The zoospore and meiospore of the aquatic phycomyceteCatenaria anguillulae

Summary The zoospore and meiospore of the aquatic phycomyceteCatenaria anguillulae (Phycomycetes, Blastocladiales, Catenariaceae) have a nuclear cap enclosing the cellular ribosomes within a double membrane, and a side body complex which is very similar to that observed in zoospores ofBlastocladiella andCoelomomyces and is structurally related to the side body complex observed in spores ofAllomyces. The structural organization of the side body complex and striated rootlet is analyzed from serial sections.The meiospore also contains an array of flattened cisternae which are in direct contact with, and appear to be derived from, the outer nuclear membrane and the backing membrane of the side body complex.The structural organization of the zoospore and meiospore ofC. anguillulae is compared to and contrasted with the structural organization observed in spores of members of theChytridiales, Blastocladiales, Monoblepharidales, andHarpochytriales. It is concluded that the structural organization of the spores of theBlastocladiales, Monoblepharidales, andHarpochytriales is similar, and affinities in spore organization can be found in some members of theChytridiales.     

Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

The meiospore ofAllomyces

Summary The arrangement of cellular organelles within the meiospore ofAllomyces macrogynus was found to be similar to the zoospore of this species, with the exception that the meiospore contains membrane enclosed electron dense reserve material which has the appearance of the gamma bodies observed in the zoospores ofBlastocladiella. The three dimensional structure of the side body complex is analyzed with serial sections and compared to homologous organelles in other members of theChytridiomycetes.     

The endobiotic thallus ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The endobiotic thallus ofPhysoderma maydis is characterized by the presence of an extremely fine rhizomycelium which passes through the host cell wall, allowing the spread of the disease, and irregularly shaped turbinate cells, which may be septate or nonseptate and which are in close association with developing resting sporangia. The formation of the resting sporangium wall is first seen as localized depositions on the rounded surface of the sporangium and only later on the flattened surface of the sporangium which will form the operculum. The substructure of the resting sporangium wall is typical for members of theBlastocladiales. The resting sporangium is contiguous with the rhizomycelium during development and is finally sealed-off from the rhizomycelium by a further deposition of wall material. After the sealing-off of the resting sporangium from the rhizomycelium the content of the sporangium is compartmentalized and the two inner wall layers are deposited. The centre of the sporangium is filled with an electron dense accretion. At the periphery of the sporangium is a layer of lipid bodies. Between the lipid bodies and the central electron dense accretion is a thin layer of cytoplasm which contains the nuclei. The outer surface of the resting sporangium is smooth.     

Synchronized development of gametophytic germlings of the aquatic PhycomyceteAllomyces macrogynus

Summary Gametophytic germlings ofAllomyces macrogynus are examined with regard to the sequence of developmental events which have been suggested to be effected by long-lived messenger RNA. During encystment of the meiospore, the cytoplasmic triplet microtubules are depolymerized, the gamma bodies are mobilized, the nuclear cap envelope is fragmented and the cap ribosomes are dispersed, the side body complex breaks into its components, and a cyst wall is deposited. All these processes take place prior to the appearance of significant amounts of protein synthesis. The first fission of the mitochondria occurs shortly after the onset of protein synthesis but before detectable RNA synthesis. The developmental sequence of gametophytic and sporophytic germlings was found to be very similar.     

The flagellar apparatus and striated rhizoplast of the zoospore ofOlpidium brassicae

Summary The flagellar apparatus and its associated structures of the zoospore ofOlpidium brassicae are described and compared with observations of other zoospores of the uniflagellatePhycomycetes. The zoospore ofO. brassicae is shown to have an extensive cone-shaped rhizoplast fused to both the functional and the vestigial kinetosomes. Three-dimensional reconstructions were made of the kinetosomal region. The vestigial kinetosome differs from the functional, as it only has triplet bundles of microtubules and it lacks a system of props. The proximal termination of the central pair of flagellar microtubules occurs within the axoneme. No terminal plate is observed. The occurrence of dictyosomes in theChytridiales, Monoblepharidales, andHyphochytriales is discussed and it is concluded that a dictyosome may be present in the encysting zoospore and the maturing zoosporangium ofO. brassicae but only vestiges of a dictyosome are to be found in the free-swimming zoospore.     

The aerobiological significance of smut spores in Tulsa,Oklahoma

Few aerobiological studies have focused on smut spores, teliospores of fungi within the order Ustilaginales, but the scientific luterature provides evidence of the potential aerobiological significance of these plant pathogens. The atmosphere in Tulsa, Oklahoma was monitored for the presence of smut teliospores using a Burkard Volumetric Spore Trap. Smut spores were identified in the atmospheric samples every day from May to October during 1991 and 1992 at concentrations that were normally below 1000 spores/m³. The peak concentration observed during this study was almost 6000 spores/m³. Daily concentrations fluctuate due to a variety of factors such as precipitation, relative humidity, percent sunshine, and the phenology of fungi in relation to their hosts. In northeastern Oklahoma, the most prevalent species of smuts in the atmosphere during the spring includeSphacelotheca occidentalis, Ustilago tritici, andU. kolleri. In the fall, spores ofU. brumivora, U. bullata, andU. maydis are more common.     

The aerobiological significance of smut spores in Tulsa,Oklahoma

Few aerobiological studies have focused on smut spores, teliospores of fungi within the order Ustilaginales, but the scientific literature provides evidence of the potential aerobiological significance of these plant pathogens. The atmosphere in Tulsa, Oklahoma was monitored for the presence of smut teliospores using a Burkard Volumetric Spore Trap. Smut spores were identified in the atmospheric samples every day from May to October during 1991 and 1992 at concentrations that were normally below 1000 spores/m³. The peak concentration observed during this study was almost 6000 spores/m³. Daily concentrations fluctuate due to a variety of factors such as precipitation, relative humidity, percent sunshine, and the phenology of fungi in relation to their hosts. In northeastern Oklahoma, the most prevalent species of smuts in the atmosphere during the spring includeSphacelotheca occidentalis, Ustilago tritici, andU. kolleri. In the fall, spores ofU. brumivora, U. bullata, andU. maydis are more common.     

Ultrastructure ofNosema marucae sp. n. (Microspora,Nosematidae), a pathogen ofMaruca testulalis (Lepidoptera,Pyralidae)

The development ofNosema marucae was followed in its host, the legume pod borerMaruca testulalis. The meronts had an irregular cell body and were binucleated, with the large diplokaryon occupying most of the cell. The sporonts contained many rough endoplastic reticula. The sporoblasts had a granular cytoplasm with a large number of ribosomes, and nuclear division and cellular cleavage were prevalent, particularly from 84 to 120 h after inoculation. Sporoblasts and young spores were evident from 108 to 144 h after inoculation.In the mature spores, the exocuticle had a rough and sculptured surface and the endocuticle thinned considerably at the apical end. The anchoring disc was thick at the point of attachment. The polar tube had 12–15 loops, and the polaroplast was multilayered with two distinct regions and occupied almost half of the spore. The diplokaryon was centrally located in the cell; the nuclear membranes of the diplokarya were separated by an electron-dense region about 0.6 nm thick.     

Membranous aggregations associated with nuclei inAspergillus flavus hyphae

Summary Different growth regions of young vegetative hyphae ofAspergillus flavus manifested structural modification of certain organelles, especially nuclei. In the apical and subapical zones the interphase nuclei had a normal, non-modified structure. In the region which was a few millimeters distant from the hyphal tip, besides normal nuclei there were some with altered morphology.Such an altered nucleus had electron lucent nuclear content and a membranous aggregate stacked against the nuclear membrane. The aggregate was made of flat, parallel membranes of smooth endoplasmic reticulum (ER). The membranous aggregate was also found in-between two nuclei and the stacking of membranes was highly orderly.     

The taxonomic significance of the mitotic spindle and nucleus-associated organelle ofEntomophaga aulicae

Summary Nuclei in protoplasts ofEntomophaga aulicae contain abundant condensed chromatin and a large central nucleolus. The metaphase spindle occupies a small eccentric area of the nucleus while the remainder of the nucleus is filled with condensed chromatin. Small portions of condensed chromatin are aligned along a broad metaphase plate and connected to the spindle poles by kinetochore microtubules. The nucleus associated organelle (NAO) is a solid barlike structure which lies at the spindle poles and is closely associated with the outer membrane of the nuclear envelope. Comparison of the nuclear characteristics ofE. aulicae with those of other members of theEntomophthorales supports the separation of theEntomophthoraceae from theBasidiobolaceae andAncylistaceae. Further comparison of details of nuclear division in theEntomophthoraceae, specifically NAO morphology, may be useful in helping to delineate evolutionary lines within the family.     

The fine structure and cytology of the association between the parasitic red algaHolmsella australis and its red algal hostGracilaria furcellata

Summary Holmsella australis Noble andKraft ms. is a colourless red algal parasite, forming whitish pustules on its photosynthetic red algal host,Gracilaria furcellata Harvey. In the infected region, host cortical tissue continues to grow and enclose the expanding pustule. Filaments of both host and parasite grow apically, the cells being connected by primary pit connections (PCs). Secondary PCs form between cells of the same species, and in addition,H. australis initiates the formation of secondary PCs with cells ofG. furcellata. All three types of secondary PC are morphologically distinct. In hostparasite PCs the surface adjoining the host cell is similar in structure to a host-host PC, while that adjoining the parasite cell has the structure of a parasite-parasite PC. The plasma membrane is continuous between the cells of the unrelated host and parasite. In addition, a cap membrane is typically produced only on the host surface, though occasionally the parasite side is enclosed by a cap membrane as well. Cap membranes are absent from parasite-parasite PCs (making them intracellular), while host-host PCs are typically extracellular, both cells producing cap membranes. The presence or absence of a cap membrane in certain positions appears to vary, and suggests that cells may be able to regulate its presence. Since transport of nutrients would be expected to occur from host to parasite cells, and between parasite cells, the morphological evidence presented here suggests the PCs may be the pathway.     

The mode of action of primycin

Primycin, an antibiotic active against Gram-positive microorganisms increased the permeability ofBacillus subtilis cell membranes when used in bacteriostatic concentrations. On addition of the antibiotic to the washed cell suspension, a dose-dependent increase in the conductivity was observed. Furthermore, an enhanced leakage of the nucleotides (measured by the³²P-ATP release from the³²P-labelled culture) could be detected.To get more information about the mechanism of the primycin-membrane interaction, the effect of the antibiotic on the ATPase activity of membrane vesicles prepared from bothBacillus subtilis andEscherichia coli B was studied. Activation was found at about 0.5 nmol antibiotic/g protein and its extent was approximately the same as with sonicated membranes used as controls. Stimulation of ATPase activity was also achieved with vesicles prewashed with 3 mM Tris-HCl buffer.Purified membrane ATPase fromBacillus subtilis could not be activated by primycin at all; above 0.3 nmol/g protein concentration the enzyme was inhibited. When acting on membrane vesicles isolated fromEscherichia coli B, inhibition without previous activation was observed, although sonication caused a substantial activation on the ATPase of these membranes.These observations confirmed our suggestion that the primary target of primycin action is the cell membrane in Gram-positive microorganisms.Abbreviations OD Optical density     

Isolation and ultrastructural identification of membranes from the fungusGilbertella persicaria

Summary Methods are described for isolating and identifying subcellular membranes from walled hyphae ofGilbertella persicaria. Differences in thickness and symmetry of membranes and in contents of vesicles were used to distinguish different types of membranes. Mitochondria, vacuoles, plasma membrane, and vesicles with attached ribosomes from homogenized germlings equilibrated at the 1.2/1.4 M interface in discontinuous sucrose gradients. Accelerated flotation in centrifuged Ficol-sucrose gradients resulted in the additional separation of the mixed membranes into three fractions: one contained predominantly intact mitochondria, another was composed of vacuoles and vesicles coated with ribosomes, and a third was enriched in plasma membranes. Based upon morphometric analysis, these fractions contained 92% mitochondria, 53% vacuoles, and 89% plasma membranes, respectively. The source of vesicles coated with ribosomes was investigated since rapidly growing hyphae ofG. persicaria contained little rough endoplasmic reticulum as compared with other classes of membranes. Reconstruction from electron micrographs of mitochondrial fragmentation and vesiculation suggested that most of the ribosome-coated vesicles originated from disrupted mitochondria rather than from rough endoplasmic reticulum. The study demonstrates the utility of ultrastructural markers to identify membranesin vitro independent of, or as an adjunct to, cytochemical and biochemical markers.     

Ultrastructure of the gametes ofCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary As part of an investigation on the developmental biology ofCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), the ultrastructure of the male and female gametes was studied. The nucleus is central and conical in shape except for a basal spur that curves back towards the large plate-like mitochondrion. A nuclear cap of ribosomes sits on the flat anterior end of the nucleus. Approximately seven lipid globules are partially embedded in the mitochondrion and are interconnected by membrane cisternae. The lipid globules are covered by a single fenestrated microbody and a backing membrane lies between the microbody and the gamete plasma membrane. The kinetosome is at the base of the nucleus and is connected to a single, posterior, whiplash flagellum. A nonkinetosomal centriole is absent. In the peripheral cytoplasm of both mating types there is a paracrystalline body of unknown composition and function. No significant ultrastructural differences were found between the male and female gametes.     

Morphological changes inEscherichia coli andBacillus cereus caused by external histone

It was found that the externally added histone changes remarkably both the surface and the internal ultrastructure of cells ofEscherichia coli. The interaction of histone with surface structures results in thickening of the inner layer of the cell wall. Cytoplasm becomes condensed, contains extensive electrontransparent zones and neither ribosomes nor the nuclear structure are differentiated. The addition of histone to germinating spores ofBacillus cereus decelerates germination and postgerminative development of this organism and changes ultrastructure of the external surface of the exosporium. The addition of Mg²⁺ ions reverting the effect of histone results in a renewal of the original ultrastruoture of the exosporium.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Ultrastructural studies on the membrane systems and cell inclusions of the filamentous prochlorophyte Prochlorothrix hollandica

The outer membrane of Prochlorothrix hollandica is covered with a network of fine fibrils on its surface and separated from the cytoplasmic membrane by an electrondense peptidoglycan layer (8 to 20 nm thick). The thylakoid membranes are arranged in stacked and unstacked regions which present four characteristic fracture faces with different numbers and sizes of intramembrane particles. Cell inclusions such as polyhedral bodies (carboxysomes), ribosomes, and polyphosphate granules were found in Prochlorothrix hollandica. Another type of cell inclusions was identified by its characteristic shape (a cylindre with conical caps) and a regular striation as gas vesicles. It is concluded that the organism is in its morphological structure similar to the cyanobacteria.Abbreviations C carboxysome - CM cytoplasmic membrane - EF_s, EF_u exoplasmic fracture face of stacked and unstacked membrane area, respectively - ES exoplasmic surface - PF_s, PF_u plasmic fracture face of stacked and unstacked membrane area, respectively - PG peptidoglycan layer - TM thylakoid membrane Dedicated to Prof. Dr. D. Peters, Hamburg, on the occasion of his 75th birthday     

Studies concerning the possible reconstitution of an active cation pump across an artificial membrane

Summary The cortical tissue of rat brain was fractionated through zonal centrifugation in a continuous sucrose density gradient, yielding a variety of morphologically distinct membrane fragments derived from nerve-end particles possessing variable levels of activity of Na, K-dependent Mg-sensitive ATPase (Na, K-ATPase) and other enzymes. Upon addition of certain of the zonal fractions, particularly those rich in the ATPase and acetylcholinesterase activities, to one side of planar artificial membranes, formed from mixtures of oxidized cholesterol and alkanes and bathed in a solution containing sodium, potassium, and magnesium ions, direct current membrane resistance fell from one to three orders of magnitude. Subsequent addition of ATP to the same side of the membrane to which the ATPase was added (thecis side) led to the development of net short-circuit current flow and open-circuit potential across the membrane (thecis side being negative with respect to thetrans side). Development of the short-circuit current and open-circuit potential is dependent upon the presence of all the substrates of Na, K-ATPase as well as that of the enzyme itself. The net current flow is inhibited and the open-circuit potential discharged by the addition of ouabain to thetrans side of the membrane, of phospholipase A to thecis side, or of trypsin to either side of the membrane. These observations provide circumstantial evidence for the reconstitution of the active cation pump across the artificial bilayer. Efforts to effect a similar reconstitution across membranes of this and other compositions employing Na, K-ATPase preparations from beef heart, beef brain, cat brain, human red cells, rabbit kidney, and rat brain microsomes failed.Career Development Awardee of the National Institutes of Health, Grant No. GM 10248.     

The mitochondrial voltage-dependent channel,VDAC, is modified asymmetrically by succinic anhydride

Summary In the accompanying paper, succinic anhydride was shown to react with the outer mitochondrial membrane channel-forming protein, VDAC, resulting in the loss of its voltage dependence. In this paper, the anhydride was added to VDAC held in a particular conformational state by means of an applied electric field. VDAC was inserted into the membranes from thecis side and the anhydride was added either to thecis ortrans side. Channels modified in the open state behaved similarly whether anhydride was added to thecis ortrans side. Modifications of VDAC in either of the two closed states did not. Modifications resulting in the loss of voltage-dependence occurred primarily when anhydride was added to the negative side of the membrane irrespective of which closed state the VDAC was in indicating that the accessibility of the gating charges alternated between thecis andtrans sides as the channel's conformation was changed from one closed state to the other. Despite the pronounced asymmetry, in general the resulting channels behaved in the same way in response to either positive or negative fields. A model consistent with the results is presented which proposes that the same gating charges are responsible for channel closure at both positive and negative fields.