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Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cisfula, and Cymbella mexicana were characterized by monosaccharide and methylation analysis, lectin-fluorescein isothiocyanate localization, and cytochemical staining. Polysaccharide was the major component of adhesives formed during cell motility, synthesis of a basal pad, and/or production of a highly organized shaft. Hot water-insoluble/hot 0.5 M NaHC0,-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64-70%) and fucosyl (32-42%) residues. I n A. longipes polymers, 2,3-, f-, 3-, and 4-linked/substituted galactosyl, f-, 3-, 4-, and 2-linked fucosyl, and t-and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTAsoluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4,/5,-, and 3,-linked/ substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized cu(l,2)-~-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Calactose residues were localized throughout the shafts of C. cisfula and capsules of A. coffeaeformis. D-Mannose and/or o-glucose, D-galactose, and a(t)-i-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorim, and Lotus tefragonolobus agglutinin, respectively. A model for diatom cell adhesive structure was developed from chemical characterization, localization, and microscopic observation of extracellular adhesive components formed during the diatom cell-attachment process.Achnanthes longipes, Cymbella cistula, Cymbella mexicana, and Amphora cofeaeformis are relatively large, fast-growing, unicellular organisms specialized in cell motility and attachment via distinct extracellular structures. These adhesive structures can be easily manipulated, observed, and isolated for the study of synthesis, transport, modification, and assembly of extracellular polymers. Modeling such systems can provide a better understanding of how plant cells adhere to surfaces and interact with their surrounding environments. Furthermore, these diatoms are a major component of marine and freshwater biofilms (Round et al., 1990) and thus cause a variety of biofouling problems (Alberte et al., 1992). Understanding how these cells adhere to surfaces will also aid in the development of new antibiofouling surfaces and of adhesives for use in marine and freshwater environments.Most diatoms attach to surfaces via polymers excreted from a slit (raphe) or apical pore field in the siliceous cell wall (frustule) (Hoagland et al., 1993). Exuded polymers are assembled into a variety of structures, such as trails (material left behind during motility), sheaths (organic matrices tightly associated with the cell wall), capsules (organic matrices loosely associated with the cell walls), and stalks (permanent attachment s...

We studied patterns of production and loss of four different extracellular polymeric substance (EPS) fractionscolloidal carbohydrates, colloidal EPS (cEPS), hot water (HW)-extracted and hot bicarbonate (HB)-extracted fractions-and community profiles of active (RNA) bacterial communities by use of Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of reverse transcription-polymerase chain reaction amplified 16S rRNA in mudflats in the Colne Estuary, United Kingdom, over two tidal emersion-immersion cycles. Colloidal carbohydrates and intracellular storage carbohydrate (HW) increased during tidal emersion and declined during tidal cover. The dynamics of cEPS and uronic acid content were closely coupled, as were the HB fraction and HB uronic acids. Changes in monosaccharide profiles of HW and HB fractions occurred during the diel period, with some similarity between cEPS and HB fractions. Increasing enzymatic release rates of reducing sugars and increased reducing sugar content were correlated with increased concentrations of colloidal carbohydrate and cEPS during the illuminated emersion period, and with the amount of HB-extracted uronic acids (the most refractory EPS fraction measured). Loss of reducing sugars was high, with sediment concentrations far below those predicted by the measured in situ release rates. T-RFLP analysis revealed no significant shifts in the overall taxonomic composition of the active bacterial community. However, 12 of the 59 terminal restriction fragments identified showed significant changes in relative abundance during the tidal cycle. Changes in the relative abundance of three particular terminal restriction fragments (bacterial taxa) were positively correlated to the rate of extracellular hydrolysis. Losses of chlorophyll a and colloidal and cEPS (up to 50-60%) occurred mainly in the first 30 min after tidal cover. About half of this may be owing to in situ degradation, with ''wash away'' into the water column accounting for the remainder.Microphytobenthic biofilms in intertidal sediments play an important role in the ecology of estuarine systems (Under-1 Corresponding author (gjcu@essex.ac.uk).

The effects of phosphate (P) limitation, varying salinity (5-65 psu), and solid media growth conditions on the polysaccharides produced by the model diatom, Phaeodactylum tricornutum Bohlin were determined. Sequential extraction was used to separate polymers into colloidal (CL), colloidal extracellular polymeric substances (cEPS), hot water soluble (HW), hot bicarbonate soluble (HB), and hot alkali (HA) soluble fractions. Media-soluble polymers (CL and cEPS) were enriched in 4-linked mannosyl, glucosyl, and galactosyl residues as well as terminal and 3-linked xylosyl residues, whereas HW polymers consisted mainly of 3-linked glucosyl as well as terminal and 2,4-linked glucuronosyl residues. The HB fraction was enriched in terminal and 2-linked rhamnosyl residues derived from the mucilage coating solubilized by this treatment. Hot alkali treatment resulted in the complete dissolution of the frustule, releasing polymers containing 2,3-and 3-linked mannosyl residues. The fusiform morphotype predominated in standard and P-limited cultures and cultures subjected to salinity variations, but growth on solid media resulted in an enrichment of the oval morphotype. The proportion and linkages of 15 residues, including neutral, uronic acid, and O-methylated sugars, varied with environmental conditions. P limitation and salinity changes resulted in 1.5-to 2.5-fold increase in carbohydrate production, with enrichment of highly branched/substituted and terminal rhamnose, xylose, and fucose as well as O-methylated sugars, uronic acids, and sulfate. The increased deoxy-and O-methylated sugar content under unfavorable environments enhances the hydrophobicity of the polymers, whereas the anionic components may play important roles in ionic cross-linking, suggesting that these changes could ameliorate the effects of salinity or P-stress and that these altered polysaccharide characteristics may be useful as bioindicators for environmental stress.

Benthic microalgae (microphytobenthos) are the dominant group of primary producers in many marine intertidal and subtidal habitats. Estuarine mudflat diatoms are thought to be major contributors of extracellular polymeric substances (EPS), which are important for sediment stabilization and in benthic food chains. Biofilms from 6 sites in the Colne estuary, UK, were fractionated to isolate biopolymers (colloidal, colloidal EPS [cEPS], low molecular weight [LMW] carbohydrates, hot water [HW] and hot bicarbonate [HB] soluble) and the same techniques were applied to diatoms cultured from these sediments. At sites dominated by benthic diatoms, colloidal carbohydrate concentration and chlorophyll a were closely related. With increasing biomass, the proportion of cEPS within the colloidal fraction decreased from 60 to 20%. Carbohydrate analysis revealed significant differences in monosaccharide and uronic acid composition of different carbohydrate fractions. Principal component analysis (PCA) of monosaccharide composition of HB polymers from both field and culture samples grouped closely along fucose and rhamnose vectors and formed 2 distinct clusters. HW and LMW fractions grouped along the glucose vector and cEPS polymers along the galactose and arabinose vectors. These data indicate that the simple relationship between colloidal carbohydrate concentration and microphytobenthic biomass in biofilms masks a high degree of potential complexity within the sediment carbohydrate pool and in the different proportions of polymeric and nonpolymeric material between different biofilms. Comparing monosaccharide composition of extracts generated using the same protocol, natural assemblages showed close relationships with unialgal cultures, confirming the important role of diatom-derived polymers in mudflat ecology. KEY WORDS: Diatoms · Biofilms · Microphytobenthos · EPS · Monosaccharide distribution · Uronic acids · Biopolymers · Fractionation Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 38: [169][170][171][172][173][174][175][176][177][178][179][180] 2005 1993, . Many benthic diatoms exhibit patterns of vertical migration which allow cells to move into the narrow photic zone present in the top few millimeters of sediment, and this diatom motility is closely linked to EPS production (Lind et al. 1997. The postulated role of EPS in the ecosystem includes biostabilisation of sediments (Paterson 1986), a carbon source for benthic and planktonic species (Decho 1990, Underwood & Smith 1998a, and in mediating species interactions (Hoagland et al. 1993).Correlations have been measured between algal biomass (chlorophyll a [chl a] concentration) and colloidal (water-soluble) carbohydrate concentrations in estuarine sediments (Underwood & Paterson 1993, Underwood & Smith 1998b. These observations led to the publication of a model describing the relationship between sediment chl a and colloidal carbohydrate concentrations, developed using data from a number of European estuar...

Plant cell walls are essential for proper growth, development, and interaction with the environment. It is generally accepted that land plants arose from aquatic ancestors which are sister groups to the charophycean algae (i.e., Streptophyta), and study of wall evolution during this transition promises insight into structure-function relationships of wall components. In this paper, we explore wall evolutionary history by studying the incorporation of pectin polymers into cell walls of the model organism Penium margaritaceum, a simple single-cell desmid. This organism produces only a primary wall consisting of three fibrillar or fibrous layers, with the outermost stratum terminating in distinct, calcified projections. Extraction of isolated cell walls with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid yielded a homogalacturonan (HGA) that was partially methyl esterified and equivalent to that found in land plants. Other pectins common to land plants were not detected, although selected components of some of these polymers were present. Labeling with specific monoclonal antibodies raised against higher-plant HGA epitopes (e.g., JIM5, JIM7, LM7, 2F4, and PAM1) demonstrated that the wall complex and outer layer projections were composed of the HGA which was significantly calcium complexed. JIM5 and JIM7 labeling suggested that highly methyl esterified HGA was secreted into the isthmus zone of dividing cells, the site of active wall secretion. As the HGA was displaced to more polar regions, de-esterification in a non-blockwise fashion occurred. This, in turn, allowed for calcium binding and the formation of the rigid outer wall layer. The patterning of HGA deposition provides interesting insights into the complex process of pectin involvement in the development of the plant cell wall.

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The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyL(3-cyano-2, 4-dich1oro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanfhes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures i n the form of stalks that elevated the diatom above the The herbicide DCB is considered a specific inhibitor of cellulose synthesis in higher plants and algae (Delmer and Amor, 1995). Disruption of plant growth in the presence of DCB has been demonstrated in cotton (Montezinos and Delmer, 1980;, tobacco (Meyer and Herth, 1978), tomato (Shedletzky et al., 1990), barley (Shedletzky et al., 1992), the charophytes Chara and Nitella (Foissner, 1992), and the green alga Vuucheria (Mizuta and Brown, 1992). At micromolar concentrations effective at inhibiting cellulose synthesis, DCB appears to have little or no short-term effect on the synthesis of noncellulosic polysaccharides, nuclear division or DNA synthesis, protein synthesis, respiration, or the in vivo labeling patterns of UDP-Glc, phospholipids, or nucleoside mono-, di-, or triphosphates (Delmer, 1987, and refs. therein).The exact mode of DCB inhibition of cellulose synthesis is not clear. DCB is thought to act primarily on the stages of cellulose synthesis involving polymerization of Glc into a P-4-linked glucan. In an attempt to delineate the mechanism of action of DCB in cotton fibers, used the photoreactive analog DCPA to identify an 18-kD DCB-binding protein thought to function as a regulatory protein for P-glucan synthesis in plants. This acidic protein was found primarily in the 100,OOOg supernatant and has not been further purified or characterized (Delmer and Amor, 1995). Labeling of UDP-Glc in vivo does not appear to be affected by DCB, indicating that its effect is at some later step in the cellulose synthesis process (Delmer and Amor, 1995). The second stage in cellulose microfibril formation involves P-4-linked glucan crystallization into cellulosic microfibrils at the PM. Mizuta and Brown (1992) have summarized the effects of DCB on the PM cellulosesynthesizing complex (the terminal complex) from higher plants and algae. These effects include a decrease or increase in the number of rosette-terminal complexes in Funaria and Triticum, respectively, and an inhibition of microfibril synthesis and assembly of Vauckeria terminal complexes.It has recently been demonstrated that DCB inhibits synthesis of ECM polymers in the noncellulosic red mil