The Effect of External Conditions on Coremium Production in Paecilomyces farinosus Bainier

Paecilomyces farinosus grew and produced coremia under laboratoryconditions on media containing wide ranges of carbon and nitrogensources. The fungus tolerated higher concentrations of complexcarbon and nitrogen sources than of simple ones. Sustained lowconcentrations of simple carbon and nitrogen nutrients favouredcoremium production. Once P. farinosus colonies had consumedsufficient nutriment, coremium production could take place withoutfurther access to external nutrients. Light was essential for initiation and development of coremia.The tips of the coremia were photosensitive throughout theirgrowth. After growth in darkness during the initial trophicphase of development, P. farinosus required exposure to lightfor at least 8 days before coremia were initiated. The isolate used produced non-coremial strains spontaneously.These usually also lacked the orange, alcohol soluble, pH sensitivepigment found in the coremial strains.     

Nutritional regulation of late meiotic events in Saccharomyces cerevisiae through a pathway distinct from initiation.

The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae, although it is not required for growth. Here we report that in stationary-phase cultures containing low concentration of glucose, cells overexpressing IME1 undergo the early meiotic events, including DNA replication, commitment to recombination, and synaptonemal complex formation and dissolution. In contrast, later meiotic events, such as chromosome segregation, commitment to meiosis, and spore formation, do not occur. Thus, nutrients can repress the late stages of meiosis independently of their block of initiation. Cells arrested at this midpoint in meiosis are relatively stable and can resume meiotic differentiation if transferred to sporulation conditions. Resumption of meiosis does not require repression of IME1 expression, since IME1 RNA levels stay high after transfer of the arrested cells to sporulation medium. These results suggest that meiosis in S. cerevisiae is a paradigm of a differentiation pathway regulated by signal transduction at both early and late stages.     

Signal transduction: external signals influence spore-number control

In budding yeast, the supply of external nutrients controls both sporulation and spore number. A new study has investigated how an external signal can be propagated internally to digitize an output of the number of spores formed.     

Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii

The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differentiate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chytridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to induce complete sporulation in B.?emersonii. We concluded that the determination of growth or sporulation in this microorganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation.     

Initiation of solvent production,clostridial stage and endospore formation in Clostridium acetobutylicum P262

Summary A minimal medium was used to investigate the triggers regulating the initiation of solvent production and differentiation in Clostridium acetobutylicum P262. The accumulation of acid end-products caused the inhibition of cell division and the initiation of solvent production and cell differentiation. Initiation only occurred with a narrow pH range. Glucose or ammonium limited cultures failed to achieve the necessary threshold of acid end-products and solvent production and differentiation were not initiated. The addition of acid end-products or ammonium to cultures containing suboptimal levels of glucose or nitrogen respectively, enhanced solvent production. Resuspension of cells in media containing the threshold level of acid end-products and residual glucose induced endospore formation. Glucose or ammonium limitation did not induce sporulation and there was a requirement for glucose and ammonium during solventogenesis and endospore formation. Initiation of solvent production and clostridial stage formation were essential for sporulation. The induction of endospore formation in C. acetobutylicum P262 differs from that in the aerobic endospore forming bacteria where sporulation is initiated by nutrient starvation.     

Use of an exchange filtration technique to obtain synchronous sporulation in an extended batch fermentation

A fermentor system with an external filtration loop has been developed to control the growth and sporulation of yeast in a single vessel. Excess growth medium, instead of being removed by centrifugation, is removed by filtration and replaced with acetate sporulation medium. The technique did give 80% sporulation after 20 hr, greatly improving the rate and degree of synchrony of sporulation and it also eliminated the contamination hazard of the conventional harvest technique, centrifugation, and resuspension of vegetative cells in sporulation medium. Furthermore it permits proper control of the environmental conditions throughout the growth, exchange, and sporulation phase. In this technique 100% recycle of biomass is achieved without any packing of the cells on the filter. This technique has wide application in the study of industrial fermentation that involves microbial differentiation such as the production of ergot alkaloids, bacitracin, and cephalosporin.     

Induction of sporulation inBlastocladiella emersonii: Influence of nutritional variables

We have studied the induction of zoosporangial sporulation inBlastocladiella emersonii by determining the influence of nutritional variables on the formation of the basal septum. This is an easy-to-monitor structure that has proved to be a useful marker of initiation of this cellular differentiation process. At any time before formation of the septum, the cells returned to the growth phase when incubated in the presence of 2% casamino acids. However, after septum formation has occurred, the cells were committed to sporulation. Certain of the amino acid components of the defined growth medium, DM₂, either prevent (tyrosine, phenylalanine, tryptophan, histidine, and threonine) or delay (valine, serine, arginine, and methionine) formation of the septum when added to the cultures together with the sporulation solution (buffered 1 mM CaCl₂). Sugars do not prevent the formation of the septum, although they can interrupt sporulation at later developmental phases. Glucosamine and acetylglucosamine can prevent formation of the discharge papilla and glucose can block completion of zoospore production. Glutamine, adenosine, and guanosine also caused long delays in septum formation. Taken together, these data favor (a) the hypothesis that amino acid starvation is one of the most important variables with respect to the induction of sporulation, and (b) the use of certain environmental chemical conditions that block sporulation at different steps as developmental tools for the study of this cellular differentiation process.     

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.     

Coordinated Cell Death in Isogenic Bacterial Populations: Sacrificing Some for the Benefit of Many?

Antibiotics are classically perceived as biological weapons that bacteria produce to hold their ground against competing species in their natural habitat. But in the context of multicellular differentiation processes, antimicrobial compounds sometimes also play a role in intraspecies competition, resulting in the death of a sub-population of genetically identical siblings for the benefit of the population. Such a strategy is based on the diversification and hence phenotypic heterogeneity of an isogenic bacterial population. This review article will address three such phenomena. In Bacillus subtilis, cannibalism is a differentiation strategy that enhances biofilm formation, prolongs or potentially even prevents full commitment to endospore formation under starvation conditions, and protects cells within the biofilm against competing species. The nutrients released by lysed cells can be used by the toxin producers, thereby delaying the full activation of the master regulator of sporulation. A related strategy is associated with the initiation of competence development under nutrient excess in Streptococcus pneumoniae. This process, termed fratricide, causes allolysis in a sub-population and is thought to enhance genetic diversity within the species. In Myxococcus xanthus, a large fraction of the population undergoes programmed cell death during the formation of fruiting bodies. This sacrifice ensures the survival of the sporulating sub-population by providing nutrients and hence energy to complete this differentiation process. The biological relevance and underlying regulatory mechanisms of these three processes will be discussed in order to extract common features of such strategies. Moreover, open questions and future challenges will be addressed.     

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.     

The role of nutrient availability in regulating root architecture

The ability of plants to respond appropriately to nutrient availability is of fundamental importance for their adaptation to the environment. Nutrients such as nitrate, phosphate, sulfate and iron act as signals that can be perceived. These signals trigger molecular mechanisms that modify cell division and cell differentiation processes within the root and have a profound impact on root system architecture. Important developmental processes, such as root-hair formation, primary root growth and lateral root formation, are particularly sensitive to changes in the internal and external concentration of nutrients. The responses of root architecture to nutrients can be modified by plant growth regulators, such as auxins, cytokinins and ethylene, suggesting that the nutritional control of root development may be mediated by changes in hormone synthesis, transport or sensitivity. Recent information points to the existence of nutrient-specific signal transduction pathways that interpret the external and internal concentrations of nutrients to modify root development. Progress in this field has led to the cloning of regulatory genes that play pivotal roles in nutrient-induced changes to root development.     

Calcium and malate are sporulation-promoting factors of Physarum polycephalum

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime mold Physarum polycephalum. The most important requirement for sporulation of Physarum is a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25 degrees C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state of Physarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.     

Protein synthesis during the differentiation of sporangia in the water mold Achlya

During the synchronous differentiation of sporangia in the absence of added nutrients, the water mold Achlya bisexualis (Coker and Couch) actively synthesized protein. Inhibition of protein synthesis at any time during the sporulation process completely inhibited further differentiation. Large changes in the rate of radioactive amino acid uptake resulted in changes in the specific activity of the cellular amino acid pool. The rate of protein synthesis was calculated from the amino acid pool specific activity and the incorporation of isotope into protein. During the 1st h after induction of the sporulation process, the rate of protein synthesis increased to two times the initial value. The amino acid precursors for this synthesis were supplied by the degradation of preexisting protein. Proteolytic enzyme activity assayed in vitro increased in proportion to the in vivo rates of protein synthesis and degradation. Differentiation was accompanied by a slight decline in dry weight of the mycelium as well as by a decrease in the protein content, whereas the relative size of the amino acid pools remained constant.     

Sporogenesis in Streptomyces melanochromogenes

The mode of spore differentiation in a strain of Streptomyces melanochromogenes was followed by analysis of ultrathin sections of sporulating aerial hyphae at various stages of sporogenesis. A special accent was laid on the formation of the sporulation septum and its alterations in the course of spore delimitation and separation. Distinct differences in formation and substructure have been observed between the cross walls of vegetative hyphae and the sporulation septa.Cross walls of vegetative hyphae are formed in a way typical for Gram-positive bacteria by a centripetal annular ingrowth of cytoplasmic membrane, on which wall material immediately is deposited. The development of the sporulation septa is characterized by the accumulation of amorphous material in addition to the newly synthesized wall layer inside the invaginating cytoplasmic membrane. This amorphous septal material will later be decomposed presumably by two lytic systems which cause the separation of the spores. The central region of the finished sporulation septum is perforated by microplasmodesmata. Spores are released by a break down of the surface sheath. The complete spores are enveloped by a twolayered cell wall and the spiny surface sheath.     

Preparation and characteristics of protoplasts from various strains of Streptomyces roseoflavus var. roseofungini]

It was shown that the variants of the roseofungin-producing organism, which differ in their differentiation, antibiotic activity, structure and cell wall composition had different sensitivity to the protoplasting factors. The protoplasting increased the population heterogeneity: in strain 1128 it was evident from an increased frequency of the secondary colonies, in variant 1-68, folding of the colonies increased and their consistency become milder, sectorial colonies and colonies with coremia formed. It was in principle possible to transform the protoplasts of S. roseoflavus var. roseofungini by plasmid DNA, which suggests that the roseofungin-producing culture may be useful in genetic engineering.     

Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoxenically on spatially heterogeneous culture media

A new in vitro experimental system was developed to study the morphogenesis of discrete regions of a single extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, growing simultaneously in six different agar-based media. The media were (i) unamended water agar (WA), (ii) WA+PO(4)(3-) (PO(4)(3-)), (iii) WA+NO(3)(-) (NO(3)(-)), (iv) WA+NH(4)(+) (NH(4)(+)), (v) WA+NH(4)(+)+MES (NH(4)(+)+MES) and (vi) minimal medium (M, complete nutrients). Each medium was amended with the pH indicator bromocresol purple. The extraradical mycelium of the fungus showed between-treatment differences in morphogenesis, architecture, formation of branched absorbing structures (BAS) and sporulation. Extraradical hyphae that developed in WA or PO(4)(3-) compartments exhibited an economic development pattern, in which runner hyphae radially extended the external colony. Extraradical hyphal growth in the NO(3)(-) compartments was characterized by increased formation of runner hyphae, BAS and spores and an alkalinization of the medium. In the two NH(4)(+)-amended media (NH(4)(+), NH(4)(+)+MES), sporulation was suppressed and considerable morphological changes were noted. These results show the plasticity of G. intraradices that lets it efficiently exploit an heterogeneous substrate.     

Catabolite-induced repression of sporulation in Bacillus subtilis

In response to nutrient limitations, Bacillus subtilis cells undergo a series of morphological and genetic changes that culminate in the formation of endospores. Conversely, excess catabolites inhibit sporulation. It has been demonstrated previously that excess catabolites caused a decrease in culture medium pH in a process that required functional AbrB. Culture medium acidification was also shown to inhibit sigmaH-dependent sporulation gene expression. The studies reported here investigate the effects of AbrB-mediated pH sensing on B. subtilis developmental competence. We have found that neither addition of a pH stabilizer, MOPS (pH 7.5), nor null mutations in abrB blocked catabolite repression of sporulation. Moreover, catabolite-induced culture medium acidification was observed in cultures of catabolite-resistant sporulation mutants, crsA47, rvtA11, and hpr-16, despite their efficient sporulation. These results suggest that AbrB-mediated pH sensing is not the only mechanism regulating catabolite repression of sporulation. The AbrB pathway may function to channel cells toward genetic competence, as opposed to other postexponential differentiation pathways.     

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.     

Protein degradation during the differentiation of eukaryotic cells: studies on the sporulation of Saccharomyces cerevisiae and on the formation of the neuromuscular junction in the chick embryo.

The role of protein degradation in cell and tissue differentiation has been investigated during the sporulation of Saccharomyces cerevisiae and during endplate formation in developing avian muscle. The results suggest that a variety of proteolytic processes as enzyme inactivation, degradation of mitochondrial membrane constituents and removal of embryonic cell surface proteins exert stringent controls over the sequence of differentiation in eukaryotic cells.