Integrated Microscopy of Extracellular Adhesives of the Marine Biofouling Alga Achnanthes Longipes (Bacillariophyceae)

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An integrated approach including TEM, SEM and LM was utilized to investigate adhesives produced by the marine fouling diatom Achnanthes longipes. Extracellular adhesive secretion during sequential attachment in living diatom cells was examined using video microscopy. A suite of cryotechniques including high pressure freezing (HPF), freeze-substitution (FS) and high resolution cryo-field-emission SEM (cryo-FESEM) were required for preservation and structural investigation of the hydrophilic polymers synthesized by A. longipes that promote attachment to ship hulls, oil rigs and other submerged structures. Living cells and associated extracellular biocomposites were rapidly cryo-immoblized and observed using a Hitachi S-900 FESEM equipped with a cryo-stage. This technique allowed resolution of details of polymer organization within the pad, shaft and collar portions of the major adhesive structure termed a stalk (Fig. 1,2). This stalk has been demonstrated to consist mainly of polysaccharide cross-linked by O-linked polypeptides. In addition to permanently adhering the diatom to the substratum, the stalk elevates A. longipes, possibly enhancing the ability to successfully compete with other biofilm dwellers.

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Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cistula, 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 NaHCO3-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64-70%) and fucosyl (32-42%) residues. In A. longipes polymers, 2,3-, t-, 3-, and 4-linked/substituted galactosyl, t-, 3-, 4-, and 2-linked fucosyl, and t- and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTA-soluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4f/5p-, and 3p-linked/substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized [alpha](1,2)-L-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Galactose residues were localized throughout the shafts of C. cistula and capsules of A. coffeaeformis. D-Mannose and/or D-glucose, D-galactose, and [alpha](t)-L-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorius, and Lotus tetragonolobus 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.
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-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes 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 in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.