Proteolysis of the replication checkpoint protein Sda is necessary for the efficient initiation of sporulation after transient replication stress in Bacillus subtilis

Cells of Bacillus subtilis actively co-ordinate the initiation of sporulation with DNA replication and repair. Conditions that perturb replication initiation or replication elongation induce expression of a small protein, Sda, that specifically inhibits the histidine kinases required to initiate spore development. Previously, the role of Sda has been studied during chronic blocks to DNA replication. Here we show that induction of Sda is required to delay the initiation of sporulation when replication elongation is transiently blocked or after UV irradiation. During the recovery phase, cells efficiently sporulated, but this required the proteolysis of Sda. The rapid proteolysis of Sda required the ClpXP protease and the uncharged C-terminal sequence of Sda. Replacing the last two residues of Sda, both serines, with aspartic acids markedly stabilized Sda. Strains expressing sdaDD from the endogenous sda locus were unable to efficiently initiate sporulation after transient replication stress. We conclude that the Sda replication checkpoint is required to delay the initiation of sporulation when DNA replication is transiently perturbed, and that the intrinsic instability of Sda contributes to shutting off the pathway. The Sda checkpoint thus co-ordinates early events of spore development, including the polar cell division, with successful completion of chromosome replication.     

The FtsEX ABC transporter directs cellular differentiation in Bacillus subtilis

A fundamental challenge in developmental biology is to elucidate the regulatory events that trigger cellular differentiation. Sporulation in the Gram-positive bacterium Bacillus subtilis serves as a simple experimental model system to address this challenge. The hallmark of sporulation is the formation of an asymmetrically positioned septum that divides the cell into unequally sized progeny. Here we describe the role of an ABC transporter, comprising the FtsE and FtsX proteins, in the initiation of spore formation. We discovered that in the absence of this transporter, entry into sporulation is delayed and an atypical symmetric septum is formed instead of a polar one. We show that this phenotype can be suppressed by artificially activating the master regulator of sporulation, Spo0A, or by activating the histidine kinases that function upstream of Spo0A. Our data indicate that the FtsEX transporter is one of the top components in the hierarchy of factors required to initiate sporulation, and thus it is essential for establishing proper temporal activation of the process.     

The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis.

Massive sporulation of Bacillus subtilis normally begins when carbon, nitrogen or phosphorus sources able to support rapid growth are no longer available. Sporulation can also be induced in exponentially growing cultures, in the presence of rapidly utilizable ammonia, glucose and phosphate if growth is partially but not completely inhibited either by inhibitors of nucleotide synthesis (hadacidin, decoyinine or 6-azauracil) or by purine deprivation in purine and especially in guanine auxotrophs. All these conditions allowing sporulation result in a decrease in the intracellular concentration of guanosine di- and tri-phosphates and usually uridine di- and triphosphates while other nucleotides decrease in some but increase in other cases. A decrease of uracil nucleotides alone, in a uracil auxotroph, does not produce massive sporulation. Our results demonstrate that the partial reduction of a guanine nucleotide, probably relative to some other compound, suffices to initiate sporulation. This reduction may always play a decisive role in the initiation of sporulation, as we have observed it under all conditions so far known to produce massive sporulation.     

The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis

Massive sporulation of Bacillus subtilis normally begins when carbon, nitrogen or phosphorus sources able to support rapid growth are no longer available. Sporulation can also be induced in exponentially growing cultures, in the presence of rapidly utilizable ammonia, glucose and phosphate if growth is partially but not completely inhibited either by inhibitors of nucleotide synthesis (hadacidin, decoyinine or 6-azauracil) or by purine deprivation in purine and especially in guanine auxotrophs. All these conditions allowing sporulation result in a decrease in the intracellular concentration of guanosine di- and triphosphates and usually uridine di- and triphosphates while other nucleotides decrease in some but increase in other cases. A decrease of uracil nucleotides alone, in a uracil auxotroph, does not produce massive sporulation. Our results demonstrate that the partial reduction of a guanine nucleotide, probably relative to some other compound, suffices to initiate sporulation. This reduction may always play a decisive role in the initiation of sporulation, as we have observed it under all conditions so far known to produce massive sporulation.     

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.     

The Arf-GTPase-activating protein Gcs1p is essential for sporulation and regulates the phospholipase D Spo14p

SPO14, encoding the major Saccharomyces cerevisiae phospholipase D (PLD), is essential for sporulation and mediates synthesis of the new membrane that encompasses the haploid nuclei that arise through meiotic divisions. PLD catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. PA stimulates Arf-GTPase-activating proteins (Arf-GAPs), which are involved in membrane trafficking events and actin cytoskeletal function. To determine if Spo14p-generated PA mediates its biological response through Arf-GAPs, we analyzed the sporulation efficiencies of cells deleted for each of the five known and potential yeast Arf-GAPs. Only gcs1delta mutants display a sporulation defect similar to that of spo14 mutants: cells deleted for GCS1 initiate the sporulation program but are defective in synthesis of the prospore membrane. Endosome-to-vacuole transport is also impaired in gcs1delta cells during sporulation. Furthermore, Arf-GAP catalytic activity, but not the pleckstrin homology domain, is required for both prospore membrane formation and endosome-to-vacuole trafficking. An examination of Gcs1p-green fluorescent protein revealed that it is a soluble protein. Interestingly, cells deleted for GCS1 have reduced levels of Spo14p-generated PA. Taken together, these results indicate that GCS1 is essential for sporulation and suggest that GCS1 positively regulates SPO14.     

Natural isolates of Saccharomyces cerevisiae display complex genetic variation in sporulation efficiency

Sporulation is a well-studied process executed with varying efficiency by diverse yeast strains. We developed a high-throughput method to quantify yeast sporulation efficiency and used this technique to analyze a line cross between a high-efficiency oak tree isolate and a low-efficiency wine strain. We find that natural variation in sporulation efficiency mirrors natural variation in higher eukaryotes: it shows divergence between isolated populations, arises from loci of major effect, and exhibits epistasis. We show that the lower sporulation efficiency of the wine strain results from a failure to initiate sporulation, rather than from slower kinetics of meiosis and spore formation. The two strains differentially regulate many genes involved in aerobic respiration, an essential pathway for sporulation, such that the oak tree strain appears better poised to generate energy from this pathway. We also report that a polymorphism in RME1 that affects sporulation efficiency in laboratory strains also cosegregates with significant phenotypic differences in our cross of natural isolates. These results lay the groundwork for the study of variation in sporulation efficiency among natural isolates of yeast.     

FluG and flbA function interdependently to initiate conidiophore development in Aspergillus nidulans through brlA beta activation.

The Aspergillus nidulans fluG gene is necessary for the synthesis of a small diffusible factor that is required for the endogenously regulated induction of asexual sporulation that takes place during the development of an air-exposed colony. Previous work established that FluG is present at nearly constant levels throughout the Aspergillus life cycle, leading to the hypothesis that FluG factor is constitutively produced and development initiates after its concentration surpasses a fixed threshold. Here we show that overexpression of fluG can overcome the developmental block normally imposed on vegetative cells in submerged culture and leads to the formation of complex conidiophores that are remarkably similar to wild-tye conidiophores made by air- exposed colonies. This fluG-induced sporulation requires the activities of other early developmental regulatory genes including, flA, flB, flC, flD, flE, and brlA. The requirement for flbA in fluG-induced sporulation is particularly interesting because overexpression of flbA can also induce sporulation in submerged culture and this flbA activity requires fluG. The interdependence of fluG and flbA activities suggests a close relationship between the products of these two genes in controlling conidiophore development. In addition to the endogenous sporulation signal provided by fluG, several environmental factors, including air exposure, carbon or nitrogen stress, and increased osmolarity, can influence developmental activation. We demonstrate that each of these signals requires the brlA beta gene, but not brlA alpha, to initiate conidiophore development. We present a model to account for the complex genetic and environmental controls leading to the activation of brlA beta and sporulation.     

The induction of sporulation in the aquatic fungus Blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl₂ with EGTA. Calcium channel blockers, CoCl₂ and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.     

Partial nucleotide limitation induces phosphodiesterase I and 5'-nucleotidase in Bacillus subtilis.

Changes in the specific activity of enzymes involved in the degradation of RNA and nucleotides were measured in Bacillus subtilis under conditions of guanine deprivation, which initiates sporulation, and uracil deprivation, which does not initiate sporulation. Whereas the specific activities of most of the enzymes studied increased by less than a factor of 3, those of 5'-mononucleotide-producing phosphodiesterase and 5'-nucleotidase increased at least eightfold under both deprivation conditions.     

Pulsed feedback defers cellular differentiation

Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable “polyphasic” positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a “timer” that operates over timescales much longer than a cell cycle.     

New cytoplasmic genetic element that controls 20S RNA synthesis during sporulation in yeast.

Under conditions that induce meiosis and sporulation in Saccharomyces cerevisiae, most strains accumulate a 20S RNA, amounting to as much as 15% of the newly synthesized RNA. The ability of cells to accumulate this new RNA species depends on a dominant genetic element that is cytoplasmically inherited, but is distinct from the other cytoplasmic elements that have been previously identified. The ability to synthesize 20S RNA does not depend on mitochondrial DNA, 2-micron DNA, the translational suppressor psi, the genetic element carrying URE3, or double-stranded killer RNA. However, all 20S- strains examined were also nonkillers, although many nonkiller strains were 20S+. This work also shows that 20S RNA accumulating is not essential for sporulation even though it is induced only by conditions that initiate sporulation. Furthermore, strains that are unable to complete meiosis are still capable of producing 20S RNA when placed under the nitrogen starvation conditions that promote sporulation.     

Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.

Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components.     

Conditional Mutants of Meiosis in Yeast

Three temperature-sensitive mutants, spo1-1, spo2-1, and spo3-1, were characterized with respect to their behavior in sporulation medium at a restrictive temperature. The time of expression of the functions defective in the mutants was determined by temperature-shift experiments during the sporulation process. In addition, each mutant was examined for the following: (i) its ability to undergo the nuclear divisions of meiosis; (ii) deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis; (iii) protein turnover; and (iv) colony-forming ability after exposure to sporulation medium. Mutant spo1-1 is defective in a function which confers a temperature-sensitive period which extends over 32% of the sporulation cycle. The temperature-sensitive period of mutant spo2-1 occupies 34% of the cycle, whereas the temperature-sensitive period of mutant spo3-1 extends over 2% of the sporulation cycle. Cytological evidence indicates that all three mutants initiate but do not complete the meiotic nuclear divisions. The DNA content of sporulation cultures of mutants spo1-1 and spo3-1 did not increase to the wild-type level; DNA synthesis in spo2-1 was normal. All three strains exhibit a loss of colony-forming ability during incubation in sporulation medium at the restrictive temperature. RNA and protein synthesis and protein turnover occur in the mutants.     

13C NMR analysis of a developmental pathway mutation in Saccharomyces cerevisiae reveals a cell derepressed for succinate dehydrogenase.

13C nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of [2-13C]acetate in a diploid strain of Saccharomyces cerevisiae homozygous for the spo50 mutation. This mutation results in failure to initiate sporulation and suppresses spd mutations (which cause derepressed sporulation). By analysing the pattern of 13C-labelling in glutamate it was deduced that the glyoxylate cycle is responsible for most of the acetate utilization and that there is very little tricarboxylic acid cycle activity. The labelling of alpha,alpha'-trehalose indicated that gluconeogenesis and the hexose monophosphate pathway operate in a similar way to the wild-type. The mutant strain has higher levels of succinate dehydrogenase than the wild-type. All of the physiological alterations caused by the spo50 mutation can be explained by this difference.     

Selection of an Asporogenous Strain of Clostridium acetobutylicum in Continuous Culture Under Phosphate Limitation

Based on the observation that cells of Clostridium acetobutylicum unable to store granulose do not initiate sporulation, a staining procedure was developed for the detection of asporogenous mutants. By application of this procedure it was shown that an asporogenous strain of C. acetobutylicum was selected in continuous culture under phosphate limitation.     

Horizontal transmission of Amblyospora albifasciati García and Becnel, 1994 (Microsporidia: amblyosporidae), to a copepod intermediate host and the neotropical mosquito, Aedes albifasciatus (Macquart, 1837)

The life cycle of Amblyospora albifasciati is characterized by three sporulation sequences involving the definitive mosquito host and a copepod intermediate host. Meiospores of A. albifasciati were infectious per os to adult females of the copepod Mesocyclops annulatus. All developmental stages in the copepod had unpaired nuclei, with sporulation involving the formation of a sporontogenic interfacial envelope and the production of a second type of uninucleate spore. These spores, formed in the ovaries of M. annulatus, were large, pyriform, and measured 10.4 x 4.8 microm. They infected Aedes albifasciatus larvae when ingested to initiate a sequence that involves schizogony and gametogony and ends with plasmogamy and nuclear association to form diplokaryotic meronts. Oval binucleate spores (9.3 x 3.1 microm) are formed in the adult mosquito and are responsible for vertical transmission to the filial generation.