NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolor

Streptomycetes have a complex morphogenetic programme culminating in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. We here compared whole genome expression of a wild-type colony of Streptomyces coelicolor forming aerial hyphae and spores with that of the chp null mutant that forms few aerial structures. This revealed that expression of 244 genes was significantly altered, among which genes known to be involved in development. One of the genes that was no longer expressed in the Δ chpABCDEFGH mutant was nepA , which was previously shown to be expressed in a compartment connecting the substrate mycelium with the sporulating parts of the aerial mycelium. We here show that expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall. Germination of spores of a nepA deletion mutant was faster and more synchronous, resulting in colonies with an accelerated morphogenetic programme. Crucially, spores of the Δ nepA mutant also germinated in water, unlike those of the wild-type strain. Taken together, NepA is the first bacterial structural cell wall protein that is important for maintenance of spore dormancy under unfavourable environmental conditions.     

Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET.     

CabC, an EF-hand calcium-binding protein, is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation in Streptomyces coelicolor

Ca(2+) was reported to regulate spore germination and aerial hypha formation in streptomycetes; the underlying mechanism of this regulation is not known. cabC, a gene encoding an EF-hand calcium-binding protein, was disrupted or overexpressed in Streptomyces coelicolor M145. On R5- agar, the disruption of cabC resulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, while cabC overexpression significantly delayed development. Manipulation of the Ca(2+) concentration in R5- agar could reverse the phenotypes of cabC disruption or overexpression mutants and mimic mutant phenotypes with M145, suggesting that the mutant phenotypes were due to changes in the intracellular Ca(2+) concentration. CabC expression was strongly activated in aerial hyphae, as determined by Western blotting against CabC and confocal laser scanning microscopy detection of CabC::enhanced green fluorescent protein (EGFP). CabC::EGFP fusion proteins were evenly distributed in substrate mycelia, aerial mycelia, and spores. Taken together, these results demonstrate that CabC is involved in Ca(2+)-mediated regulation of spore germination and aerial hypha formation in S. coelicolor. CabC most likely acts as a Ca(2+) buffer and exerts its regulatory effects by controlling the intracellular Ca(2+) concentration.     

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.     

Allelic Differences within and among Sister Spores of the Arbuscular Mycorrhizal Fungus Glomus etunicatum Suggest Segregation at Sporulation

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF are made up of coenocytic networks of hyphae through which nuclei and organelles can freely migrate. In this study, we investigated the possibility of a genetic bottleneck and segregation of allelic variation at sporulation for a low-copy Polymerase1-like gene, PLS. Specifically, our objectives were (1) to estimate what allelic diversity is passed on to a single spore (2) to determine whether this diversity is less than the total amount of variation found in all spores (3) to investigate whether there is any differential segregation of allelic variation. We inoculated three tomato plants with a single spore of Glomus etunicatum each and after six months sampled between two and three daughter spores per tomato plant. Pyrosequencing PLS amplicons in eight spores revealed high levels of allelic diversity; between 43 and 152 alleles per spore. We corroborated the spore pyrosequencing results with Sanger- and pyrosequenced allele distributions from the original parent isolate. Both sequencing methods retrieved the most abundant alleles from the offspring spore allele distributions. Our results indicate that individual spores contain only a subset of the total allelic variation from the pooled spores and parent isolate. Patterns of allele diversity between spores suggest the possibility for segregation of PLS alleles among spores. We conclude that a genetic bottleneck could potentially occur during sporulation in AMF, with resulting differences in genetic variation among sister spores. We suggest that the effects of this bottleneck may be countered by anastomosis (hyphal fusion) between related hyphae.     

Autophagy during conidiation and conidial germination in filamentous fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)-AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.     

Regulation of Streptomyces development: reach for the sky!

Streptomyces coelicolor is characterized by a complex life cycle and serves as a model system for bacterial development. After a feeding substrate mycelium has been formed, this filamentous bacterium differentiates by forming aerial hyphae that septate into spores. The bld cascade regulates initiation of aerial growth, whereas the whi genes control spore formation. Recent findings indicate the existence of another regulatory pathway that operates after aerial hyphae have started to grow into the air, which we call the sky pathway. This pathway controls the expression of the chaplin and rodlin genes. These genes encode proteins that assemble into a rodlet layer that provides surface hydrophobicity to aerial hyphae and spores.     

Production and localization of restrictocin in Aspergillus restrictus.

The production and secretion of restrictocin (a cytotoxin that cleaves ribosomal RNA) by cultures of the fungus Aspergillus restrictus was investigated. Previous studies have indicated that restrictocin production in liquid culture coincides with the appearance of differentiated cell structures. A study of the correlation between the appearance of differentiated structures and restrictocin production was conducted with A. restrictus grown on agar medium. Restrictocin was found to be associated with the cell mass of the agar-grown culture (in contrast to liquid cultures), and was first observed when aerial hyphae emerged. Restrictocin levels increased until the time of conidiation, after which they fell off sharply. No restrictocin could be found in the agar medium. The presence of restrictocin upon and within various cell structures was determined by immunofluorescent laser microscopy. This study showed that restrictocin became localized to the conidiophores and phialides during the process of conidiation. Prior to this, restrictocin was found within the hyphae in localized concentrations that may correspond to secretory vesicles.     

OBSERVATIONS ON THE CHROMATINIC BODIES OF Streptomyces coelicolor

Colonies of Streptomyces coelicolor growing on cellophane and impression preparations from sporing colonies were stained for chromatin by the methods of Feulgen, DeLamater (1951), and Piéchaud (1954). The chromatinic bodies of the substrate hyphae have a great variety of configurations. During the development of the spores, elongated chromatinic structures in the young aerial hyphae separate into a number of subunits and a single round chromatinic body is included in each spore.     

Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor

In the filamentous bacterium Streptomyces coelicolor , the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 μm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 μm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants ( whiG , whiH and whiB ), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.     

MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores

MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.     

Spore dispersal of a resupinate ectomycorrhizal fungus, Tomentella sublilacina, via soil food webs

Patterns of fungal spore dispersal affect gene flow, population structure and fungal community structure. Many Basidiomycota produce resupinate (crust-like) basidiocarps buried in the soil. Although spores are actively discharged, they often do not appear to be well positioned for aerial dispersal. We investigated the potential spore dispersal mechanisms of one exemplar of this growth form, Tomentella sublilacina. It is a widespread ectomycorrhizal fungus that sporulates in the soil organic horizon, can establish from the spore bank shortly after disturbance, but also can be a dominant species in mature forest stands. We investigated whether its spores could be dispersed via spore-based food webs. We examined external surfaces, gut contents and feces from arthropod fungivores (mites, springtails, millipedes, beetles, fly larvae) and arthropod and vertebrate predators (centipedes, salamanders) from on and around T. sublilacina sporocarps. Spore densities were high in the guts of many individuals from all fungivore groups. Centipede gut contents, centipede feces and salamander feces contained undigested invertebrate exoskeletons and many apparently intact spores. DAPI staining of spores from feces of fungivores indicated that 7-73% of spores contained intact nuclei, whereas spores from predators had lower percentages of intact nuclei. The spiny spores often were lodged on invertebrate exoskeletons. To test the viability of spores that had passed through invertebrate guts we used fecal droppings of the millipede Harpaphe haydeniana to successfully inoculate seedlings of Pinus muricata (Bishop pine). These results indicate the potential for T. sublilacina spore dispersal via invertebrates and their predators in soil food webs and might help to explain the widespread distribution of this species. It is likely that this is a general mechanism of dispersal for fungi producing resupinate sporocarps, indicating a need to develop a fuller understanding of the linkages of soil food webs and spore dispersal.     

Seasonality of vesicular-arbuscular mycorrhizae in sedges in a semi-arid tropical grassland

Vesicular-arbuscular mycorrhizal (VAM) colonization and spore numbers in the rhizosphere of Cyperus iria L. and C. rotundus L., growing in a semi-arid tropical grassland, was studied during the 1993 and 1994 monsoons. In addition, climatic and chemical properties of the soils were determined in order to investigate their influence on mycorrhizal variables. VAM fungal association in the sedges was confirmed by plant- and root-trap culture techniques. The soil nutrients exhibited seasonal variations, but were highly variable between years. Intercellular hyphae and vesicles with occasional intraradical spores characterized mycorrhizal association in sedges. Dark septate fungi also colonized roots of sedges. Temporal variations in mycorrhizal colonization and spore numbers occurred, indicating seasonality. However, the patterns of mycorrhizal colonization and spore numbers were different during both the years. The VAM fungal structures observed were intercellular hyphae and vesicles. Changes in the proportion of root length with VAM structures, total colonization levels and spore numbers were related to climatic and edaphic factors. However, the intensity of influence of climatic and soil factors on VAM tended to vary with sedge species.     

The gamma body: A vesicle generating structure

Gamma bodies, which are present in the sporangia and gametangia of Allomyces and in its spores, are interpreted as constituting vesicle generating structures. During spore cleavage the mobilization–decay of the gamma bodies leads to vesicle formation; the vesicles appear to fuse to form the axonemal and plasma membrane of the spore. Vesicle formation by the gamma bodies during spore cleavage can be perturbed by phosphate buffer which leads to the formation of myelin–figure arrays of membranes, or by colchicine and benomyl which give rise to large vacuolar structures after gamma body decay. During the motile period of the spores of Allomyces , mobilization of the gamma bodies leads to vacuole formation and the resulting vacuoles fuse with the plasma membrane of the spore and by this means maintain the osmotic balance of the spore. During spore encystment the gamma body decays and forms vesicles which fuse with the plasma membrane of the cyst; these vesicles appear to be instrumental in chitin wall synthesis.     

Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.     

Acidic vesicles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita

Localization and movement of organelles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita, were observed using a combination of fluorescent probes and laser-scanning confocal microscopy. Dense, evenly distributed acidic vesicles were visible in germ tubes and extraradical hyphae using DIC with the fluorescent acidotropic probe LysoTracker. These vesicles were distinct from both tubular vacuoles stained with DFFDA and lipid bodies stained with BODIPY 493/503 or Nile Red. Tubular vacuole bundles appeared to be influenced by the bidirectional cytoplasmic streaming of acidic vesicles and lipid bodies. Movement of the acidic vesicles occurred bidrectionally at different rates. The size and distribution of lipid bodies were variable. Based on our observations, the function of these organelles is discussed in relation to nutrient translocation in arbuscular mycorrhizas. Abbreviations: AM – arbuscular mycorrhiza; DAPI – 4′,6-diamidino-2-phenylindole; DIC – differential interference contrast; BODIPY 493/503 – 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; DMSO – dimethyl sulfoxide; FITC – fluorescein isothiocynate; caboxy-DFFDA – Oregon Green 488 carboxylic acid diacetate.     

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.     

A sensitive method for examining whole-cell biochemical composition in single cells of filamentous fungi using synchrotron FTIR spectromicroscopy

Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids at about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.     

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.     

Organization of Organelles within Hyphae of Ashbya gossypii Revealed by Electron Tomography

Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.