septation were inhibited to a similar degree when either rifampin or chloramphenicol was added at different times before the second hour into sporulation but that penicillin preferentially blocked septation. We interpret these results as indicating that the syntheses of the gene products for both septation and sigma E activation occur at approximately the same time in development but that synthesis of an intact septum is unlikely to be a prerequisite for the formation of sigma E. We observed that penicillin could not only block septation but, depending on the time of its addition, could also inhibit both the activation of sigma E and the synthesis of its precursor. The basis of this effect is unknown, but it is not due to an overall disruption of protein synthesis. The incorporation of [35S] methionine by the sporulating cultures was unaffected by penicillin treatment. A time course study of the effects of rifampin and chloramphenicol treatments on sigma E levels revealed that both the synthesis of sigma E and its disappearance from sporulating cultures is inhibited by these antibiotics. This suggests that ongoing macromolecular synthesis is required for the turnover of sigma E.     

Acid-Soluble Nucleotides in an Asporogenous Mutant of Bacillus subtilis

An asporogenous mutant of Bacillus subtilis Sp(-)H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t(2) in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP-N-acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t(2). No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed.     

Chemical and Morphological Studies of Bacterial Spore Formation : III. The Effect of 8-Azaguanine on Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.     

Cell Cycle Dependency of Sporulation in Saccharomyces cerevisiae

The study of sporulation in Saccharomyces cerevisiae is complicated by the fact that not all cells in the population complete sporulation and that the kinetics of development of those which do are not synchronous. By separating vegetative cells by zonal rotor centrifugation into fractions of increasing cell volume and hence progressive stages of the vegetative cell cycle, it was possible to observe sporulation of more homogeneous, synchronous populations. The capacity of S. cerevisiae to complete sporulation is low for small single cells at the beginning of the cell cycle and is greatest for large budded cells about to divide. The capacity of a cell to complete sporulation thus appears to be directly related to the stage in the vegetative cell cycle from which it was taken. The use of synchronously sporulating cultures made it possible to examine very early decision events leading to the commitment of a cell to sporulation. In addition, differences in the capacity of a mother and daughter cell produced by cell scission were examined.     

Protein degradation during sporulation ofBacillus megaterium: Effect of actinomycin D

The first acceleration of protein degradation in cells ofBacillus megaterium was found at the stage 0–I of sporulation, the second one at the stage II–III, where the sporulation process became irreversible. These accelerations were reduced by actinomycin D inhibiting RNA and protein syntheses by more than 95%. In the presence of the antibiotic, only 8% of prelabeled proteins were degraded. Actinomycin D did not lower either the concentration of ATP or the proteolytic activity in the homogenate prepared from sporulating cells. This indicates that the inhibition of protein catabolism by actinomycin D was not owing to the absence of ATP or proteolytic enzymes. Actinomycin probably inhibited an unknown step preceding the proteolytic attack of the protein molecules during sporulation, because it had no significant effect on proteolysis during vegetative growth.