Heterocysts with Reduced Cell Walls in Populations of Cycad Cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. In addition to heterocysts with the typical ultrastructure, the cyanobiont microcolonies also contained altered heterocysts with reduced cell walls, which might dominate in all regions of the coralloid roots. The altered heterocysts represented a protoplast enclosed in a heterocyst-specific envelope with additional layers. Some heterocysts contained an additional reticular protoplast-enclosing sheath below the heterocyst-specific envelope, whereas the other heterocysts contained an additional electron-opaque outer layer. The substance of the inner sheath of the former heterocysts resembled the polysaccharides of mucilage, which fills the intercellular space, whereas the electron-opaque outer layer of the latter heterocysts probably had a protein nature. The substances that constitute the sheath and the outer layer are likely to be synthesized intracellularly and then released with the aid of membrane-bounded vesicles or by ruptures in the cytoplasmic membrane.     

Evidence that the barrier to the penetration of oxygen into heterocysts depends upon two layers of the cell envelope

Mutants of Anabaena sp. PCC 7120 with O₂-sensitive acetylene-reducing activity were studied to identify envelope components that contribute to the barrier limiting diffusion of oxygen into the heterocyst. Mutant strain EF114, deficient in a heterocyst-specific glycolipid, reduced acetylene only under strictly anaerobic conditions. Analysis of in vivo O₂ uptake as a function of dissolved pO₂ showed that EF114 has lost the low affinity, diffusion-limited respiratory component associated with heterocysts in wild-type filaments. The low affinity respiratory activity was also lost in EF116, a mutant in which the cohesiveness of the outer polysaccharide layer was reduced. Restoration of aerobic nitrogen fixation in a spontaneous revertant of EF116 and in a strain complemented with cosmid 41E11 was associated with restoration of the low affinity component of respiratory activity. The results provide evidence that the barrier to diffusion of gas into heterocysts depends upon both the glycolipid layer and the polysaccharide layer of the heterocyst envelope.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

The primary walls of cotton fibers contain an ensheathing pectin layer

Summary Cotton fiber walls (1–2 days post anthesis) are distinctly bilayered compared to those of nonfiber epidermal cells, with a more electron-opaque outer layer and a less electron-opaque, more finely fibrillar inner layer. When probed with antibodies and affinity probes to various saccharides, xyloglucans and cellulose are found exclusively in the inner layer and de-esterified pectins and extensin exclusively in the outer layer. Ovular epidermal cells that do not differentiate into fibers have no pectin sheath, but are labelled throughout with antixyloglucan and cellulase-gold probes. Middle lamellae between adjacent cells were clearly labelled with the antibodies to de-esterified pectins, however. Similarly, cell walls of leaf trichomes have a bilayered wall strongly enriched in pectin, whereas other epidermal cells are not bilayered and are pectin poor. These data indicate that one of the early markers of fiber and trichome cells from other epidermal cells involves the production of a pectin layer. The de-esterified pectins present in the ensheathing layer may allow for expansion and elongation of the fiber cells that does not occur in the other epidermal cells without such a sheath or may even be a consequence of the elongation process.     

Morphological and ultrastructural changes in vegetative cells and heterocysts of Anabaena variabilis grown with fructose.

The morphology and ultrastructure of Anabaena variabilis grown in medium with and without 40 mM fructose were compared. Vegetative cells and young heterocysts in fructose-supplemented medium were significantly larger, were filled with glycogen granules, and had fewer thylakoids. Developing heterocysts contained large numbers of glycogen granules well into mature stages, and envelope formation was precocious. As heterocysts enlarged in fructose medium, their shape became more broadly oblong compared with the more rectangular heterocysts in fructose-free medium.     

Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.     

Novel ATP-driven pathway of glycolipid export involving TolC protein

Upon depletion of combined nitrogen, N(2)-fixing heterocysts are formed from vegetative cells in the case of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. A heterocyst-specific layer composed of glycolipids (heterocyst envelope glycolipids (HGLs)) that functions as an O(2) diffusion barrier is deposited over the heterocyst outer membrane and is surrounded by an outermost heterocyst polysaccharide envelope. Mutations in any gene of the devBCA operon or tolC result in the absence of the HGL layer, preventing growth on N(2) used as the sole nitrogen source. However, those mutants do not have impaired HGL synthesis. In this study, we show that DevBCA and TolC form an ATP-driven efflux pump required for the export of HGLs across the Gram-negative cell wall. By performing protein-protein interaction studies (in vivo formaldehyde cross-linking, surface plasmon resonance, and isothermal titration calorimetry), we determined the kinetics and stoichiometric relations for the transport process. For sufficient glycolipid export, the membrane fusion protein DevB had to be in a hexameric form to connect the inner membrane factor DevC and the outer membrane factor TolC. A mutation that impaired the ability of DevB to form a hexameric arrangement abolished the ability of DevC to recognize its substrate. The physiological relevance of a hexameric DevB is shown in complementation studies. We provide insights into a novel pathway of glycolipid export across the Gram-negative cell wall.     

Heterocyst development and diazotrophic metabolism in terminal respiratory oxidase mutants of the cyanobacterium Anabaena sp. strain PCC 7120

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N(2) fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.     

Boron Requirement for Envelope Structure and Function in Anabaena PCC 7119 Heterocysts

The effects of boron deficiency on the ultrastructure and envelopecomposition of heterocysts in the filamentous cyanobacteriumAnabaena PCC 7119 were studied. Microscopic examination of boron-deficientcultures showed changes in heterocyst morphology. When thesecells were isolated and their glycolipid content determined,this specific component of the laminated layer of the heterocystenvelope was found to be lacking. The evidence presented supportsthe view that boron plays an essential role in the structureand function of the heterocyst envelope. Key words: Anabaena, boron, heterocysts, nitrogenase, oxygen-protection     

Ultrastructural studies of heterocyst induction by neo-peptone in Anabaena cylindrica

Abstract An ultrastructural study has been performed to elucidate the effect of active polypeptide(s) from neo-peptone on heterocyst induction in Anabaena cylindrica [1]. There was an immediate aggregation of A. cylindrica cells and a clumping of filamentous appendages in the mucilaginous sheath on the addition of active polypeptide(s) from neo-peptone. However, there was no change in the cell wall and cell membrane ultrastructure. An increase in cell length, contortion and disintegration of thylakoids, disappearance of polyphosphate bodies and an accumulation of polyglucose bodies were observed after 18 h of treatment. The double heterocysts induced show a normal heterocyst ultrastructure with well-developed polar nodules between the heterocysts and the vegetative cells, as well as between two heterocysts.
It appears that the inductive effect of active polypeptide(s) from neo-peptone is mediated through their specific binding to filamentous appendages in the mucilaginous sheath.     

Distribution of nuclear pores and perinuclear dense substances in spermatocytes I of some oedionychina fleabeetles

The nuclear envelope of growing postpachytene spermatocyte I differs notably in structure between the fleabeetles Omophoita cyanipennis and Oedionychus bicolor. The former species shows a more conventional structure with an even and probably random distribution of nuclear pores, and a strongly electron-opaque layer of fibrogranular material (FM) separated from the outer nuclear membrane by an intermediate layer of about 40 nm thickness. Peripherally from the FM layer, a continuous corona of granular dense material (GM) is accumulated around the nucleus. In Oedionychus, all nuclear pores are clustered in “nuclear sieves”, i.e., shallow, cup-like indentations of the nuclear envelope. The sieves are filled with an electron-opaque substance resembling the FM of the Omophoita spermatocytes. This substance is kept at a distance of about 40 nm from the outer nuclear membrane. GM is produced only at the sieves, and is thus discontinuous. The sieves with their contents are called nuclear sieve complexes (NSC).     

Ultrastructural features of the constricted region of dividing plastids

Summary The ultrastructure of the constricted region of dividing plastids of spinach, bean, turnip, tobacco, and wheat has been studied. In these species, an electron-opaque, ring-like structure (RS) girdles the constricted region of plastids in advanced stages of division. The RS is a compound entity composed of two concentric rings of electron-opaque materials; one on the stromal face of the inner membrane and the other on the cytoplasmic face of the outer membrane. It was concluded that the compound nature of the RS is highly conserved in angiosperms being present in some cereal grasses and in plants representing four different orders of dicotyledonous plants. Evidence indicating that the electron-opaque materials of the RS are integrated into the envelope membranes was also provided and it was suggested that the envelope in the region of the RS may have unique properties. For spinach, it was also noted that plastids with deeply constricted necks tend to have RSs with lower volumes than those from wider necks and that endoplasmic reticulum was frequently present in the cytoplasm of the constriction region.Abbreviations RS ring structure - ER endoplasmic reticulum     

THE ZYGOSPORE WALL OF CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE): MORPHOGENESIS AND EVIDENCE FOR THE PRESENCE OF SPOROPOLLENIN1

Chlamydomonas monoica Strehlow is being developed as a model for genetic analysis of zygospore morphogenesis, and many relevant mutant strains are available. To provide the basis for interpreting the ultrastructural phenotypes of zygospore mutants, an analysis of wall morphogenesis in wildtype zygospores of C. monoica was undertaken. Following synthesis of a thick, fibrous, primary zygote wall, granular material accumulated between the plasma membrane and the primary zygote wall and aggregated into a repetitive array of electron-opaque fibrous stripes. A new wall layer, the outer layer of the secondary zygospore wall, first appeared as segments with a fibrous outer surface overlying a well-defined band of electron-translucent material. These segments gave rise to an intact sheath adjacent to the plasma membrane. Beneath this sheath, electron-opaque material (forming the inner layer of the secondary zygospore wall) accumulated unevenly and forced the surface sheath to undulate, creating a pattern of peaks and valleys that was exposed to the external environment 4 rupture and release of the primary zygote wall. The zygospore wall included material resistant to degradation by potassium hydroxide, 2-aminoethanol, and acetolysis, but it was destroyed by exposure to chromic acid. These characteristics, in combination with the autofluorescence of untreated zygospore walls and their failure to stain with phloroglucinol, suggest that sporopollenin may be responsible for many of the resistant properties associated with the mature zygospore of Chlamydomonas.     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Atypical cell forms overproducing extracellular substances in population of cycad cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural changes indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was proteinous. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by channels in the cytoplasmic membrane to form, respectively, a slimy extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.     

Atypical Cell Forms Overproducing Extracellular Substances in Populations of Cycad Cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural properties indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was protein-like. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by ruptures in the cytoplasmic membrane to form, respectively, a mucilagious extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.     

Predicted glycosyl transferase genes located outside the HEP island are required for formation of heterocyst envelope polysaccharide in Anabaena sp. strain PCC 7120

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.     

Transmission-scanning electron microscopic observations of selected Eikenella corrodens strains.

The morphology of Eikenella corrodens 333/54-55 (ATCC 23834) and two human periodontal lesion isolates, strains 470 and 373, was examined by transmission and scanning electron microscopy. All strains exhibited a cell envelope characteristic of gram-negative bacteria. Staining with ruthenium red and alcian blue revealed a loosely organized fibrous slime layer associated with the outer surface of the outer membrane. Slime "stabilization" was achieved by incubation of cells with antisera prepared against whole cells of the Eikenella strains. The stabilized slime appeared as a thick, electron-opaque layer juxtaposed to the outer membrane. Negative staining and heavy metal shadow-casting revealed an interwoven network of fibrils approximately 4 nm in diameter. These fibrils appeared to represent subunits of a larger fibril. Scanning electron microscopy after antibody slime stabilization confirmed the presence and location of the slime layer.