Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Studies on Chlorella protoplasts

Protoplasts of Chlorella saccharophila (Krüger) Nadson were obtained by cellulase digestion of the microfibrillar inner compount of the cell wall after the resistant outermost layer had been scratched with sea sand. The absence of the cell wall was demonstrated immunologically, electron microscopically and by staining, thus confirming the protoplastic nature of the treated cells. After transfer to an enzyme-free medium regeneration of a thin cell wall was observed. The regeneration of the cell wall obviously followed the same steps as does the cell wall development of the autospores. At least 50% of the protoplasts were able to form colonies when plated on a suitable agar medium.     

Polymorphism in gloeotaenium (Chlorococcales,Chlorophyceae)

Morphological variations of Gloeotaenium loitlesbergarianum Hansgirg were studied both in cultures and in nature. In cultures, the alga exhibits considerable variation in the number of cells per colony, ranging from unicells to colonies with more than four cells. The characteristic band was also absent in cultures. In nature, colonies resembling the culture material of Gloeotaenium also occur. The morphology of the alga varies depending on the nature and composition of the nutrients available. The study shows that Gloeotaenium may exhibit polymorphism in nature as well.     

Identification of 4-O-methyl-D-xylose as a constituent of the cell wall of Chlorella vulgaris

From the cell wall of a strain of Chlorella vulgaris a sugar was isolated after acid hydrolysis and was identified as 4-O-methyl-D-xylose by the following criteria: (i) mass spectroscopy of its alditol acetate revealed characteristic primary fragments with m/e 117 and m/e 261, and, when one deuterium atom was substituted at C-1, with m/e 262 instead of m/e 261; (ii) after demethylation with BCl₃, xylose was identified as its parent sugar by chromatographic methods; (iii) L-iditol: NAD 5-oxidoreductase (sorbitol dehydrogenase) catalyzed the oxidation of its alditol, but not of 4-O-methyl-L-xylitol. 4-O-Methyl-D-xylose amounted to approx. 10% of the cell walls' dry weight or 1.6% of the cells' dry weight.     

Regeneration and maturation of daughter cell walls in the autospore-forming green alga<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis> (Chlorophyta,Trebouxiophyceae)

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17–21 nm) emerged as a small, but complete, autospore.     

Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae)

The most recent revision of the genus Chlorella, based on biochemical and SSU rDNA analyses, suggested a reduction to a set of four "true" spherical Chlorella species, while a growing number of morphologically different species such as Micractinium (formerly Micractiniaceae) were found to cluster within the clade of "true"Chlorella. In this study, the generic concept in Chlorellaceae to Chlorella and Micractinium was evaluated by means of combined SSU and ITS-2 rDNA sequence analyses and biotests to induce development of bristles on the cell wall. Molecular phylogenetic analyses of Chlorella and Micractinium strains confirmed their separation into two different genera. In addition, non-homoplasious synapomorphies (NHS) and compensatory base changes (CBC) in the secondary structures of SSU and ITS-2 rDNA sequences were found for both genera using this approach. The Micractinium clade can be differentiated into three different genotypes. Using culture medium of the rotifer Brachionus calyciflorus, phenotypic plasticity in Chlorella and Micractinium was studied. Non-bristled Micractinium cells developed bristles during incubation with Brachionus culture medium, whereas Chlorella did not produce bristles. Grazing experiments with Brachionus showed the rotifer preferred to feed on non-bristled cells. The dominance of colonies versus solitary cells in the Micractinium culture was not correlated with the "Brachionus factor". These results suggest that morphological characteristics like formation of bristles represent phenotypic adaptations to the conditions in the ecosystem.     

Observations in Hemichloris antarctica Tschermak-Woess & Friedmann (Chlorophyceae) and the occurrence of a second Hemichloris species, Hemichloris polyspora n. sp

The chlorococcalean genus Hemichloris is characterized by the possession of two chloroplasts per vegetative cell. The occurrence of a second species of the genus is reported (H. polyspora sp. nov.). Just as H. antarctica it grows cryptoendolithically in sandstone in Southern Victoria Land, Antarctica. In H. antarctica propagation by two autospores prevails over four, whilst in the new species H. polyspora in general four or eight (rarely 16 or 32) autospores are produced and Borodinella-stages do occur typically. Sexuality and zoosporulation do not exist in both species. Internal structures of chloroplasts can be observed by light microscopy more regularly in H. polyspora than in H. antarctica and under various conditions. Investigations of both Hemichloris species by transmission electron microscopy show them to go back to more or less extended assemblages of plastoglobuli. In both species the plastoglobuli are arranged around tubular inflations of thylakoids and apparently attached to the thylakoids. Keeping the cultures for three (even up to seven) months without light makes them survive and causes coming forth of the chloroplast structure throughout.     

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae)

Scenedesmus obliquus, strain 633, which synthesizes ketocarotenoids and sporopollenin, also forms pink-red-colored cell walls. Both the cell walls left over after autospore liberation and those from homogenates of disrupted green cells have similar carotenoid pigmentation. Canthaxanthin, astaxanthin, an unidentified ketocarotenoid, and lutein were found as integral cell wall components. They are bound to the outer (trilaminar) layer of the complete cell wall which also contains sporopollenin.Abbreviations CWH complete cell walls isolated from the homogenates - CWM maternal cell walls accumulated in the medium - KC ketocarotenoid - SC secondary carotenoids - SP sporopollenin     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Species-specific ability ofChlorella strains (Chlorophyceae) to form stable symbioses withHydra viridis

46 strains ofChlorella, identified by physiological and biochemical characters, were examined for their ability to form stable symbioses with aposymbioticHydra viridis. It was found to be a species-specific characteristic. Among the 15 taxa studied, onlyC. saccharophila var.ellipsoidea, C. saccharophila var.saccharophila, C. fusca var.vacuolata, C. kessleri, C. luteoviridis, andC. protothecoides formed stable symbioses withHydra viridis. Among the 11 known physiological and biochemical characters of theseChlorella species, only acid tolerance seems to be correlated with symbiosis: All symbiotic species are capable of growing at or below pH 4.0.     

Production and nutritional quality of the rotifer Brachionus plicatilis fed marine Chlorella sp. at different cell densities

The effect of different cell densities of marine Chlorella sp. on the growth rate, doubling time and production of the rotifer Brachionus plicatilis was investigated. A significant increase in rotifer production was achieved at a density of 50 × 10⁶ Chlorella cells ml^–1. The nutritional quality of rotifers grown at different concentrations of Chlorella is discussed.     

Development of cell wall appendages inAcanthosphaera zachariasi (Chlorococcales): Kinetics,site of cellulose synthesis and of microfibril assembly,and barb formation

Summary The investigation of the formation of cell wall appendages inAcanthosphaera by means of light and electron microscopy and by the use of dyes which interfere with microfibril assembly resulted in several observations which are helpful to an understanding of the formation of normal cell walls. The barbs are built up in the ER, pass through the Golgi apparatus, and are extruded exocytotically after cytokinesis, a remarkable example of the secretion of a structured product. Each cellulose microfibril in a spike develops in a distinct pit of the plasmalemma. The pits are aggregated in a pit field, generating one spike, and are closely adjacent to a basal vesicle which might have morphogenetic and/or regulatory functions. The pits are the site of cellulose synthesis; here the plasmalemma is conspicuously thickened. As shown directly and by the application of Calcofluor white and Congo red, the microfibrils assemble at a certain distance from the plasma membrane,i.e. cellulose synthesis and microfibril assembly are separated by a gap. It is discussed whether single glucan chains or small bundles of them are released from the plasmalemma. The elongation rate of the spikes indicates that about 1000 glycosidic linkages per glucan chain per minute are formed.     

Synthesis of the inner cell wall layer of the chlamydomonad flagellate,Gloeomonas kupfferi

Summary An antibody to the inner wall layer ofGloeomonas kupfferi was isolated and used in a developmental analysis of cell wall processing, secretion and extracellular assembly. The focus of the processing of this matrix layer is the endomembrane system, in particular the Golgi apparatus (GA) and contractile vacuole (CV). During interphase, inner wall materials are processed in the GA, packaged in trans face vesicles and transported to the CV, the final internal depository of wall precursors until release to the cell surface. During cell division, significant changes occur in the inner wall layer processing. Early on in cytokinesis, the GA does not label with our antibody, suggesting that other wall layers are being processed. In later stages of cytokinesis, the GA changes in morphology and begins to produce inner wall layer materials. These wall precursors are shuttled to the CV where they are released around the daughter cell protoplasts. The first wall layer that is formed around daughter cells is the crystalline median wall layer. Once assembled, the inner wall layer condenses upon the crystalline layer and grows in size.     

The cell wall of the chlamydomonad flagellate,Gloeomonas kupfferi (Volvocales,Chlorophyta)

Summary The large unicellular flagellate,Gloeomonas kupfferi, has recently been used as an important tool in chlamydomonad cell biology research, especially in studies dealing with the structure and function of the endomembrane system. However, little is known about the main secretory product, the cell wall. This study presents structural, chemical and immunological information about this wall. This 850–900 nm thick matrix is highly elaborate and consists of three distinct layers: an inner stratum (325 nm thick) consisting of tightly interwoven fibers, a medial crystalline layer consisting of 22–23 nm subunits and an outer wall layer (500 nm thick) of outwardlyradiating fibrils. Rapid freeze-deep etch analysis reveals that the 35–40 nm fibers of the outer layer form a quasi-lattice of 160 nm subunits. The outer wall can be removed from whole pellets using the chelator, CDTA. The medial wall complex can be solubilized by perchlorate. SDS-gel electrophoresis reveals that the perchlorate soluble-material consists of five high molecular weight glycoproteins and five major low molecular weight glycoproteins. The electrophoretic profile is roughly similar to that ofChlamydomonas reinhardtii. Antibodies were successfully raised against the outer wall component and were shown to label the outer wall layer.     

Late type of daughter cell wall synthesis in one of the Chlorellaceae, <Emphasis Type=Italic>Parachlorella kessleri</Emphasis> (Chlorophyta,Trebouxiophyceae)

Autosporulation is a common mode of propagation for unicellular algae. Autospore-forming species of Chlorellaceae, Chlorella vulgaris Beijerinck, C. sorokiniana Shihira et Krauss, C. lobophora Andreyeva, and Parachlorella kessleri (Fott et Nováková) Krienitz et al. have glucosamine as the main constituent of their rigid cell wall. Recent phylogenetic analyses have showed that the Chlorellaceae divided into two sister groups: the Chlorella-clade and the Parachlorella-clade. We compared the cell wall structure and synthesis of the daughter cell wall in the four species by electron microscopy using rapid freezing and freeze substitution methods. The cell wall of C. vulgaris, C. sorokiniana, and C. lobophora consisted of an electron-dense thin layer with an average thickness of 17–20, 22, and 19 nm, respectively. In these three species, daughter cell wall synthesis occurred on the outer surface of the plasma membrane in the early cell-growth phase. The cell wall of P. kessleri, however, was electron-transparent and 54–59 nm in thickness. Ruthenium red staining of P. kessleri indicated that ruthenium-red-specific polysaccharides accumulated over the outer surface of the plasma membrane. Immunoelectron microscopic observation with an anti--1, 3-glucan antibody and staining with wheat germ agglutinin (WGA) indicated that the cell wall contained -1, 3-glucan and WGA specific N-acetyl--D-glucosamine. In P. kessleri, daughter cell wall synthesis began after successive protoplast division. The daughter cell wall synthesis during autosporulation in the four species of Chlorellaceae can be classified into two types—the early and the late types.     

An autolysin dissolves the inner cell wall layer of the algaPediastrum (Hydrodictyaceae)

Summary An autolysin produced by young colonies ofPediastrum frees them from the vesicle in which they are formed within 12 hours of release of zoospores from the parent cell. The polysaccharide vesicle is derived from the inner wall layer of the parent cell. Refrigeration delays vesicle disintegration; boiling stops it completely. A purified, lyophilized extract of the vesicle fluid added to boiled vesicled colonies removes the vesicle in 2 hours with the release of reducing sugars and polysaccharides.Biogel P2 and P10 chromatography of the products following incubation of the enzyme preparation and wall showed no more than 1% oligosaccharides; the remaining carbohydrates had a molecular weight of several thousand daltons. Analyses of isolated vesicle wall material (70–85% of the dry weight) showed mannose accounting for approximately 50% of the dry weight, with none of the other neutral sugars present (fucose, xylose, galactose and glucose) representing more than 3%. Uronic acids account for 20–25% of the wall weight, and proteins less than 2%.Pediastrum colonies are thus freed from the vesicles in which they are formed by the action of an autolysin they produce. The autolysin acts on the vesicle wall material to generate reducing sugars and cause it to disintegrate into its constituent polysaccharides.     

Cytoplasmic reorganization accompanies the deposition of a bipolar cell wall in the large-celled red alga Anotrichium tenue

The filamentous red alga Anotrichium tenue C. Aghard (Naegeli) (formerly Griffithsia tenuis C. Aghard; Baldock, 1976, Aust. T. Bot. 24, 509–593) has large (1–2 mm long), cylindrical, multinucleate cells that exhibit a daily, cyclic redistribution of chloroplasts. Chloroplasts accumulate in the mid-region of each growing cell during the day; consequently, filaments appear banded with a light apical end-band, a dark mid-band and a light basal end-band in each growing cell. Chloroplasts disperse at night so that the bands are no longer visible and the cells appear evenly pigmented. Anotrichium tenue also has a type of cell elongation, known as bipolar band growth, in which new material is added to the microfibrillar part of the wall in bands located at the apical and basal poles of elongating cells. This site of wall growth corresponds to the position of the light-colored end-bands present during the day. Here we examine the structural relationship between the cytoplasmic bands and the wall-growth bands. Our results show that, in addition to the previously described bipolar wall bands, there is a non-microfibrillar wall band in the mid-region of the cell. This wall component apparently branches from near the top of the microfibrillar outer wall and terminates near but not at the bottom of the cell. It contains nodules of sulphated polysaccharide material secreted from a band of vesicles, which co-localize with the chloroplasts in the mid-band. The outer wall appears to enclose the entire cell. Nuclei do not redistribute with the chloroplasts or wall vesicles into the mid-band but remain evenly distributed throughout the cytoplasm. Each wall component grows by a different mechanism. We show that two types of wall growth, diffuse and the bipolar-type of tip growth, occur in the same cell and we propose that the observed segregation of the cytoplasm supports localized growth of the unique inner wall component. Additionally, we show that A. tenue is an excellent model for study of the role and mechanism of cytoplasmic compartmentalization and cell polarity during plant cell growth.We wish to thank Dr. Richard Cloney (University of Washington and Dr. Tom Schroeder (Friday Harbor Laboratories, Friday Harbor, Wash.) for helpful discussions and critical review of this work. We also thank Dr. Susan Waaland (University of Washington) for sharing her original observations on the chloroplast banding phenomenon in Anotrichium tenue. We are grateful to the Friday Harbor Laboratories for the use of their space and facilities. This research was supported by funds from the Washington Sea Grant Program (awarded to J.R.W.) and by the Developmental Biology Training Grant, predoctoral fellowship, National Institutes of Health, No. HD07183 to A.W.S.     

A novel type of teichoic acid from the cell wall of Bacillus subtilis VKM B-762

The cell wall of Bacillus subtilis VKM B-762 contains, along with 1,5-poly[4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)ribitol phosphate], a novel type of glycopolymer involving three types of inter-monomeric bonds in the repeating unit, viz., amide, glycosidic and phosphodiester:Such a structural pattern of natural glycopolymers has been hitherto unknown. This polymer represents a novel type of teichoic acids.     

Cytochemical and biochemical observations on the cell wall of the spore ofGlomus epigaeum

Summary The cell wall of the spore ofGlomus epigaeum Daniels and Trappe, which has fibrillar subunits regularly arranged in arcs, was studied ultrastructurally and biochemically.The periodic acid/thiocarbohydrazide/silver proteinate (PATAg) reaction for polysaccharide location (Thiéry 1967) and the silver methenamine reaction for protein location (Swift 1968) were performed on whole spores, progressively alkaline-extracted and autoclaved spores, and untreated and alkaline-extracted cell wall fractions. The cytochemical results and those obtained from frozen sections indicated that the fibrils forming the main structure of the outer and inner wall consist of chitin. Quantitative determinations showed that chitin is the most important component (47%) of the alkali-insoluble residue and represents 27.2% of the whole cell wall fraction. It occurs predominantly as the acetylated form. Cytochemical and biochemical observations showed that the matrix surrounding the fibrils is made of alkali-soluble, PATAg positive polysaccharides (4.98% of the whole cell wall fraction). Monomers were identified by gas liquid chromatography as being -lactone of glucuronic acid, and glucose, rhamnose and mannose. Alkali-soluble proteins are an important part of the matrix, being spread mostly throughout the inner wall and constituting a large portion (55.1 %) of the alkali-soluble fraction.From the results we derive a model in which the chemical components are interconnected to build up a macromolecular network, in agreement with electron-microscopic observations.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.