Adenine Nucleotide Changes Associated with the Initiation of Sporulation in Bacillus subtilis

At the end of the exponential growth phase of Bacillus subtilis, there is a decrease in the energy level of the cell, whether expressed as adenosine triphosphate concentration or adenylate energy charge. Phosphate limitation of exponentially growing cells produces a similar decrease in the energy level of the cell, and sporulation is derepressed in the presence of 10 mM glucose. A reduction in the tryptophan concentration of the medium during phosphate limitation of the tryptophan auxotroph B. subtilis 168 prevented the decrease in energy charge. Cells do not sporulate under these conditions. Energy charge values of 0.30 to 0.35 found during sporulation do not lead to cell death.     

On the formation of crystal proteins during sporulation in Bacillus thuringiensis var. thuringiensis

The sporulation potential of Bacillus subtilis as a function of position in the cell cycle was determined by transferring cells from growth medium to sporulation medium at various times during growth. Growth was induced by incubating heat-activated spores in rich medium or by diluting stationary phase vegetative cultures with fresh growth medium. The results supported earlier observations that sporulation potential is cell cycle dependent. The rise in sporulation potential was studied by exposing cultures to the inhibitors of cell wall and protein synthesis, vancomycin and chloramphenicol. The delay in the appearance of the peak of sporulation potential caused by these inhibitors compared with the reported lack of effect of nalidixic acid, indicates that the appearance of sporulation potential requires synthesis of a macromolecular component other than deoxyribonucleic acid. The effect of nalidixic acid in preventing the decline of the sporulation potential was compared with the effect of high temperature on a mutant temperature sensitive for the initiation of DNA replication. It was found that prevention of chromosome completion with nalidixic acid maintained a high sporulation potential, whereas prevention of chromosome re-initiation in the temperature sensitive mutant did not affect the decline in sporulation potential as the cells enter stationary phase.Abbreviations NAL Nalidixic acid - HPUra 6-(p-hydroxyphenylazo)-uracil - VAN Vancomycin - CAM Chloramphenicol - BHI Brain heart infusion broth - c.f.u. Colony forming units     

Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.

The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.     

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σ^E因子控制的cry1Aa基因表达。     

Bicarbonate is a stimulus in the inter-species induced sporulation of strict anaerobic Syntrophomonas erecta subsp. sporosyntropha

Previously Syntrophomonas species had been described as the bacteria those did not form spores, however, in our previous studies, a newly isolated S. erecta subsp. sporosyntropha JCM13344^T was found to form spores in the co-culture with methanogens, while not in mono-culture or in co-culture with sulfate reducer. In this study, we examined the sporulation stimulus conferred by methanogens in the co-culture. By reducing bicarbonate in mono-culture and sulfate-reducing co-culture, we could induce S. erecta subsp. sporosyntropha JCM13344^T to form spores, so that bicarbonate at lower concentration was determined as another stimulus for sporulation. Based on the substrate degradation by strain JCM13344^T in different concentration of bicarbonate vs at different pHs, it was suggested that bicarbonate could stimulate the sporulation by mediating a nutrient deprivation but not pH drop. To further confirm the sporulation potential of this group of bacteria, spo0A fragments were amplified from strain JCM13344^T as well as all the recognized Syntrophomonas species, confirming that they were members of the spore-forming group. Since sporulation is a kind of response of spore-forming bacteria to environmental stresses, the observation in this work implies that stresses can be created even between the mutual beneficial partners, in this case, inducing sporulation.     

Decrease of Fluorescence Intensity After the K Step in Chlorophyll a Fluorescence Induction is Suppressed by Electron Acceptors and Donors to Photosystem 2

Chlorophyll a fluorescence induction measured by a fluorometer with a high temperature stressed plant material shows a new K step which is a clear peak due to fast fluorescence rise and subsequent decrease of fluorescence intensity. We focused on an explanation of the decrease of fluorescence after the K step using artificial electron acceptors and donors to photosystem 2 (PS2). Addition of the artificial electron acceptors or donors suppressed the decrease of fluorescence after the K step. We suggest that the decrease mainly reflects (by more than 81 %) an energy loss process in the reaction centre of PS2 which is most probably a nonradiative charge recombination between P680⁺ (oxidised primary electron donor in PS2) and a negative charge stored on either Pheo⁻ or Q_A ⁻ (reduced primary electron acceptor of PS2 and reduced primary quinone electron acceptor of PS2, respectively). We suggest that the energy loss process is only possible when the inhibition of both the donor and the acceptor sides of PS2 occurs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Changes in adenine-nucleotide content in the apical bud of Sinapis alba L. during floral transition

The total adenylate pool of the apical buds of vegetative plants of Sinapis alba L. continuously grown in short days fluctuates over a 24-h cycle with the minimum occurring at the end of the dark period. In the buds of plants induced to flower by a single long-day treatment, total adenylate pool increases above the control level 16 h after the start of the long day, resulting mainly from a rise in ATP and ADP contents. This occurs 6 h after the increase in the soluble carbohydrate content previously shown to occur in the apical buds of plants induced to flower (Bodson 1977, Planta 135, 19–23). A transient rise of the energy charge occurs 22 h after the start of the inductive long day.Abbreviations LD long day - SD short day     

Role of the adenylate system and glycolytic flux in the control of protein synthesis in isolated rat lung cells

(1) Glucose stimulates the incorporation of amino acids into protein in lung cells isolated by digestion of the lung stroma with collagenase. This effect reflects mainly an increase in protein synthesis since no effect of glucose had been found to the uptake of amino acid precursors and, although glucose decreases the rate of intracellular proteolysis by 15%, this effect cannot account for the increased incorporation of radioactivity into proteins. Furthermore, glucose did not induce any significant change in the intracellular content of valine. (2) For glucose to act on protein synthesis, it must be glycolyzed since its stereoisomer, L-glucose, which is not metabolized by lung cells, has no effect. (3) The mechanism of glucose action does not seem to be related simply to variations of cellular ATP content or energy charge. The following arguments seem to support this conclusion: (i) glucose does not bring about significant variations in the concentration of reactants of the adenylate system; (ii) the increase in protein synthesis induced by glucose in energy-depleted cells correlates with a rise in ATP content and energy charge; however, adenosine, which increases ATP levels in a form quantitatively similar to glucose, is unable to affect protein synthesis: (iii) glucose also accelerates the incorporation of amino acids into proteins in adenosine-treated lung cells in which the ATP concentration was almost double that of the control and the energy charge was considerably elevated, ruling out the possibility that a rise in the steady-state concentration of ATP and/or energy charge alone could be responsible for the acceleration of protein synthesis. (4) It can be concluded that the effect of glucose in increasing protein synthesis in lung cells is dependent on some signal arising from its breakdown and not to variations in the concentration of reactants or energy charge of the adenylate system.     

Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum

Concentrations of adenine and pyridine nucleotides and the associated charge values were examined in extracts of mycelium of Penicillium notatum during vegetative growth and reproductive development promoted by the addition of Ca²⁺ (10 mmol dm^-3). The significant increase in adenylate energy charge promoted by Ca²⁺ was due to a fall in intracellular AMP and a concomitant rise in ATP concentration. Intracellular concentrations of NADH and NAD fell within 1 h of the addition of Ca²⁺. The catabolic reduction charge was unchanged by Ca²⁺ whilst the anabolic reduction charge increased in Ca²⁺-induced mycelium due to lowered intracellular NADP concentration. Reduced concentration of NADPH in Ca²⁺-induced mycelium, relative to the vegetative controls, lowered the phosphorylated nucleotide fraction. The results are discussed in relation to metabolic economy during morphogenesis in P. notatum.     

Hydrolytic Enzymes and Sporulation in Bacillus intermedius

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II–IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.     

Transcellular ion currents during sporangium development in the water mould Achlya bisexualis

Changes in the pattern of electric currents that accompany the transformation of growing hyphae of Achlya bisexualis into sporangia have been examined. When hyphae were transferred to a non-nutrient buffer, they continued to extend for several hours and then gave rise to sporangia. Throughout this process, current (positive charge) flowed into the apical region that corresponds approximately to the future sporangium. The current ceased after the crosswall appeared. The sporangium then remained electrically quiescent, except for a brief intense burst of outward current at the 'homogeneous' stage of spore cleavage. The inward current during sporangium formation largely represents an influx of protons. Addition of nitrate abolished the flow of electric current with little effect on sporulation. The late burst of outward current is most probably an artefact, generated by the discharge of salts from the sporangial vacuole. The transcellular electric current apparently plays no role in sporangium formation or in spore cleavage. Calcium ions, however, are required and may traverse the plasma membrane.     

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T₀) or at T₃, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

Enhancement of sporulation in species of Bipolaris, Curvularia, Drechslera, and Exserohilum by growth on cellulose-containing substrates

Nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum were compared for sporulation on agar media and for enhancement of sporulation by growth on four cellulose-containing substrates (index card, filter paper, cheesecloth, cotton fabric). On two natural and one synthetic agar media, sporulation varied from profuse to nonexistent among three isolates of each species. Growth of all species on cellulose substrates resulted in large and significant increases in sporulation. Growth on index card pieces often provided the greatest increases, but no single substrate was superior for all species, and significant substrate × isolate interactions were observed within species. Overlay of filter paper onto whole colonies in agar plates resulted in 2 to 18-fold increases in sporulation for eight of nine species and production of spores in sufficient quantity for most experimental purposes. Overlay of soil dilution plates with filter paper to promote sporulation of colonies enabled detection of B. spicifera, B. hawaiiensis, C. lunata, and E. rostratum at relatively low population levels (≤1.3 × 10³ colony-forming units per gram of soil) in samples of a naturally infested soil. Results indicate that enhancement of sporulation by growth of species of Bipolaris, Curvularia, Drechslera, and Exserohilum on cellulose substrates may facilitate (i) their identification in culture, (ii) production of spores at relatively high concentrations, and (iii) detection and enumeration of these fungi in soil.     

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.     

Correlation among turnover of nucleic acids,ribonuclease activity and sporulation ability of Saccharomyces cerevisiae

The turnover of nucleic acids and changes in ribonuclease activity during sporulation of Saccharomyces cerevisiae were studied. In the sporulating strains, 37–58% of vegetatively synthesized RNA were degraded during the sporulation process. The degree of degradation of vegetative RNA was proportional to the sporulation ability. In the non-sporulating strains, the degradation of vegetative RNA was less than 28% in the sporulation medium. Accompanied by the degradation of vegetative RNA, a ribonuclease activity increased several times during sporulation. We have found a close relation among the sporulation rate, the degree of the degradation of vegetative RNA and the increase in ribonuclease activity in the sporulation medium, using cells of which sporulation ability was repressed by changing the age or carbon source in various degrees.     

The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A.

SinR is a pleiotropic DNA binding protein that is essential for the late-growth processes of competence and motility in Bacillus subtilis and is also a repressor of others, e.g., sporulation and subtilisin synthesis. In this report, we show that SinR, in addition to being an inhibitor of sporulation stage II gene expression, is a repressor of the key early sporulation gene spo0A. The sporulation-specific rise in spo0A expression at time zero is absent in a SinR-overproducing strain and is much higher than normal in strains with a disrupted sinR gene. This effect is direct, since SinR binds specifically to spo0A in vitro, in a region overlapping the -10 region of the sporulation-specific Ps promoter that is recognized by E-sigma H polymerase. Methyl interference and site-directed mutagenesis studies have identified guanine residues that are important for SinR recognition of this DNA sequence. Finally, we present evidence that SinR controls sporulation through several independent genes, i.e., sp0A, spoIIA, and possibly spoIIG and spoIIE.     

Resolving the excited state equilibrium of peridinin in solution

The carotenoid peridinin is abundant in the biosphere, as it is the main pigment bound by the light-harvesting complexes of dinoflagellates, where it collects blue and green sunlight and transfers energy to chlorophyll a with high efficiency. Its molecular structure is particularly complex, giving rise to an intricate excited state manifold, which includes a state with charge-transfer character. To disentangle the excited states of peridinin and understand their function in vivo, we applied dispersed pump-probe and pump-dump-probe spectroscopy. The preferential depletion of population from the intramolecular charge transfer state by the dump pulse demonstrates that the S(1) and this charge transfer state are distinct entities. The ensuing dump-induced dynamics illustrates the equilibration of the two states which occurs on the time scale of a few picoseconds. Additionally, the dump pulse populates a short-lived ground state intermediate, which is suggestive of a complex relaxation pathway, probably including structural reorientation or solvation of the ground state. These findings indicate that the unique intramolecular charge transfer state of peridinin is an efficient energy donor to chlorophyll a in the peridinin-chlorophyll-protein complex and thus plays a significant role in global light harvesting.