Adhesion molecules from the diatom Phaeodactylum tricornutum (Bacillariophyceae): genomic identification by amino-acid profiling and in vivo analysis.

Cell adhesion molecules (CAMs) are important in prokaryotes and eukaryotes for cell-cell and cell-substratum interactions. The characteristics of adhesive proteins in the model diatom Phaeodactylum tricornutum were investigated by bioinformatic analysis and in vivo characterization. Bioinformatic analysis of the protein coding potential of the P. tricornutum genome used an amino-acid profile that we developed as a new system to identify uncharacterized or novel CAMs. Putative diatom CAMs were identified and seven were characterized in vivo, by generation of transgenic diatom lines overexpressing genes encoding C-terminal yellow fluorescent protein (YFP) fusion proteins. Three of these selected genes encode proteins with weak similarity to characterized proteins, a c-type lectin and two fasciclins, whereas the others are novel. The resultant cell lines were investigated for alterations in their adhesive ability. Whole cell-substratum adhesion strength was measured in a fully turbulent flow chamber, while atomic force microscopy was used to quantify the relative frequency of adhesion, as well as the length and strength of single molecules in the secreted mucilage. Finally, quartz crystal microbalance analysis characterized the visco-elastic properties and interaction of the mucilage-substratum interface. These combined studies revealed a range of phenotypes affecting adhesion, and led to the identification of candidate proteins involved in diatom adhesion. In summary, our study has for the first time combined bioinformatics and molecular physiological studies to provide new insights into diatom adhesive molecules.

Novel whole cell adhesion assays of three isolates of the fouling diatom Amphora coffeaeformis reveal diverse responses to surfaces of different wettability.

Whole cell, strength of adhesion assays of three different isolates of the fouling diatom Amphora coffeaeformis were compared using a hydrophilic surface viz. acid washed glass (AWG), and a hydrophobic surface viz. a self assembled monolayer (SAM) of undecanethiol (UDT). Assays were performed using a newly designed turbulent flow channel that permits direct observation and recording of cell populations on a test surface. Exposure to continuous shear stress over 3 h revealed that the more motile isolate, WIL2, adhered much more strongly to both test surfaces compared to the other two strains. When the response of the isolates to shear stress after 3 h was compared, there was no significant difference in the percentage of cells removed, irrespective of surface wettability. Cells of the three isolates of A. coffeaeformis varied significantly in their response to different surfaces during initial adhesion, indicating the presence of a wide range of 'physiological races' within this species.

The quartz crystal microbalance: a new tool for the investigation of the bioadhesion of diatoms to surfaces of differing surface energies.

Diatoms are a major component of the biofoul layer found on modern low-surface-energy, 'foul release' coatings. While diatoms adhere more strongly to hydrophobic, as opposed to hydrophilic, surfaces, surprisingly little is known of the chemical composition of their adhesives. Even less is known about the underlying processes that characterize the interaction between the adhesive and a given surface, including those of differing wettability. Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we examined differences in the viscoelastic properties of the extracellular adhesives produced by the marine diatoms Amphora coffeaeformis Cleve and Craspedostauros australis Cox interacting with surfaces of differing wettability; 11-mercaptoundecanoic acid (MUA) that is hydrophilic and 1-undecanethiol (UDT) that is hydrophobic. While the overall delta f/delta D ratios were slightly different, the trends were the same for both diatom species, with the layer secreted upon UDT to be more viscoelastic and far more consistent over several experiments, compared to that on MUA which was less viscoelastic and demonstrated far more variability between experiments. While the nature of the parameter shifts for C. australis were the same for both surfaces, A. coffeaeformis cells settling upon UDT illustrated significant positive f and D shifts during the initial stages of cell settlement and adhesion to the surface. Further experiments revealed the parameter shifts to occur only during the initial adhesion of cells upon the pristine virgin UDT surface. The mechanism behind these parameter responses was isolated to the actin-myosin/adhesion complex (AC), using the myosin inhibitor 2,3-butanedione 2-monoxime (BDM) to remove the cells ability to 'pull' on adhesive strands emanating from the cell raphe. The observations made herein have revealed that adhesives secreted by fouling diatoms differ significantly in their interaction with surfaces depending on their wettability, as well as illustrating the unique mechanics behind the adhesion of A. coffeaeformis upon hydrophobic surfaces, a mechanism that may contribute significantly to the cells success in colonizing hydrophobic surfaces.

Divalent cations stabilize the aggregation of sulfated in the adhesive nanofibers of the biofouling diatom Toxarium undulatum.

A species of marine diatom, Toxarium undulatum, has emerged as a problematic biofouler of contemporary environmentally benign marine coatings. Previous analyses by atomic force microscopy (AFM) showed the cell-substratum adhesive of this alga contained macromolecules with a modular protein backbone assembled into nanofibers in which the domains of the macromolecules folded and unfolded in a co-ordinated manner. In the present study, we investigated further the composition and properties of the adhesive. A combination of energy dispersive X-ray analysis (EDXA) and Fourier transform infrared (FTIR) spectroscopy showed that the adhesive contained mainly protein, carbohydrate, sulfate, calcium, and magnesium. AFM demonstrated that EDTA treatment of native T. undulatum adhesive resulted in rapid disruption of the adhesive nanofiber (ANF) structure but ANFs were restored by subsequent treatment (within 1 h) with solutions containing divalent cations. Prolonged exposure to EDTA (≥18 h) led to cell detachment. The soluble EDTA extract was separated from the cells, dialyzed, concentrated, and analyzed further. The extract had a protein-to-carbohydrate-to-sulfate weight ratio of 1.0 : 0.2 : 0.9 and contained a single, high-molecular-mass (>220 kDa) band by SDS-PAGE which was visualized by Stains-All® but not by Coomassie blue, indicating that it was a highly anionic macromolecule. The most abundant amino acids in the extract were glycine (22 mol%), aspartic acid/aspartamine (14 mol%), and histidine (11 mol%). The adhesive contained 11 neutral sugars dominated by mannose (50 mol%) and xylose (29 mol%). On the basis of these data, we propose that the ANFs of T. undulatum are composed of sulfated high-molecular-mass glycoproteins cross-linked by calcium and magnesium ions. The cross-linking enables domains of adjacent protein backbones to unfold and re-fold in register.

Utilizing QCM-D to characterize the adhesive mucilage secreted by two marine diatom species in-situ and in real-time.

The quartz crystal microbalance with dissipation monitoring (QCM-D) was used to monitor the deposition of adhesive extracellular polymeric substances (EPS) employed by the marine biofouling diatoms Craspedostauros australis Cox and Amphora coffeaeformis Cleve during initial adhesion and subsequent motility. Upon injection into the QCM chamber, initial negative frequency (f) shifts and positive dissipation (D) shifts were measured that correlated to cells impacting and adhering to the QCM sensor surface. Following this "initial adhesion" response, f continued to decrease while D increased logarithmically. Rather than the result of any cell morphological alterations at the substrate surface, the shifts were correlated to the time-dependent deposition of EPS upon the substrate surface as a result of cellular motility, or gliding. Experiments utilizing comparable cell concentrations of the diatom species C. australis and A. coffeaeformis revealed significant differences between the parameter responses recorded, where A. coffeaeformis produced Deltaf and DeltaD values of -32 Hz and 6.6, and C. australis produced values of -82 Hz and 42, respectively, after 20 h post-inoculation. The viscoelastic properties of the adhered EPS adlayer from both species were examined via a Deltaf/DeltaD plot, providing reproducible signature "ratio" values for each species that likely correlate to differences in EPS interactions with the substrate that may be associated directly to differences in the fouling potential of the two species. There is a distinct lack of knowledge regarding the chemical nature of the adhesive polymers engaged, and few quantitative techniques are applicable to the study of diatom EPS. We propose that QCM-D may be a useful tool in identifying differences in the EPS employed by diatoms of different fouling potential.