The Plasma Membrane of Myxosporidian Valve Cells: Freeze Fracture Data

Freeze fracturing of Myxosporidian spores reveals the occurrence of a continuous layer of transmembrane particles all over the surface area of the valve cells which form the spore envelope. These particles are densely packed all over the P face membrane. Due to their polygonal outline, their diameter (6-7 nm) and their central core, they resemble the particles forming the connections of gap junctions which metabolically couple the neighboring cells in animal tissues. In the present report, the role of the transmembrane particles is still hypothetical. However, they might represent a membrane structural specialization of the spores which are submitted to osmotic variations of the fluid external medium. Furthermore similar transmembrane particles are observed at the level of the septate junction which seals the valve cells. In this occurrence, they are arranged in a series of 40 double rows parallel to the suture of the spore envelope. These findings support the view that Myxosporidia are Metazoa and raise the problem of their origin.     

Microsporidian Spore Discharge and the Transfer of Polaroplast Organelle Membrane into Plasma Membrane

Electron microscopic examinations of Glugea hertwigi and Spraguea lophii spores indicated the presence of a single plasma membrane; however, this membrane remained in the spore during the discharge of the sporoplasm from the spore. Although discharged spores retained the old plasma membrane, the extruded sporoplasms acquired a new plasma membrane. In order to determine where the new plasma membrane came from, we used two fluorescent probes with membrane affinities. The markers were tested on unfired and discharged spores. The probe, N-phenyl-1-naphthylamine (NPN), labeled the polaroplast membrane in addition to the apolar groups in the posterior vacuoles of unfired spores. After spore discharge, NPN label disappeared from the spore ghosts except for a slight fluorescence on residual plasma membranes. Much of the NPN-labeled membrane reappeared after spore discharge on the outer envelope of discharged sporoplasms. The probe chlorotetracycline (CTC) labeled calcium-associated membranes of spore polaroplasts. During spore discharge, the CTC fluorescence shifted from the polaroplast organelle of unfired spores to the outer envelope of discharged sporoplasms. These results indicate that the polaroplast organelle may provide the new plasma membrane for discharged microsporidian sporoplasms.     

Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition

Myxococcus xanthus is a Gram-negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation-independent remodelling of the rod-shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co-polymerase 2a (OPX/PCP-2a) machinery. Conversion of the capsule-like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo or two other genetic loci encoding homologues of polysaccharide synthesis enzymes fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel Gram-negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition.     

Germination proteins in the inner membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster

Dormant bacterial spores are extraordinarily resistant to environmental insults and are vectors of various illnesses. However, spores cannot cause disease unless they germinate and become vegetative cells. The molecular details of initiation of germination are not understood, but proteins essential in early stages of germination, such as nutrient germinant receptors (GRs) and GerD, are located in the spore inner membrane. In this study, we examine how these germination proteins are organized in dormant Bacillus subtilis spores by expressing fluorescent protein fusions that were at least partially functional and observing spores by fluorescence microscopy. We show that GRs and GerD colocalize primarily to a single cluster in dormant spores, reminiscent of the organization of chemoreceptor signalling complexes in Escherichia coli. GRs require all their subunits as well as GerD for clustering, and also require diacylglycerol addition to GerD and GRs' C protein subunits. However, different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. We predict that the clusters represent a functional germination unit or 'germinosome' in the spore inner membrane that is necessary for rapid and cooperative response to nutrients, as conditions known to block nutrient germination also disrupt the protein clusters.     

Internal valves in populations of Meridion circulare from the A'er Mountain region of northeastern China: Implications for taxonomy and systematics

A population of Meridion circulare var.circulare (Greville) C.A.Agardh from Inner Mongolia was found to produce Innenschalen or internal spores.Examination of this population with light and scanning electron microscopy showed morphological differentiation between vegetative and spore morphologies.Vegetative valves typically bear costae and one rimoportula at the headpole.Spores lack costae and have two rimoportulae,one at the headpole and the other at the footpole.There is plasticity in the production of valve morphologies,and a variety of vegetative valve and spore combinations are evident.This population of M.circulare var.circulare has initial valves of over 90μm in length,and all of the initial cells encountered are acostate and bear two rimoportulae.These observations suggest that either spores are the product of sexual reproduction,or that initial valves may be produced parthenogenetically in Meridion.Spores as products of the sexual process have not been reported in diatoms previously,and parthenogenesis in Meridion was reported previously but discounted in other published reports.The plasticity of valve morphologies expressed in M.circulare var.ciculare,between vegetative valves and spores (and back) across a short temporal period suggests that diatoms can alter their cell wall structure dramatically and quickly in response to external variables.     

Structure and Development of a Marine Actinosporean, Sphaeractinomyxon ersei n. sp. (Myxozoa)

ABSTRACT This is the first ultrastructural study of the development of a marine actinosporean and of a species belonging to the genus Sphaeractinomyxon Caullery & Mesnil, 1904. S. ersei n. sp. is described from a limnodriloidine oligochaete, Doliodrilus diverticulatus Erséus, 1985, from Moreton Bay. Queensland, Australia. Development is asynchronous, there being all stages from two-celled pansporoblasts through to mature spores present simultaneously within a host. Spores develop in groups of eight within pansporoblasts in the coelom and when mature are located also in the intestinal lumen. The primordial spore envelope and sporoplasm develop separately in the pansporoblast until the polar filament is formed within the polar capsule and the capsulogenic cell cytoplasm has begun to degrade. The sporoplasm then enters the spore through a separated valve junction. Mature spores are triradially symmetrical with three centrally located polar capsules and a single binucleate sporoplasm with about 46 germ cells. Swellings or projections of the epispore do not occur when spores exit the host and contact sea water.     

Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the Plasmodium Wall and of Sporogenesis*

SYNOPSIS Ultrastructure of the plasmodium wall and of sporogenesis were studied in Myxosoma funduli Kudo infecting the gills of Fundulus kansae (Garman). Plasmodia were located within the lamellar tissues adjacent to sinuses and capillaries. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite ectoplasm. The plasmodium membrane was covered by a surface coat of almost uniform thickness which prevented direct parasite-host cell contact. Numerous generative cells and cell aggregates, representing early stages of spore development, were seen in immature plasmodia. Later stages of spore development, including mature spores, were observed in older plasmodia. Sporogenesis was initiated by envelopment of one generative cell, the sporont, by a 2nd, nondividing cell, the envelope cell. The sporont and its progeny proceeded through a series of divisions until there were 10 cells, all compartmentalized within the envelope cell. Subsequently, the 10 cells became structurally differentiated and arranged into two 5-celled spore-producing units, each consisting of 1 binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Observations of later developmental stages revealed the major events of capsulogenesis, valvogenesis, and sporoplasm maturation, which occurred concomitantly during spore construction.     

Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores

Dormant Bacillus subtilis spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the gerA family of operons, of which the gerB operon is a member. The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane. In this study we have used Western blot analysis to determine the precise location of GerBA, a gerB-encoded receptor protein, in various spore fractions. GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles. GerBA was also solubilized from the P100 fraction with detergent but not with high salt. These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane. Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane. Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore. We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of gerB operon expression and with the putative receptor function of the proteins encoded by the gerB operon.     

Internal valves in populations of Meridion circulare from the A’er Mountain region of northeastern China: Implications for taxonomy and systematics

Abstract A population of Meridion circulare var. circulare (Greville) C.A. Agardh from Inner Mongolia was found to produce Innenschalen or internal spores. Examination of this population with light and scanning electron microscopy showed morphological differentiation between vegetative and spore morphologies. Vegetative valves typically bear costae and one rimoportula at the headpole. Spores lack costae and have two rimoportulae, one at the headpole and the other at the footpole. There is plasticity in the production of valve morphologies, and a variety of vegetative valve and spore combinations are evident. This population of M. circulare var. circulare has initial valves of over 90 μm in length, and all of the initial cells encountered are acostate and bear two rimoportulae. These observations suggest that either spores are the product of sexual reproduction, or that initial valves may be produced parthenogenetically in Meridion. Spores as products of the sexual process have not been reported in diatoms previously, and parthenogenesis in Meridion was reported previously but discounted in other published reports. The plasticity of valve morphologies expressed in M. circulare var. ciculare, between vegetative valves and spores (and back) across a short temporal period suggests that diatoms can alter their cell wall structure dramatically and quickly in response to external variables.     

Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them. METHODS AND RESULTS: Spores of B. subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective alpha/beta-type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway. Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins. Spores prepared at a higher temperature were also much more resistant to these agents. Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite- and chlorine dioxide-treated spores much more readily released DPA upon a subsequent normally sub-lethal heat treatment than did untreated spores. Hypochlorite-killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+-DPA, and these spores could not be recovered by lysozyme treatment. Chlorine dioxide-treated spores also did not germinate with Ca2+-DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients. However, while germinated chlorine dioxide-killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties. CONCLUSIONS: Hypochlorite and chlorine dioxide do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat. Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane. While chlorine dioxide-killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide.     

Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene-aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol-sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 degrees C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.     

Characteristics of the cytodifferentiation of omega-particle symbiotic bacteria from the micronucleus of Paramecium caudata clone M1-48]

The structure of the omega-particle-bacteria, growing in the micronucleus of Paramecium caudatum (Ciliata, Protozoa), was studied by electrom microscopy in the course of their life cycle. The cytoplasm of the spindle-shaped vegetative cells contains a large number of dense particles and transparent regions comprising the fibrillar material. Such cells, via several intermediate stages, are transformed into elongated twisted cells that are regarded as spores. The spore consists of two parts: homogeneous, and that containing the membrane system and rounded light bodies. The membranes are often double and connected with the fibrils. The cell wall is constructed, during all stages, of the outer membrane layer and the inner electron-dense layer.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

Dielectric Study of the Physical State of Electrolytes and Water Within Bacillus cereus Spores

Dielectric measurements revealed that dormant spores of Bacillus cereus have extremely low conductivities at high frequencies (50 MHz) and so must contain remarkably low concentrations of mobile ions both within the core and in the surrounding integuments. Activation, germination, and outgrowth were all accompanied by increases in conductivity of the cells and their suspending medium, and this result indicated that intracellular electrolytes had become ionized and leaked from the spores. High-frequency dielectric constants of spores were consistent with normal states for cell water. These values increased during successive stages of development from dormant spore to vegetative bacillus, and they could be directly related to increases in cell water content. In all, the results refuted a model of the dormant spore involving freely mobile, ionized electrolytes and supported a model involving electrostatically bound electrolytes.     

A novel Cdc20-related WD-repeat protein, Fzr1, is required for spore formation in Schizosaccharomyces pombe

Ste9/Srw1 which shows sequence homology to Hctl from budding yeast, is an activator of the anaphase-promoting complex (APC) in the fission yeast Schizosaccharomyces pombe. By homology search of the S. pombe genome, we identified the gene fr1+, which encodes the protein with the highest homology to Ste9 among five Cdc20-like proteins. Like Ste9, Fzr1 contains seven WD-repeats in its C-terminal region. In spite of this structural similarity, however, overproduction of either of these proteins cannot complement mutants lacking the other. fzr1+ is transcribed exclusively during meiosis and sporulation, suggesting that it plays a role in these processes. In fact, the fzr1 disruptant formed aberrant asci, which contained only one or two mature spores, though meiotic nuclear divisions proceeded with kinetics similar to wild type, and meiotic segregation of chromosomes was normal. Structural alteration of spindle pole bodies, which is a prerequisite for the formation of the forespore membrane, occurred normally in fzr1delta during the second meiotic division. Localization of spore rim marker proteins fused to green fluorescent protein showed that nascent prespores were irregularly shaped, small in size and few in number in fzr1delta cells compared to wild-type cells. Furthermore, electron microscopy revealed that the outer layer of the spore walls was often missing in fzr1delta spores. These results show that Fzr1 is specifically involved in the assembly of the spore envelope and also in spore maturation. Fzr1, a structural homolog of the APC regulator, therefore plays an important role in spore morphogenesis.     

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.     

Expression of glycoprotein gIII-human decay-accelerating factor chimera on the bovine herpesvirus 1 virion via a glycosyl phosphatidylinositol-based membrane anchor.

Mutants of bovine herpesvirus 1 that express a truncated envelope glycoprotein gIII or a gIII-human decay-accelerating factor (hDAF) chimeric protein (gIII.hDAF) were employed to evaluate the function of the transmembrane and cytoplasmic domains of the gIII molecule. Truncated gIII (i.e., lacking the transmembrane and cytoplasmic region) was readily released from infected cells and was not detected on mature virus particles. In contrast, replacement of the transmembrane and cytoplasmic domains with the carboxyl-terminal portion of hDAF restored the expression of gIII on the membranes of infected cells as well as on virion surfaces. The presence of the gIII.hDAF chimera on virus particles was also associated with normal gIII function, i.e., the mediation of virus attachment and penetration. The gIII-hDAF chimera, which is present on both infected cell surfaces and virions, could be cleaved by a phosphatidylinositol-specific phospholipase C, indicating that it was anchored in the membrane via glycosyl phosphatidylinositol. Our results from this study suggest that the transmembrane and cytoplasmic regions of the gIII molecule serve as a general membrane anchor, but they do not contain structural signals required for the specific assembly of envelope proteins into mature virions.     

The impact of urban and forest fires on the airborne fungal spore aerobiology

The occurrence of airborne fungal spores is driven by several biogeographic and climatic factors. In addition, the occurrence of fires near fungal ecosystems seems to play an important role on the aerobiology of fungal spores. Wildfires are prevalent throughout the world and particularly so in several South European countries with Mediterranean climate. The present survey aimed at analysing the impact of urban and forest fires on the airborne fungal spore content of Madeira Island. Data suggest that after a fire occurrence, the concentration of fungal spores tends to increase in the air, peaking on the 10th day after a fire event. It is likely that fire-induced convections promote release of fungal spores from their natural habitat and that the soil heating conditions could trigger the germination of fungi colonizing post-fire plant residues and soil. It is also possible that the registered low precipitation and wind intensity could help increase the concentration of fungal spores in the atmosphere during 1–2 weeks. These findings need further research, not only at a regional but also at a larger scale, in order to clarify the specific impact of fires on such biological particles and the possible consequences on public health.     

Microsporidian Spore Envelope Keratins Phosphorylate and Disassemble During Spore Activation

The microsporidian spore stage of the nerve parasite, Spraguea lophii, consists of outer envelope stabilized in part by keratins, including K4 and K13. The nonepidermal K4 and K13 keratins were found only in the spore envelope and were absent in the internal microsporidian sporoplasm. At the time of spore activation, the keratin-based outer spore envelope assemblage dissociated and became phosphorylated when the spores were placed in the presence of labeled ATP. Verapamil or lanthanum, agents which block S. lophii spore activation, also blocked spore envelope keratin disassembly and phosphorylation when the spores were incubated in activation medium with labeled ATP. However, after the removal of the verapamil or lanthanum, the spores regained the capacity to activate in discharge medium and the keratin analogues appeared to dissociate and phosphorylate.     

Synthesis of alpha-glucans in fission yeast spores is carried out by three alpha-glucan synthase paralogues, Mok12p, Mok13p and Mok14p

Fission yeast possesses a family of (1,3)-alpha-glucan synthase-related genes; one of them, mok1+/ags1+, plays an essential function in morphogenesis during vegetative growth. Here we show that three mok1+ paralogues -mok12+, mok13+ and mok14+- are required for sporulation to succeed, acting at different stages of the spore wall maturation process. Mutation of mok12+ affected the efficiency of spore formation and spore viability. Deletion of mok13+ does not affect spore viability but the spores showed reduced resistance to stress conditions. mok14Delta mutant spores failed to accumulate the amylose-like spore wall-specific polymer. mok12+, mok13+ and mok14+ expression was restricted to sporulating cells and the proteins localized to the spore envelope but with different timing. mok11+ was also induced during the sporulation process although its deletion did not show apparently a sporulation defect. In vegetative cells, beta-glucans are more abundant than alpha-glucans (55% versus 28%). In spores, the situation was the opposite, alpha-glucans accounted for 46% while beta-glucans were approximately 38% of the total polysaccharides. We found at least two types of alpha-glucan polymers, Mok12p and Mok13p, were involved in the synthesis of the greater part of alpha-glucan in the spores envelope, a polymer that is mainly digested with alpha-1,3 glucanase, while Mok14p, homologous to starch synthases, was required for the synthesis of the iodine-reactive polymer that is made of alpha-1,4 glucose residues.