Fungal spores: dormancy, germination, chemical composition, and role in biotechnology (review)

This review is focused on one of the stages of ontogenesis distinctive by its particular tolerance to the action of unfavorable factors and ability to retain the genomic material for a long period of time, i.e., fungal spores. The major part is devoted to the characterization of the specific stage typical for spores, which is called dormancy. Data are presented characterizing the carbohydrate and lipid composition of spores, with special attention being paid to the role of carbohydrate protectors, in particular, trehalose and mannite, as well as to the role of rafts in the process of sporogenesis. The role of special compounds called autoinhibitors and autostimulators in the process of exit from dormancy is discussed. The final section deals with the role of spore seeding material in biotechnological processes. Data on the correlation between the chemical composition of spores, their ability to remain dormant, and the germination process are considered. Special biotechnological approaches are presented for the first; they allow for the preservation of the germinating ability of spores, intensification of sporogenesis, changes in the ratio of final fermentation products, and an increase in their yield.     

Changes in lipid composition and carbohydrate composition of Aspergillus niger conidia during germination

Data on the lipid composition and carbohydrate composition of Aspergillus niger conidia make it possible to characterize the individual germination stages and differentiate between the conidia capable of germination and those that lost the germination capacity. The following criteria are proposed: the ratio of phosphatidylcholine and phosphatidylethanolamine, the ratio of mannitol and arabitol, and the levels of sterols and free fatty acids. The role of these compounds in the biochemical background of cell transition from dormancy to active metabolism and their use as indices of the quality of inocula in biotechnological processes are discussed.     

Changes in Lipid Composition and Carbohydrate Composition of Aspergillus niger Conidia during Germination

Data on the lipid composition and carbohydrate composition of Aspergillus niger conidia make it possible to characterize the individual germination stages and differentiate between the conidia capable of germination and those that lost the germination capacity. The following criteria are proposed: the ratio of phosphatidylcholine and phosphatidylethanolamine, the ratio of mannitol and arabitol, and the levels of sterols and free fatty acids. The role of these compounds in the biochemical background of cell transition from dormancy to active metabolism and their use as indices of the quality of inocula in biotechnological processes are discussed.     

Effect of trehalose on the viability of sporangiospores of the mucorous fungus <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

Sporangiospores of Blakeslea trispora are in a state of exogenous dormancy, and water is the key factor controlling their germination. A wide range of carbohydrates, ammonium salts, and yeast extract had a weak stimulating effect (less than 50%) on spore germination, whereas amino acids could significantly inhibit this process. Cultivation of B. trispora on sporogenous sucrose- and trehalose-containing media (S and T spores, respectively) resulted in significant changes in spore formation, as well as in the chemical composition of spores and their viability. In the presence of trehalose, the amount of spores increased twofold; spore viability during storage increased as well. All changes in the carbohydrate composition of the cytosol and in the composition of the spore membrane lipids indicated that the dormancy of T spores was deeper than that of S spores, which has a favorable effect on their viability.     

Effect of carbohydrate composition of the cytosol of Aspergillus niger conidia on their viability during storage

It is shown that the rate of sporogenesis, the appearance of low-molecular-weight thiols, and the activation of carbohydrate synthesis in spores adversely affected the viability of Aspergillus niger conidia during storage. Conversely, the prevalence of trehalose over mannitol and the absence of glycerol, erythritol, and glucose in carbohydrate composition facilitated viability of conidia. The data obtained are discussed in regard to the biochemical criteria that may be used to characterize quiescent state of fungi and retaining the viability of the inoculum.     

Effect of Carbohydrate Composition of the Cytosol of Aspergillus niger Conidia on Their Viability During Storage

It is shown that the rate of sporogenesis, the appearance of low-molecular-weight thiols, and the activation of carbohydrate synthesis in spores adversely affected the viability of Aspergillus niger conidia during storage. Conversely the prevalence of trehalose over mannitol and the absence of glycerol, erythritol, and glucose in the carbohydrate composition facilitated the viability of conidia. The data obtained are discussed with regard to the biochemical criteria that may be used to characterize the quiescent state of fungi and retaining the viability of the inoculum.     

The effect of carbohydrates on the sporogenesis of Clostridium perfringens and Bacillus anthracis

The authors studied the effect of a number of carbohydrates on the sporogenesis of Clostridium perfringens and Bacillus anthracis (vaccine strain STI) as probable soil factors capable of influencing the duration of survival of these causative agents in the external environment. Differences in the effect of the same sugars on the formation of spores by these microorganisms and clearly expressed sporogenesis-inhibiting effect of glucose (and also of lactose in clostridia) have been demonstrated. The analysis of the peculiarities of sporogenesis under unadjusted and stabilized pH values provides a basis for regarding the "glucose effect" as repression of sporogenesis in the given causative agent, but not as inhibition resulting from considerable acidification of the culture medium. This is essential for the soil conditions characterized by high buffer capacity. The ecological value of substances of carbohydrate nature consists in their important role in the energetics and trophicity of microbial coenoses of the soil which cannot fail reflecting on the fate of pathogenic microorganisms in the soil.     

The effect of growth medium on B. anthracis Sterne spore carbohydrate content

The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides. We have investigated the carbohydrate composition of B. anthracis Sterne spores produced using four different medium types formulated with different sources of medium components. The amount of rhamnose, 3-O-methyl rhamnose and galactosamine was found to vary significantly between spores cultured using different medium formulations. The relative abundance of these monosaccharides compared to other monosaccharides such as mannosamine was also found to vary with medium type. Specific medium components were also found to impact the carbohydrate profile. Xylose has not been previously described in B. anthracis spores but was detected at low levels in two media. This may represent residual material from the brewery yeast extract used to formulate these two media. These results illustrate the utility of this method to capture the impact of growth medium on carbohydrate variation in spores. Detecting carbohydrate profiles in B. anthracis evidentiary material may provide useful forensic information on the growth medium used for sporulation.     

On the nature of sporogenesis in some aerobic bacteria

Washed vegetative cells of various species of aerobic spore-forming bacteria sporulate abundantly when shaken in distilled water in air. The spores thus formed possess the same heat resistance as spores formed in a complete growth medium. Various factors influencing sporogenesis in water are described. Glucose in low concentration completely suppresses sporogenesis under these conditions and the suppression is relieved by the presence of ammonia as an exogenous source of nitrogen. Various amino acid and purine antimetabolite analogues inhibit sporogenesis and their inhibitory effects are completely reversed by much smaller amounts of the corresponding metabolites. Sporogenesis is thus regarded as a de novo synthesis of spore proteins from preexisting endogenous (enzyme) proteins. Cells low in protein fail to sporulate and the capacity of the cell to adaptively attack maltose and trehalose is strongly interfered with after the cell is irreversibly committed to sporulation, but not before that. Evidence is advanced supporting the hypothesis that sporogenesis is an endogenous process which commences when the supply of exogenous energy and carbon is depleted. It utilizes low molecular weight nitrogenous substances liberated by the degradation of preexisting enzyme proteins of the vegetative cell. Sporogenesis and adaptive enzyme formation are regarded as competitive synthetic processes, both utilizing endogenous enzyme proteins. The events of sporogenesis suggest that this process may be an adaptive protein synthesis, analogous to adaptive enzyme synthesis.     

Biofuels – challenges & chances: How biofuel development can benefit from advanced process technology

The presented article highlights the process of biofuel production with a special focus on bioethanol. After a short introduction to the “problems” of biofuels – the “first generation” biofuels (in regards to their competition to feed and food production) and the “second generation” biofuels (in regards to the required more complex process technology) − the different steps in the process from natural resources towards the final product are presented and the underlying biotechnological challenges discussed: the pre-treatment of the natural resources followed by the biotechnological processes of hydrolysis and fermentation. Topics such as enzyme screening for efficient or even multi-step hydrolysis as well as microbial strain selection under process conditions and the optimization of the anaerobic fermentative conversion of the saccharides to bioethanol are discussed. Optimizing the production of bioethanol to be competitive with petrochemical fuels is the main challenge for the underlying process development.     

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.     

Microcycle sporogenesis in some streptomycetes without shift down treatment

Conditions for microcycle sporogenesis in two streptomycete strains without shifting the culture were found. The sporulation in Streptomyces granaticolor took place after 24 h of cultivation. The dry mass was increasing till 32 h probably due to production of a hydrophobic substance resembling fibrous sheath of aerial hyphae and spores. Ultrathin section of microcycle spores are presented.     

A comparison of tapetal structure and function in pteridophytes and angiosperms

This article is based on a survey of literature with particular reference to the structure and functioning of the tapetum during sporogenesis in angiosperms and pteridophytes. Some original observations onSchizaea pectinata andSelaginella japonica are also included. In the past, superficial similarities in the structure and function of pollen grains and spores have dominated the research approach, but patterns of development and the functioning of the tapetum during sporogenesis in angiosperms and pteridophytes are shown to differ quite significantly and raises questions relating to homologies. It is suggested that direct comparisons may no longer be appropriate.     

Carbohydrate Metabolism During Ascospore Development in Yeast

Carbohydrate metabolism, under sporulation conditions, was compared in sporulating and non-sporulating diploids of Saccharomyces cerevisiae. Total carbohydrate was fractionated into trehalose, glycogen, mannan, and an alkali-insoluble fraction composed of glucan and insoluble glycogen. The behavior of three fractions was essentially the same in both sporulating and non-sporulating strains; trehalose, mannan, and the insoluble fraction were all synthesized to about the same extent regardless of a strain's ability to undergo meiosis or sporulation. In contrast, aspects of soluble glycogen metabolism depended on sporulation. Although glycogen synthesis took place in both sporulating and non-sporulating strains, only sporulating strains exhibited a period of glycogen degradation, which coincided with the final maturation of ascospores. We also determined the carbohydrate composition of spores isolated from mature asci. Spores contained all components present in vegetative cells, but in different proportions. In cells, the most abundant carbohydrate was mannan, followed by glycogen, then trehalose, and finally the alkali-insoluble fraction; in spores, trehalose was most abundant, followed by the alkali-insoluble fraction, glycogen, and mannan in that order.     

Induction of Accelerated Sporangial Lysis by Basic Peptide Antibiotics. A Novel Method of Preparation of Free Spores of Bacilli

An accelerated release of free spores from sporangia of Bacillus cereus NCIB-8122 and Bacillus subtilis SMYW was induced by the addition of the basic peptide antibiotics, polymyxin B or colistin (100 μg/ml), to sporangia formed in liquid Bactopeptone medium. Destruction of sporangial cell walls of B. cereus prelabelled with ³H-4-diaminopimelic acid commenced shortly after the addition of either antibiotic, the label being gradually released into the medium. Normal free spores were released following the addition of antibiotics to sporangia containing refractile spores (stages IV-V of sporogenesis). Earlier additions induced the lysis of both compartments of the sporangium, accompanied by the release of already-synthesized dipicolinic acid and alreadyaccumulated ⁴⁵calcium. The heat resistance and germination ability of spores released in the presence of the antibiotics were the same as those of control spores released by long-term spontaneous lysis of sporangia. Similar effects of the antibiotics were observed with B. subtilis SMYW. Results obtained were used firstly for fast preparation of relatively clean free spores and secondly for the characterization of the developmental stage of sporogenesis at which the spore becomes independent of the maternal cell. It reaches this property at the end of stage IV and during stage V.     

Metabolic Requirements for Microcycle Sporogenesis of Bacillus megaterium

Spores of Bacillus megaterium QM B1551 germinated, elongated, and resporulated (microcycle sporogenesis) in simple chemically defined media which permitted no cell division. The second-stage spores thus produced were heat-stable and required heat activation for germination. The original amount of spore deoxyribonucleic acid tripled before completion of the cycle. Acetate and a small amount of a tricarboxylic acid cycle intermediate were the minimal organic metabolic requirements for microcycle sporogenesis. During this cycle, germinated cells oxidized acetate only after a delay, whether or not glucose was initially present. Spores that were germinated in the absence of a carbon source first oxidized an endogenous substrate, and then developed the ability to oxidize acetate.     

Spores before sporophytes: hypothesizing the origin of sporogenesis at the algal-plant transition

Fossil spores from mid-Ordovician deposits (475 million yr old) are the first indication of plants on land and predate megafossils of plants by 30-50 million yr. Sporopollenin-walled spores distinguish land plants from algae, which typically have heavy-walled zygotes that germinate via meiosis into motile or protonemal cells. All land plants are embryophytes with spores produced by the sporophyte generation. It is generally assumed that retention of the zygote and delay in meiosis led to matrotrophic embryo development and intercalation of the diploid sporophyte before spore production. However, new data on the cell biology of sporogenesis in extant bryophytes suggest that spores were produced directly from zygotes in protoembryophytes. The mechanism of wall transfer from zygote to meiospores was a three-phase heterochrony involving precocious initiation of cytokinesis, acceleration of meiosis, and concomitant delay in wall deposition. In bryophyte sporogenesis, cytokinesis is typically initiated in advance of meiosis, and quadrilobing of the cytoplasm is followed by development of a bizarre quadripolar spindle that assures coordination of nuclear distribution with predetermined spore domains. This concept of the innovation of sporogenesis at the onset of terrestrialization provides a new perspective for interpreting fossil evidence and understanding the evolution of land plants.     

Effect of polymyxins on Bacillus polymyxa sporogenesis

Bacillus polymyxa var. Ross. producing polymyxin M and Bacillus polymyxa 153 producing polymyxin B form spores during submerged cultivation when the rate of biosynthesis of antibiotic peptides is low and when the production of antibiotics is over. However, sporogenesis is stimulated if polymyxins are added at the early stage of cultural growth. Inhibition of the synthesis of antibiotics suppresses the formation of spores. Substances other than polymyxins do not exhibit such a specific effect on sporogenesis. The fact that the culture requires endogenous polymyxins which are most effective in the period prior to the appearance of spores in the culture suggests the regulatory action of these peptides at the stage between vegetative growth and spore formation in Bacillus polymyxa.     

MEIOSIS IN VITRO: SPOROGENESIS IN CULTURED FERN PLANTS

Sporangia containing mature, functional spores can be induced to form on pinnae of Todea barbara sporophytes maintained in nutrient culture. Meiosis is initiated in the sporogenous cells and proceeds to completion in vitro with the formation of viable spores. Sporangial development, meiosis and the production of spores occur in plants which exhibit signs of senescence. Fertile leaves are characterized by a reduction in chlorophyll concentration and total cellular nitrogen when compared to sterile leaves. The data suggest that the process of sporogenesis may be correlated with a reduction in metabolic activity of the leaf.