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.     

The Fine Structure of Streptomyces coelicolor : I. The Cytoplasmic Membrane System

Colonies and spore suspensions of Streptomyces coelicolor were fixed by the method of Kellenberger, Ryter, and Séchaud (1958) and embedded in methacrylate or araldite. Thin sections were cut with an A. F. Huxley microtome and examined in a Siemens' Elmiskop I. At all stages of development the hyphae of Streptomyces coelicolor have an extensive membranous component in the cytoplasm. The membranes are continuous with the plasma membrane and have a variety of configurations at different places in the hyphae. Tubular structures, vesicles, and parallel stacks of membranes are seen. In some areas concentric layers of membranes form whorled structures which are particularly frequent in the region of developing cross-walls and within maturing spores. In the spores membranous structures often lie embedded in the nuclear material. In disintegrating hyphae the intracytoplasmic membranes round off into small vesicles and remain when the rest of the cytoplasmic structure has gone. In the absence of typical mitochondria and other cytoplasmic membranous structures it is possible that the membranous component of the cytoplasm of Streptomyces coelicolor may perform the functions of the endoplasmic reticulum and/or the mitochondria of higher cells.     

Composition and Ultrastructure of Streptomyces venezuelae

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.     

Mycelial differentiation and spore formation by Streptomyces brasiliensis in submerged culture.

Streptomyces brasiliensis ATCC 23727 showed extensive sporulation when cultured in a liquid medium containing galactose and glutamic acid as carbon and nitrogen sources. Sporogenic hyphae formed under these conditions were morphologically similar and developmentally equivalent to aerial hyphae and metamorphosed into chains of spores by following a sequence of ultrastructural changes similar to that observed during growth on solid media. In addition, our electron microscopy study revealed two previously unrecognized aspects of hyphal development in streptomycetes: the formation of sporogenic hyphae was always preceded by changes in the structure of the nucleoid, and the sheath that characteristically covered these hyphae was not deposited coincidently with wall formation in the apical growing portion of the hypha.     

Characterization of Laurencia arbuscula spore mucilage and cell walls with stains and FITC-labelled lectins

In most red algae, spores are liberated without a cell wall, within a sheath of mucilage that is responsible for its primary attachment. Utilizing fluorescent-labelled lectins, we identified carbohydrate residues and their location in the mucilage and cell walls of spores of Laurencia arbuscula. Cell wall formation and mucilage composition were studied with Calcofluor, Toluidine Blue (AT-O), Alcian Blue (AB) and periodic acid-Schiff (PAS). In the mucilage, we identified α-d-mannose, α-d-glucose, N-acetyl-glucosamine, N-acetyl-galactosamine and β-d-galactose. All sugar residues were found in the cell wall, in the spore body rather than in the rhizoid, which suggests that the residues may be related to initial substrate adhesion. A cell wall is produced soon after the spore's attachment, beginning with a deposition of cellulose around the spore, as indicated by Calcofluor. A polarization of the cell wall triggers the process of germination. The cell-wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while neutral polysaccharides were positive to PAS.     

The surface structure of spores and aerial hyphae in Streptomyces viridochromogenes

Summary The fine structure of the surface membrane which covers the cell walls of spores and aerial hyphae of S. viridochromogenes was investigated by means of electron microscopy of air-dried whole mounts, thin sections, negative stainings and freeze-etchings. The delicate sheath of the spores, which is responsible for the surface features, exhibits conspicuous cone shaped protrusions called spines. They are composed of 5–12 rodlike units, each apparently consisting of a bundle of fine fibrils. Non-sporulating hyphae have no spines; their surface membrane shows an irregular pattern consisting of long tubules.     

Composition of the Cellular Envelopes of Anabaena cylindrica

Comparative chemical analyses were made of the walls of vegetative cells, heterocysts, and spores, and of the mucilage of Anabaena cylindrica. The wall of the vegetative cell is composed predominantly of amino compounds, with a mannose-rich carbohydrate component comprising only 18% of the dry weight. In contrast, 62% of the heterocyst wall and 41% of the spore wall is carbohydrate. The carbohydrate moieties of the heterocyst wall and spore wall are similar in that the ratio of glucose, mannose, galactose, and xylose is approximately 75:20:3:4 in both walls. It appears that, during the differentiation of a vegetative cell into either a spore or a heterocyst, a glucose-rich wall polysaccharide is produced that is different from the polysaccharide component of the wall of the vegetative cell and of the sheath. In the case of the heterocyst, the wall was estimated to account for approximately 52% of the dry weight of the whole cell.     

A Highly Redundant Gene Network Controls Assembly of the Outer Spore Wall in S. cerevisiae

The spore wall of Saccharomyces cerevisiae is a multilaminar extracellular structure that is formed de novo in the course of sporulation. The outer layers of the spore wall provide spores with resistance to a wide variety of environmental stresses. The major components of the outer spore wall are the polysaccharide chitosan and a polymer formed from the di-amino acid dityrosine. Though the synthesis and export pathways for dityrosine have been described, genes directly involved in dityrosine polymerization and incorporation into the spore wall have not been identified. A synthetic gene array approach to identify new genes involved in outer spore wall synthesis revealed an interconnected network influencing dityrosine assembly. This network is highly redundant both for genes of different activities that compensate for the loss of each other and for related genes of overlapping activity. Several of the genes in this network have paralogs in the yeast genome and deletion of entire paralog sets is sufficient to severely reduce dityrosine fluorescence. Solid-state NMR analysis of partially purified outer spore walls identifies a novel component in spore walls from wild type that is absent in some of the paralog set mutants. Localization of gene products identified in the screen reveals an unexpected role for lipid droplets in outer spore wall formation.     

Otospora bareai, a new fungal species in the Glomeromycetes from a dolomitic shrub land in Sierra de Baza National Park (Granada, Spain)

A new fungal species of the Glomeromycetes was isolated from the rhizosphere of Pterocephalus spathulatus and Thymus granatensis, two rare endemic plants growing on dolomite in the Sierra de Baza (Granada, southern Spain). The fungus was propagated in pot cultures of Sorghum vulgare and Trifolium pratense for 4 y and it is described here on the basis of the spores found in nature and formed in pot cultures. Its brown spores (140-210 microm diam) form laterally on a persistent, brown stalk (=neck) of a sporiferous saccule. They have two walls without ornamentation: a brown, three- to four-layered outer wall and a hyaline two- to three-layered inner wall. The unique combination of spore formation and spore wall structure does not fit with any of the known fungal genera. Spore formation is similar to that of Acaulospora spp. and Archaeospora trappei, but Acaulospora spp. has three spore walls with a characteristic "beaded" wall, and the outer wall of Ar. trappei is simple, thin, hyaline and only bilayered. Spore wall structure of the new fungus is similar to that of Entrophospora infrequens, however this fungus forms its spores internally, inside the hyphal stalk of the sporiferous saccule. Molecular analyses of the small subunit of the ribosomal gene phylogenetically place the new fungus next to Diversispora spurca, which forms one-walled glomoid spores (i.e. terminally on hyphae). Based on these analyses we place the new fungus into a new genus in the family Diversisporaceae under the epithet Otospora bareai.     

Changes in the surface layer (sheath) of the cell wall of streptomycetes during sporulation

Fine structural changes of the sheath, occurring during spore formation in strains Streptomyces finlayi ATCC 23 340 and Streptomyces coeruleorubidus FBUA 328 were investigated by means of electron microscopy of air-dried whole mounts and thin sections. The results suggest that in the strains the process of sporulation is not strictly synchronized spatially with the molecular arrangements and re-arrangements (formation of hairy or spiny surface ornaments) occurring outside the wall of the sporulation hyphae, in the sheath. On the contrary, the intensity of transformation induction in the sheath may show a gradually decreasing tendency from the tip of the sporulating hypha towards its sterile basal part, resulting in the formation of both ornamented and smooth spores in the same chain. This suggests that the morphogenetic changes occurring in the sheath during spore formation are probably controlled by a "functional centre", located near the tip of the sporulating hypha, and this centre is perhaps indentical with the cell unit at the tip.     

Fine structure of the cell walls of Polyporus myllitae Cke. et Mass.

Following a brief survey of the wall structure of the vegetative hyphae of a number of basidiomyeetes, attention is focused upon Polyporus myllitae Cke. et Mass. After removal of the outer amorphous layer by various chemical treatments, the underlying surface is seen to consist of an interwoven network of microfibrils. There is no evidence of any preferred angle of orientation. However, on what is believed to be the inner surface of the hyphal wall, microfibrils show a strong tendency towards a transverse orientation. The resulting structure is compatible with the multi-net concept of cell wall growth of Houwink and Koelofsen. There is no obvious change in microfibril orientation in passing along a hypha towards its tip. Electron-opaque cross-walls partition hyphae, sometimes separate a branch from a parent hypha, and occur in clamp connections. The cross-wall consists of microfibrils underlying, or embedded in, an amorphous matrix. They are circularly arranged around a single central pore which has a thickened rim.     

Ultrastructural study of galacturonic acid distribution in some pathogenic fungi using gold-complexed Aplysia depilans gonad lectin

Aplysia gonad lectin, isolated from the mollusc Aplysia depilans, was successfully conjugated to colloidal gold and used for ultrastructural detection of galacturonic acids in some pathogenic fungi. These sugar residues were found to occur in the fibrillar sheath surrounding hyphal cells of Ascocalyx abietina and in intravacuolar dense inclusions of this fungus spores. In hyphae and spores of Ophiostoma ulmi, galacturonic acids were detected mainly in the outermost wall layers. In contrast, these saccharides appeared associated with the innermost wall layers and especially the plasma membrane of Verticillium albo-atrum cells. Galacturonic acids were found to be absent in cells of Fusarium oxysporum f.sp. radicis-lycopersici and Candida albicans. These cytochemical data indicate therefore that a heterogeneity in wall composition exists between ascomycete fungi. The significance of the presence of galacturonic acids in the cell walls of certain fungi is still open to question.     

Spore Adhesion and Cell Wall Formation in Gelidium Floridanum (Rhodophyta, Gelidiales)

The attachment of spores to a substratum is essential for their germination and, therefore, to the completion of the life cycle of the red algae. In most red algae, spores are liberated without a cell wall, within a sheath of mucilage which is responsible for their primary attachment. Utilizing fluorescent-labeled lectins, we identified carbohydrate residues and their locations in the mucilage and cell walls of spores of Gelidium floridanum. Cell wall formation and mucilage composition were studied with calcofluor, toluidine blue (AT-O), alcian blue (AB) and periodic acid-Schiff (PAS). In the mucilage we identified α-D mannose, α-D glucose, β-D-galactose, N-acetyl-glucosamine and N-acetyl-galactosamine. The first two sugar residues were not found in the cell wall of the germ tube but they were present on the rhizoid’s cell wall indicating their importance to substrate adhesion. A cell wall is produced soon after the spore’s attachment, beginning with a polar deposition of cellulose and its gradual spread around the spore as indicated by calcofluor. The cell wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while cellulose microfibrills were positive to PAS. A polar disorganization of the cell wall triggers the process of germination. As spores are the natural form of propagation of Gelidium, the understanding of the mechanisms of spore attachment may contribute to the cultivation of this valuable seaweed.     

A study of the fine structure of ascosporogenesis in Saccobolus kerverni

Summary Observations of ascospore fromation in KMnO₄-fixed Saccobolus kerverni apothecia with the electron microscope reveal the following sequence. Ascus formation is preceded by the development of croziers whose fine structure differs little from that of vegetative hyphae. Following fusion of the two nuclei in the ascus mother cell, the resultant ascus elongates, and two large vacuoles appear, first below and later above the fusion nucleus. These vacuoles soon occupy dominant positions at the tip and bottom of the ascus and assume a flocculent appearance. Nuclear blebbing occurs during meiosis, mitosis, and the subsequent spore delimitation process in the central cytoplasmic portion of the ascus. Each spore initial is surrounded by two membranes, the plasma and investing membranes, between which the spore wall is deposited in two layers, an inner primary wall and an outer secondary wall. Following primary wall deposition the spores clump; secondary wall deposition begins outside the primary wall at the places where the spores are contiguous. Interdigitation of these walls and disappearance of the investing membranes in the sutures lead to the envelopment of all eight ascospores in a common secondary wall. A flocculent material in the epiplasmic vacuoles aggregates around the mature spore balls.Based on a portion of a dissertation presented to the Faculty of the Graduate School of the University of Texas in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Micro-autoradiographic studies on host-parasite interactions

Tritium labeled uredospores of Uromyces phaseoli were produced be feeding the host, Phaseolus vulgaris, with ³H-orotic acid. These spores were allowed to germinate on and to penetrate into a bean leaf. 24 hrs after inoculation, the bean rust had formed the first haustorium. All fungal structures, including the fungus walls, were heavily labeled. No label could be detected in the cells that had come into contact with the hyphae. In the infected host cell, the haustorium was labeled heavily, but the sheath around the haustorium and the host cell remained free of label. These results indicate that no detectable amounts of label leach from the bean rust into the host at this stage of infection although it is known that the rust takes up many metabolites. Since the sheath remains free of label and all fungal structures are evenly labeled, it is concluded that the sheath is formed by the host.     

Cytology and ultrastructure of interactions between <Emphasis Type="Italic">Ustilago esculenta</Emphasis> and <Emphasis Type="Italic">Zizania latifolia</Emphasis>

Ustilago esculenta is a biotrophic smut fungus that parasitizes Zizania latifolia, an edible aquatic vegetable of the southern China region. Infection results in swelling of the upper parts of the Z. latifolia culm which are called jiaobai and have a unique flavor and delicacy and are popular among Chinese. The infection process of Z. latifolia by U. esculenta was investigated with light and electron microscopy. Distribution of hyphae was uneven in plants; hyphae were mainly present in the swollen upper parts (jiaobai), the nodal regions of mature culms and old rhizomes and buds or shoots. Hyphae were rare in the internodes of mature culms and were fewer in the internodes of old rhizomes. All new buds produced on the nodes of culms and rhizomes were infected by hyphae in November before and in March after overwintering. The hyphae grew into the buds from the parent nodes via intervascular tissues only or via parenchyma tissues and vascular bundles. Hyphae extended within and between the host cells and frequently formed hyphal aggregations or clusters, not only in the mature tissues but also in developing tissues. The typical interface between the fungal hyphal wall and invaginated host plasma membrane comprised a sheath. The sheath surrounding a hyphae comprised an outer electron-opaque matrix and an inner electron-dense layer. The electron-opaque matrix layers were thicker in jiaobai tissues, ranging from 0.28 to 0.85 μm. The electron-dense hyphal coatings were more conspicuous in the young buds or shoots and mature culms than in the jiaobai. The intercellular hyphae caused large cavity formation between the cells or rupture of host cell walls, for gaining entry into host cells. The broken host cell wall fused with the electron-opaque matrix of the hyphal sheath as an interactive interface. The teliospore wall and wall ornamentation development was the same in postmature jiaobai tissues with sporadic sori and in the huijiao (jiaobai tissues containing the massive sori), but a sheath enveloping the teliospore was more transparent in the process of teliospore development in the jiaobai than in the huijiao.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

Studies of aureobasidium pullulans (de Bary) Arnaud

Summary The dark pigmentation ofAureobasidium pullulans is due to melanin that stains the walls and may be heavily encrusted on the surface of older cells. The pigment granules can be shaken off ultrasonically. The melanin is quite impervious to ultraviolet light.Most strains ofA. pullulans observed secrete a viscous mucin that encloses hyphae and spores in a matrix. In both hyphae and yeastlike blastospores, cross walls develop centripetally, and in many cases a pore is left in the cross wall, rendering possible protoplasmic movement and a heterokaryotic condition, which may account for the variability of the species. These wall pores are not like those found in Basidiomycetes.The blastospores contain droplets of lipid, and usually have but one nucleus though the few larger spores may have two or rarely three nuclei. The blastospores are not borne on stipules but bud from the parent cell in a manner similar to yeasts and leave behind a bud scar.     

Hyphal wall growth in Neurospora crassa and Geotrichum candidum.

Growth of the walls of hyphae of Neurospora crassa and Geotrichum candidum was studied using longitudinal and serial transverse sectioning methods. Rigidification of the hyphal wall below the extension zone did not appear to involve the gross formation of a secondary wall since the transition from extensible to non-extensible wall was not associated with an increase in thickness. However, behind the extension zone the walls leading hyphae of N. crassa increased in thickness until eventually they attained a thickness which was up to five times that of the tip wall. A hypothesis of hyphal wall growth is proposed.     

Development of mestome sheath cells in leaves ofAegilops comosa var.thessalica

Summary The development of mestome sheath cells ofAegilops comosa var.thessalica was studied by electron microscopy. Anatomical and cytological observations show that this grass belongs to the C₃ or non-Kranz plants. In the asymmetrically thickened walls of mestome sheath cells a suberized lamella is present. This lamella is deposited asynchronously. In the midrib and the large lateral bundles it appears first in the outer and inner walls and usually later in the radial walls. In the small lateral bundles its appearance is delayed in the inner walls of those cells situated on the xylem side. At maturity the suberized lamella is observed in all cell walls; however, in the small lateral bundles it is partly or totally absent from the walls of some cells situated on the xylem side. Tertiary wall formation is asynchronous as well, for it generally follows the deposition pattern of the suberized lamella.During the development of the mestome sheath cells microtubules show marked changes in their number and orientation, being fewer and longitudinal during suberin deposition. Dictyosomes are very active and may be involved in primary and tertiary wall formation. Endoplasmic reticulum cisternae are abundant and partly smooth, while plasmalemmasomes may function to reduce the plasmalemma extension. However, cytoplasmic structures that are clearly involved in suberin synthesis could not be identified.Suberized lamellae react strongly with silver hexamine. This is probably due to post-fixation with osmium tetroxide.On the basis of structural characteristics the mestome sheath may be regarded as an endodermis (cf., alsoFahn 1974). The significance of this view for water and assimilate exchange between the mesophyll and the bundle is discussed.This report represents a portion of a doctoral dissertation.