PAPER                                                                                     ...
are several reports of protein being co-extracted with the extra-      detaching and be reversibly unfolded and refolded t...
sequentially in 10 mM EDTA solution and distilled water and           Following initial observations, the piezo-head was r...
Assay methods                                                       Table 1 Constituent atoms (atom%) of the background si...
Fig. 2 (A) FTIR spectrum of a Toxarium undulatum adhesive pad on a CaF2 substratum. (B) The second derivative spectrum of ...
Table 3 Mean values (Æ%SE) of parameters for ANF I curves analyzed
Table 5 Constituent monosaccharides (mol%) of extractsa obtained
from Toxarium undulatum adhesive pads.

Monosaccharideb  ...
Table 6 Constituent amino acids of the EDTA extracta obtained from      species of Navicula, Nitzschia, Tropidonies, and A...
a chlorophye alga, Prasiola linearis.53 These sawtooths were          structure of the sulfated glycoproteins. The present...
Provasoli-Guillard centre for culture of marine phytoplankon, West         49 K. E. Cooksey, Appl. Environ. Microbiol., 19...
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Soft Matter Algal Adhesive


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Analysis of the adhesive used by the only organism able to stick to the new coating used on the hulls of US Navy vessels!

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Soft Matter Algal Adhesive

  1. 1. PAPER | Soft Matter Divalent cations stabilize the aggregation of sulfated glycoproteins in the adhesive nanofibers of the biofouling diatom Toxarium undulatum Anthony Chiovitti,a Philip Heraud,b Tony M. Dugdale,a Oliver M. Hodson,a Roger C. A. Curtain,c Raymond R. Dagastine,cd Bayden R. Woodb and Richard Wetherbee*a Received 9th October 2007, Accepted 13th December 2007 First published as an Advance Article on the web 24th January 2008 DOI: 10.1039/b715455k 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-AllÒ 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. Introduction by colonizing organisms when the vessel is stationary is easily removed by shear when the vessel begins moving. These coatings Biofouling of submerged artificial substrata, such as ship hulls, have proved effective against biofouling by a range of vertebrates occurs at great expense to government and industry in the and macroalgae, however, they are still susceptible to extensive form of reduced operational efficiency and periods of inactivity fouling by microorganisms,6 and, in particular, a class of phyto- while the vessel is cleaned.1,2 Marine antifouling coatings tradi- plankton known as the diatoms. tionally have relied upon toxic strategies, such as the incorpora- Diatoms are unicellular microalgae of the class Bacillariophy- tion of copper or organotin biocides into the paint formulation.3 ceae (division Ochrophyta), best known for their ornate silica cell Although these have proved generally effective, the adverse walls.7 In general, the silica cell walls comprise two similar but impact of the biocides leaching into the environment has neces- unequally-sized halves that fit together in the manner of a petri sitated a shift away from the traditional coatings. For example, dish. There are broadly two subclasses of diatoms defined by a worldwide ban has been placed on the use of tributyltin hydride cell wall symmetry.7 These are the centrics, which have radial in marine coatings and this ban becomes fully enforceable in symmetry and are mainly planktonic, and the pennates, which 2008.4 In their place, a new generation of environmentally benign have bilateral symmetry and are mainly benthic. The benthic marine coatings that exploit low surface energy to reduce adhe- diatoms are of most concern to biofouling owing to their attach- sion has become popular.5 The philosophy behind these coatings ment and motility on surfaces. Many studies, including those of is not that they resist fouling entirely but that the biofilm formed important biofouling diatoms such as the Amphora or Ach- nanthes species,8–11 indicate that the adhesives of diatoms are a School of Botany, University of Melbourne, Victoria 3010, Australia. composed mainly of heterogeneous polyanionic polysaccharides E-mail: (see ref. 12 for a review). Furthermore, studies of natural and b Centre for Biospectroscopy, Monash University, Clayton, Victoria 3800, cultured populations of diatoms show that their secreted poly- Australia saccharides contribute significantly to biofilm composition and c Department of Chemical and Biomolecular Engineering, University of Melbourne, Victoria 3010, Australia stability (see ref. 13 for a review). Consequently, recent research d Particulate Fluids Processing Centre, University of Melbourne, Victoria into the biochemistry of the secreted extracellular polymers of 3010, Australia diatoms has primarily focused on polysaccharides.14–16 There This journal is ª The Royal Society of Chemistry 2008 Soft Matter, 2008, 4, 811–820 | 811
  2. 2. are several reports of protein being co-extracted with the extra- detaching and be reversibly unfolded and refolded to give cellular polysaccharides of diatoms but the proteins and their essentially the same sawtooth pattern for hundreds of cycles. relationship with the extracellular matrix have been investigated The sawtooth curves were fitted by the wormlike chain (WLC) only rarely.11,17,18 However, there is evidence from atomic force model, giving a low average persistence length of 0.036 nm microscopy (AFM) and proteolytic studies for some diatom that, combined with a high average force for unfolding species that indicate that proteins or glycoproteins participate (0.8 nN), indicated that the ANFs were comprised of polymer in adhesion between the cell and the substratum (see ref. 19 for chains that were aligned in parallel and unfolded and refolded a review). This emerging view is supported by studies of the synchronously.20 Subsequent AFM analyses demonstrated that adhesives of the biofouling diatom, Toxarium undulatum. the T. undulatum adhesive pads were populated by at least three T. undulatum is a major fouler of the marine antifouling classes of ANF (named I–III), as well as ‘‘oligomers’’ and rarely coatings based on polydimethyl siloxane elastomer, often detected single modular proteins.22 ANF I–III and oligomers becoming the dominant species on such coatings.20 This is gave distinctive sawtooth patterns with idiosyncratic average unusual not only because T. undulatum is a relatively insignifi- force values, average interpeak forces, and persistence lengths, cant component of the native marine flora but also because but all were related by similar interpeak distances, which were molecular phylogenetic studies indicate it is apparently a member the same as those of the rarely detected single molecules, indica- of the centric diatoms rather than the pennate diatoms whose ting they were discrete supramolecular assemblies composed of morphology and benthic lifestyle it emulates.21 T. undulatum cells varying numbers of the same polymer. When probed with are capable of moving, or gliding, over the substratum surface AFM, regular sawtooth patterns were also recorded from the and ultimately achieving sessile adhesion by secreting a mucila- extracellular mucilage of pennate diatom, Phaedactylum tricor- ginous pad from between defined cell wall structures (termed nutum,23 suggesting that diatom adhesives may share some ‘‘girdle bands’’) at the cell apices (Fig. 1A).21 AFM analysis of common mechanical properties. the mucilaginous pad of T. undulatum identified adhesive nano- Although the composition of the T. undulatum adhesive was fibers (ANFs) which gave reproducible sawtooth patterns that not investigated in detail, the multi-cycle sawtooth patterns for were the characteristic AFM fingerprint of modular proteins.20 bridged ANF were abolished by treatment with protease, The AFM curves showed that the first ca. 200 nm portion of demonstrating that protein constituted the polymer backbone.20 the ANFs readily unfolds without application of significant force In addition, the adhesive pads were labelled with concanavalin and is probably located near the anchor point for the adhesive, A, demonstrating that carbohydrates containing glucose and/ whereas the remainder comprises up to 27 domains, each succes- or mannose were present in the adhesive.20 In the present study, sively unfolding to increase the length of the nanofiber by ca. we sought to further characterize the composition of the 34 nm. The ANFs have efficient self-healing properties since T. undultaum adhesives using a combination of physical and they could bind and remain bridged to the AFM probe without chemical approaches. Materials and Methods Algal cell culture T. undulatum cells were initially isolated into clonal culture from panels coated with IntersleekÒ 425 (International Coatings, Akzo Nobel, Gateshead, United Kingdom) in Port Phillip Bay, Victoria that were provided by the Defence Science and Techno- logy Organization (DSTO), Melbourne, Victoria, Australia. The standard culture conditions for T. undulatum entailed growth under static conditions in sterile K medium plus silicates (K + Si)24 inside a growth cabinet equipped with cool white and Grolux fluorescent lights (Sylvania, Munich, Germany) at 16 C with a 12 h : 12 h light–dark cycle. Prior to each experi- ment, axenic cultures were obtained by growing cells in K + Si medium containing 0.1 mg mLÀ1 streptomycin sulfate and 100 u mLÀ1 sodium penicllin G followed by subculture into sterile K + Si medium without antibiotics. Energy dispersive X-ray analysis (EDXA) Cells were inoculated onto chromic acid-washed glass coverslips and grown for 7 or for 21 days under standard culture conditions Fig. 1 (A) SEM of two Toxarium undulatum cells attached to the sub- in tissue culture petri dishes. The slides were soaked briefly in stratum by a common adhesive pad at their apices. (B) Overlay of ED distilled water to remove salts and dried in vacuo over silica gel spectra recorded for the adhesive pad (broken line) shown in panel A at room temperature. For EDTA-treated slides, EDTA solution and the background silica substratum (continuous line). The spectra (0.5 M, pH 8.2) was added to the culture to a concentration of have been normalized to the Si peak. 10 mM and the cells incubated for 1 h prior to soaking the slides 812 | Soft Matter, 2008, 4, 811–820 This journal is ª The Royal Society of Chemistry 2008
  3. 3. sequentially in 10 mM EDTA solution and distilled water and Following initial observations, the piezo-head was removed, then drying as above. The dried slides were evaporatively carbon and 0.5 M EDTA solution (pH 8.2) was added to the K + Si coated and examined by scanning electron microscopy (SEM) medium to a final concentration of 10 mM and gently mixed using an Oxford Instruments Isis model microanalysis system using a micropipettor. The piezo-head was returned and deflec- (accelerating voltage 20 keV) equipped with a Si(Li) ATW tion versus distance curves were recorded on adhesive pads for Pentafet Detector (139 eV resolution). X-Ray spectra were up to 60 min after addition of the EDTA. Curves were also collected at 0–20 keV. The Isis Quant analysis package (ZAF recorded using higher trigger points (10 nm or 20 nm deflection), model) was used to process the data obtained. This package a surface dwell time of up to 2 s, or increased scan distances to was previously calibrated with standards of known composition. ensure the effects of the EDTA on the adhesive were consistent over a range of investigative conditions. After these observations were recorded, the piezo-head was again removed, and the K + Fourier transform infrared (FTIR) spectroscopy Si + EDTA solution was thoroughly rinsed out and replaced Cells were inoculated onto CaF2 (3 mm thick) or ZnSe (2 mm with fresh K + Si medium fortified with both 15 mM CaCl2 thick) substrata (Crystran Ltd., Poole, Dorset, UK) in tissue and 15 mM MgCl2. The piezo-head was returned to its position culture petri dishes and grown for 20 h under standard culture and a final set of deflection versus distance curves were recorded. conditions, except that the cells were incubated in sterile-filtered In a time-course experiment, separate Petri dishes containing seawater instead of K + Si medium. The CaF2 substratum with attached T. undulatum were treated with K + Si + EDTA for attached T. undulatum cells was soaked briefly in Milli-Q water 2 h, 3 h, 6 h, 12 h, and 24 h. Adhesive pads were probed at the to remove salts and dried in vacuo over silica gel at room tempe- end of each time point, immediately before and after replacement rature. The ZnSe substratum with attached cells was first rinsed of the K + Si + EDTA with K + Si medium fortified with 15 mM in Milli-Q water, then one half of the slide was immersed in K + Ca2+ and 15 mM Mg2+. Si medium containing 10 mM EDTA for 45 min prior to being rinsed again in Milli-Q water and dried in vacuo as above. Isolation of pads Spectra were recorded on a Varian (Varian Inc., Palo Alto, CA) FTS 7000 series FTIR spectrometer equipped with a 64 Â T. undulatum cells from axenic cultures were inoculated onto 64 element focal plane array (FPA, Varian) detector and a model chromic acid-washed coverslips inside conical flasks and cultured UMA600 microscope (Varian) in transmission mode. Infrared under standard conditions for periods ranging from 4 to 7 weeks. spectral maps were constructed from 64 Â 64 simultaneously At harvest, the spent culture medium was decanted and the recorded spectra at a pixel size of 5.5 Â 5.5 mm2 between 900 coverslips with attached cells were washed twice with fresh K + and 4000 cmÀ1 at a resolution of 6 cmÀ1 with 128 co-added scans. Si medium. The coverslips were re-immersed in K + Si medium The spectral data hypercube was pre-processed by taking the containing EDTA (10 mM) and allowed to stand for 18 h during second derivative using a Saviztky–Golay smoothing function which most cells detached. The coverslips were recovered from (13 points) and vector normalizing spectra between 1800–1000 culture and rinsed briefly in distilled water to remove salts and cmÀ1 to account for differences in sample thickness. The images most remaining cells. Microscopic examination of representative were constructed using Cytospec (Cytospec Inc., New York, coverslips revealed that 95% of cells were removed by this NY) version 1.2 IR imaging software. FPA images were method, but mucilaginous adhesive pads remained on the contrasted showing either silicate or sulfate concentration, coverslips, although their morphology appeared altered. determined from the integrated area of the stretching bands from 1110–1050 cmÀ1 and 1240–1200 cmÀ1, respectively. Images EDTA extracts and maps were contrasted using the ‘‘Jet’’ colour scheme T. undulatum cells were grown and treated with EDTA as available in Cytospec. described for isolation of adhesive pads above. The K + Si medium containing the EDTA was recovered, centrifuged to Atomic force microscopy (AFM) pellet detached cells, and the supernatant transferred to dialyze in a 6–8 kDa MWCO membrane against two changes of 10 mM Cells were inoculated into tissue culture petri dish containing EDTA solution (pH 8.2) and then exhaustively against distilled K + Si medium and returned to standard culture conditions water. Aliquots of the dialyzed EDTA extract were concentrated for 2 days prior to analysis. The petri dish was positioned on on a Savant Speed Vac (model SC110A) or by lyophilization the stage of an Asylum MFP-3D atomic force microscope followed by reconstitution in an appropriate volume of water (Asylum Research, Santa Barbara, CA) equipped with an and stored at À20 C until analyzed. To confirm that T. undulatum oxide-sharpened ‘‘V’’-shaped Si3N4 cantilever (typical radius of was the origin of the putative anionic macromolecule, one curvature 20 nm; Park Scientific Instruments, Sunnyvale, T. undulatum culture was inoculated into K + Si medium contai- CA) with a measured spring constant of 21.96 mN mÀ1.25 The ning antibiotics and grown under axenic conditions (as described cantilever tip was guided over an adhesive pad with an optical above) for 5 days prior to harvesting by EDTA treatment. microscope. Deflection versus distance curves were recorded in contact mode with a trigger point set to 5.00 nm deflection before Polyacrylamide gel analysis the cantilever was retracted from the surface. Typical scan conditions were 5.00 nm trigger point, 2.0 mm scan distance, Extracts were separated by SDS-PAGE.26 The gels were stained 0.5 Hz scan rate and no dwell. The tip velocity varied between sequentially with Coomassie Blue and Stains AllÒ (Sigma, St 1.50 and 2.00 mm sÀ1 for the range of scan distances and rates. Louis, MO). This journal is ª The Royal Society of Chemistry 2008 Soft Matter, 2008, 4, 811–820 | 813
  4. 4. Assay methods Table 1 Constituent atoms (atom%) of the background silica substra- tum and of Toxarium undulatum adhesive pads on the substratum. 27 Carbohydrates were assayed by the method of Dubois et al. using glucose as the standard. Protein was assayed by the Native EDTA-treated bicinchoninic acid (BCA; Pierce, Rockford, IL) following the a b c Atom Substratum Adhesive Substratumd Adhesivee manufacturer’s protocol and using BSA as the standard. Sulfate was assayed by the BaCl2 turbidimetric method of Tabatabai28 as O 62.7 57.9 62.7 53.8 modified by Craigie et al.29 using K2SO4 to construct the Na 4.5 3.4 4.4 6.4 Mg — 1.7 — — standard curve. Phosphate was assayed by the method of Itoh Al 1.9 1.8 1.9 2.1 et al.30 using KH2PO4 to construct the standard curve. Si 25.0 24.4 25.0 29.1 P — 0.1 0.1 0.1 S — 3.5 — 1.4 Constituent monosaccharide analysis K 2.8 3.1 2.9 3.5 Ca — 1.0 — — Neutral monosaccharides in lyophilized EDTA extracts or Ti 1.3 1.3 1.2 1.5 samples of dried, ground coverslips with isolated pads were Fe — 0.1 — — converted to their corresponding alditol acetates by trifluoroace- Zn 1.8 1.7 1.8 2.1 tic acid-hydrolysis, reduction with NaBD4, and acetylation.31 a The atom% of C and N were not calculated owing to configurational The alditol acetates were separated, identified, and quantified constraints (see text for details). b Data represent average of 3 replicates. c by gas chromatography-mass spectrometry (GC-MS) as Data represent average of 6 replicates. d Data represent average of 2 described previously.14 replicates. e Data represent average of 5 replicates. Symbols: — ¼ not detected. Constituent amino acid analysis Amino acids were converted to their phenylthiocarbamoyl second-derivative spectrum is shown in Fig. 2B and the peak (PTC) derivatives following hydrolysis in 6 N HCl containing assignments are presented in Table 2. The prominent amide I 0.1% phenol for 16 h and separated by HPLC on a Pico-Tag band at 1638 cmÀ1 and the amide II band at 1545 cmÀ1 of the column (Millipore Corporation, Waters Chromotagraphy spectra confirmed the presence of protein in the adhesive. The Division, Milford, MA).32 The PTC-amino acids were identified positions of amide I bands in FTIR spectra of proteins are by their retention times and quantified using response factors sensitive to secondary structure.33 The 1638 cmÀ1 band in the determined from commercial standards (amino acids standards spectra of T. undulatum pads, together with the minor absorption H, Pierce, Rockford, IL). band at 1685 cmÀ1 indicated that the protein was dominated by b-sheets.33–35 An additional minor absorption band at 1667 cmÀ1 Results indicated some b-turn structure.35 By contrast, the amide I band in FTIR spectra of the diatom cells was dominated by absorp- EDXA of T. undulatum adhesive mucilage tion at around 1645 cmÀ1 (data not shown), indicating a predo- Representative ED spectra of dried, native adhesive pads of minance of a-helix structure, most likely arising from the T. undulatum and the background recorded on an acid-washed abundant Rubisco protein inside the cells.36 coverslip are presented in Fig. 1B. The relative proportions of Two strong absorption bands were observed at 1215 cmÀ1 and the elements in the background and the adhesive pads are pre- 1255 cmÀ1 in the FTIR spectra of the T. undulatum pads sented in Table 1. There was a complex collection of elements (Fig. 2B). Absorption in this region is typically associated with in the adhesive pads that were also present in the background. sulfate and phosphate moieties.37,38 Spectral mapping of These included Si, O, Na, Al, K, Ti, and Zn, which are typical the 1215 cmÀ1 band demonstrated that it was localized to the elements of borosilicate glass and were therefore interpreted as adhesive pads (Fig. 2D) and was clearly discernible from the components of the coverslip used as the substratum. ED spectra diatom cells, which could be distinguished by the 1090 cmÀ1 of the pads and the background were normalized to the Si band band corresponding to the silica in the cell walls (Fig. 2E).39 In derived predominantly from the silica substratum. The elements line with EDXA data, which demonstrated that S was present that were present in the adhesive pads and absent in the back- and P was essentially absent in the pads of T. undulatum ground were C, S, Ca, and Mg. O also increased in the adhesive (Fig. 1B, Table 1), the 1215 cmÀ1 and 1255 cmÀ1 bands were pad relative to the background when allowance was made for the assigned to the symmetric and antisymmetric stretching of O contributing to the silica substratum to which the pads were sulfate groups, respectively (Table 1).37 Unlike the 1215 cmÀ1 adhered. Only traces of P occurred in the spectra of both the band, absorption at 1255 cmÀ1 was also observed in FTIR spec- background and the adhesive pads. The EDXA system was tra of the diatom cells (data not shown) and most likely arose insensitive to N due to absorption by the detector window. from asymmetric stretching of phosphate esters in cellular proteins, nucleic acids, and low-molecular-mass metabolites.40 Three bands at 1165 cmÀ1, 1135 cmÀ1, and 1095 cmÀ1 in the FTIR spectroscopy FTIR spectra of adhesive pads were assigned mainly to ring FTIR spectra recorded of dried, native adhesive pads of T. undu- vibrations of sugars (Table 2), indicating the presence of latum grown on CaF2 and ZnSe substrata gave essentially the carbohydrates. Additional minor bands in the FTIR spectra same FTIR spectra. A representative FTIR spectrum of the of the adhesive pads (Fig. 2B, Table 2) indicated the presence native pads on CaF2 is presented in Fig. 2A. The corresponding of carboxylate anions. 814 | Soft Matter, 2008, 4, 811–820 This journal is ª The Royal Society of Chemistry 2008
  5. 5. Fig. 2 (A) FTIR spectrum of a Toxarium undulatum adhesive pad on a CaF2 substratum. (B) The second derivative spectrum of that shown in panel A. The peaks labelled 1–11 were assigned according to reference data (see Table 2). (C) Light micrograph of two T. undulatum cells attached by a common adhesive pad. (D) FTIR spectral map of the sulfate concentration in the pad and cells shown in panel C determined from the integrated area of the symmetric SO2 stretching band between 1240–1200 cmÀ1 in second derivative, vector normalized spectra, with red indicating the strongest absorbance and blue the weakest. (E) FTIR spectral map of the silicate concentration in the pad and cells shown in panel C determined from the integrated area of the symmetric Si–O stretching band between 1110–1050 cmÀ1 in second derivative, vector normalized spectra, with red indicating the strongest absorbance and blue the weakest. Atomic Force Microscopy (AFM) to the ANFs reported previously for T. undulatum and they represented the classical fingerprint of unfolding a modular Frequent, reproducible sawtooth curves were recorded in protein.20,22 The curves classified as type-I ANFs were fitted contact mode on the native adhesive pads of live T. undulatum with the WLC model and contained an average of ca. 22 peaks cells in K + Si medium (Fig. 3A). These curves corresponded with an average distance of 36.2 nm separating each peak and Table 2 Assignments of absorbance bands in second-derivative FTIR an average peak force of 554.6 pN (Table 3). These were compa- spectra recorded for the dried adhesive pads of Toxarium undulatum rable to the curves recorded previously on T. undulatum adhesive grown on CaF2 and ZnSe substrata. pads,20,22 although the average peak force was lower than earlier estimates for type-I ANFs by the WLC model (872 pN Æ 4.8%).22 Peaka Wavenumber/cmÀ1 Assignmentb Reference Curves were also recorded on native pads showing irregular 1 1638 n(C]O) in secondary 63,64 detachment events (Fig. 3B). These occurred with more frequency amides, referred to as than reported in the earlier papers mainly because contact mode amide I band AFM was used in the current investigation, whereas the earlier 2 1600–1590 nas(COOÀ) 65,66 3 1545 n(C–N) and d(N–H) from 63,64 work used the more sensitive ‘‘fly fishing’’ method.41 amides, referred to as In the 20–60 min following the addition of 10 mM EDTA, amide II band a divalent cation chelator, to the K + Si medium, AFM force 4 1450 das(CH3), das(CH2), and 40,67,68 ds(C–OH) curves of the adhesive pads lacked the typical sawtooth pattern 5 1420 ns(COOÀ) 66 of the ANFs (Fig. 3C). Instead, these curves displayed irregular 6 1370 ds(CH3) and ds(CH2) 67 detachments extending to over 1.0 mm with adhesive forces 7 1320 n(C–H) and d(N–H) of 69 generally #200 pN, substantially weaker than those of the native proteins, referred to as amide III band ANFs and had a substantially different appearance to the 8 1255 nas(S]O) 37 irregular detachments events recorded on untreated native 9 1215 ns(S]O) 37,70 pads (cf. Fig. 3B). The curves recorded on the EDTA-treated 10, 11 1290–960 Mainly n(C–O–C), 38,67,71,72 pads also indicated that the mucilage had become softer since n(C–C–C), and n(C–C–O) the extension and retraction curves did not overlap while in a Numbers correspond to the peaks labelled in Fig. 2B. b Symbols: nas ¼ contact with the pad (Fig. 3C). These observations demonstrated asymmetric stretch; ns ¼ symmetric stretch; das ¼ asymmetric deformation (bend); ds ¼ symmetric deformation (bend). that addition of EDTA to the growth medium disrupted the structure and properties of the T. undulatum adhesive mucilage. This journal is ª The Royal Society of Chemistry 2008 Soft Matter, 2008, 4, 811–820 | 815
  6. 6. Table 3 Mean values (Æ%SE) of parameters for ANF I curves analyzed by the wormlike chain model. K + Si K + Si + Ca + Mg ANF I as a proportion of total 65.5 67.5 sawtooth curves (%) Number of curves analyzed 19 27 Average peak force/pN 554.6 (Æ1.3%) 536.9 (Æ1.4%) D Force, trough to next peak/pN 173.1 (Æ2.5%) 131.3 (Æ1.8%) Average final peak force/pN 762.3 (Æ3.0%) 703.3 (Æ4.6%) Average first peak force/pN 407.5 (Æ3.8%) 418.4 (Æ2.6%) Distance to first peak/nm 211.2 (Æ11.3%) 328.5 (Æ6.3%) Distance to last peak/nm 934.3 (Æ5.8%) 1082.3 (Æ3.0%) Distance between peaks/nm 36.2 (Æ2.8%) 37.5 (Æ1.0%) Persistence length, q/pm 42.3 (Æ5.5%) 39.9 (Æ4.0%) Average number of peaks 21.8 (Æ3.6%) 22.4 (Æ2.6%) Table 4 Proportions of curves detected by AFM as recorded adhesive events. Number Non-specific Sawtooth Medium of curves attachments (%) curves (%) K + Si 434 71 29 K + Si + 10 mM EDTA 479 100 0 K + Si + 15 mM Ca2+ + 15 299 60 40 mM Mg2+ When the K + Si medium containing EDTA was flushed out and replaced with K + Si medium fortified with 15 mM of Ca2+ and 15 mM Mg2+ ions, sawtooth curves comparable to those of native pads were again detected (Fig. 3D). The propor- tion of the attachment events recorded as sawtooth curves (Table 4) and the features of the curves classified as type-I ANFs were also similar to those of native pads (Table 3). These observations indicated that the structure and properties of the ANFs were restored by removing the EDTA and replenishing the medium with divalent cations. In a separate experiment, T. undulatum adhesive pads were incubated for 2 h, 3 h, 6 h, 12 h, 18 h, or 24 h in K + Si medium containing EDTA and then returned to K + Si medium contain- ing 15 mM Ca2+ and 15 mM Mg2+ for analysis. Increasing exposure time to EDTA decreased the proportions of attach- ment events detected as sawtooth curves to 1%–2% after the divalent cations were replenished (data not shown) for time points of 2 h to 6 h. Sawtooth curves were not detected at all following $12 h exposure to EDTA, although non-specific adhesion and detachment events were still recorded. Analysis of EDTA-treated adhesive Fig. 3 AFM force curves recorded on Toxarium undulatum adhesive ED spectra recorded of EDTA-treated and dried T. undulatum pads. (A) A typical sawtooth curve recorded on a native adhesive pad. adhesive pads showed that C and S were still present but the (B) An irregular force curve recorded on a native adhesive pad. (C) A Ca and Mg had been eliminated (Table 1). However, FTIR representative curve recorded on an adhesive pad 45 min after treatment spectra recorded of EDTA-treated and dried T. undulatum with EDTA. (D) A typical sawtooth curve recorded on an adhesive pad adhesive pads on a ZnSe substratum (data not shown) were after treatment with EDTA and subsequent treatment with K + Si essentially identical to those of dried, native pads. The EDXA medium enriched with 15 mM MgCl2 and CaCl2. In all force curves, and FTIR data therefore indicated that divalent cations had the advancing and retracting curves are indicated by grey and black been removed by EDTA treatment and that sulfated organics arrows, respectively. had been left behind in the residual adhesive material on the substratum, but the secondary structure of the protein 816 | Soft Matter, 2008, 4, 811–820 This journal is ª The Royal Society of Chemistry 2008
  7. 7. Table 5 Constituent monosaccharides (mol%) of extractsa obtained from Toxarium undulatum adhesive pads. Monosaccharideb Adherent mucilage EDTA extract Glc 4 * Gal 4 8 Man 50 47 Xyl 29 30 Ara 1 3 Rib tr 1 Rha 6 4 Fuc 4 6 3,4-diMeMan 1 1 3-/4-MeGal 1 — a Carbohydrate extracts. Adherent mucilage ¼ adhesive pads isolated on coverslips by detaching cells with EDTA (data represent average of 4 replicates). EDTA extract ¼ medium recovered from EDTA-treated cultures (data represent average of 2 replicates). b Monosaccharides: Glc ¼ glucose; Gal ¼ galactose; Man ¼ mannose; Xyl ¼ xylose; Ara ¼ arabinose; Rib ¼ ribose; Rha ¼ rhamnose; Fuc ¼ fucose; 3,4-diMeMan ¼ 3,4-di-O-methylmannose; 3-/4-MeGal ¼ 3-O-methylgalactose and 4-O-methylgalactose. Symbols: tr ¼ trace (0.8 mol%); – ¼ not detected. * ¼ Glc accounted for 56 mol% of total sugars in the EDTA extract; Glc is excluded and the calculation of the proportions of the remaining sugars are normalized to Fig. 4 10% SDS-PAGE of the molecule extracted in the EDTA-treated 100 mol% (see text for details). medium from Toxarium undulatum pads. The gel was sequentially stained with Coomassie blue and Stains AllÒ. component of the EDTA-treated material was not appreciably different to that of the native adhesive. SDS-PAGE analysis of an aliquot of this EDTA-treated Prolonged exposure to EDTA (standing undisturbed for $18 medium revealed a single, sharp band with a molecular mass h) resulted in detachment of the cells from the coverslips (95% that exceeded the 220 kDa marker of the standard proteins of cells). However, hydrated adhesive mucilage remained on the (Fig. 4). The molecule stained blue with Stains AllÒ, a cationic surface of the coverslips, although the morphology of this carbocyanine dye, but could not be visualized with Coomassie material appeared altered compared with that of native adhesive blue at loadings of up to 25 mg of protein. This staining specific- pads; i.e., the firm ‘‘doughnut’’ shape of standalone native ity also has been observed for certain sulfated glycosamino- adhesives gave way to irregularly shaped smears (data not glycans, phosphorylated or sialic acid-rich glycoproteins, and shown). In order to obtain a biochemical signature for the lipophosphoglycans and indicated that the macromolecule in adhesive material, the constituent neutral sugars of the adhesive the extract was highly anionic.42–45 left behind following EDTA treatment were analyzed by crush- The amino acids in the EDTA extract were analyzed (Table 6). ing the dried coverslips and acid-hydrolyzing the material in The most abundant amino acids were Gly (22 mol%), Asp/Asn situ. Eleven neutral sugars were detected (Table 5), including (14 mol%), His (11 mol%), and Glu/Gln (7 mol%). At the pH three hexoses, three pentoses, two 6-deoxyhexoses, and three of sea water (8.2), the His residues (pKa $6) would be essentially O-methylated hexoses. The dominant neutral sugars were unionized. The total basic residues (Lys, Arg) therefore mannose and xylose, together accounting for 79 mol% of the accounted for just 3 mol% of the total. Hydroxylated amino total sugars (Table 5). The high level of mannose was consistent acids (Ser, Thr, Tyr, Hyp), representing potential sites of O-gly- with the previously reported labelling of native T. undulatum can attachment, comprised 10 mol% of total amino acids. adhesives with the lectin, concanavalin A.20 Composition of the EDTA-medium extract Discussion The EDTA-treated medium was separated from the detached The EDXA and FTIR data demonstrated that the native cells by centrifugation, dialyzed, concentrated, and analyzed adhesive pads of T. undulatum consist mainly of protein, carbo- further. The estimated weight ratio for protein : carbohydrate : hydrate, and sulfate together with Ca2+ and Mg2+ ions. The sulfate (as SO3Na) in the extract was 1.0 : 0.2 : 0.9. Phosphate importance of the divalent cations in maintaining the integrity was assayed but not detected in the extract. The constituent of the adhesive was demonstrated by treatment with EDTA. neutral sugars of the extract comprised a high level of glucose AFM analysis showed that EDTA treatment resulted in rapid (56 mol% of total sugars). However, if glucose was excluded disintegration of the nanostructure of the adhesive and that from the calculation of the relative proportions of the sugars, the adhesive nanostructure could be recovered by flushing out the constituent sugar profile of the extract essentially matched the EDTA and replenishing the divalent cations. Longer that of the adhesive deposited on the substratum (Table 5). These treatments with EDTA ($2 h), however, resulted in decreased results confirmed that the adhesive material was present in the recovery of the adhesive nanostructure and eventually ($18 h) EDTA-treated medium, together with a soluble glucan. led to cell detachment. SDS-PAGE of the extract derived by This journal is ª The Royal Society of Chemistry 2008 Soft Matter, 2008, 4, 811–820 | 817
  8. 8. Table 6 Constituent amino acids of the EDTA extracta obtained from species of Navicula, Nitzschia, Tropidonies, and Auricula.48 While Toxarium undulatum. these studies proved that extraction of divalent cations caused the diatom adhesives to fail, the effect on the structure and Amino Acid Mol% physical properties of the adhesive remained unknown. Gly 22 The present study shows that cations contribute to the compo- Ala 6 sition of the T. undulatum adhesive and that they must regulate Val 2 the higher order structure of the adhesive. The AFM data Leu 5 Ile 5 demonstrated that the regular sawtooth patterns were lost Phe 2 following treatment with EDTA and were largely replaced by Tyr 2 irregular unbinding events suggestive of the adhesion and Met 3 Cys 5 detachment of entangled polymers. AFM also demonstrated Pro 5 that removing the EDTA and re-incubating disrupted T. undula- Hyp 2 tum adhesive pads in medium fortified with Ca2+ and Mg2+ ions Ser 4 recovered the original sawtooth patterns. This reversibility is Thr 2 His 11 analogous to the solution behaviour of high-molecular-mass Lys 2 sulfated polysaccharides, which form viscous solutions at rela- Arg 1 tively low polymer concentrations in salt-free media as a result Asnb 14 Glnb 7 of loose chain entanglement but form firm colloidal gels in the presence of divalent cations through ionic cross-linking.51 From a EDTA extract ¼ same as described in Table 2 (data represent average persistence-length calculations based on the WLC model, of 3 replicates). b Asn ¼ Asp + Asn; Gln ¼ Glu + Gln. Dugdale et al.22 estimated that the type-I ANFs of T. undulatum comprised up to 30 individual modular proteins, yet these proteins synchronously unfolded and refolded as a cohesive dialyzing the EDTA-treated medium showed that it contained unit within the ANF. In that context, the most straightforward a macromolecule with very high molecular mass (220 kDa, interpretation of the data in the present study is that the high- Fig. 4). The staining specificity of Stains AllÒ and Coomassie molecular-mass sulfated glycoprotein is the fundamental blue indicated that the anionic groups were covalently bound to adhesive polymer and the divalent cations assist the aggregation this macromolecule. The composition of the extract in which the of the sulfated glycoproteins into ANFs. The cations presumably macromolecule was derived (protein, carbohydrate, and high screen negative charges and bridge sulfate groups on adjacent levels of sulfate but no phosphate) was consistent with the compo- glycoproteins in the ANFs. Cationic bridges also could be formed nents of the native diatom adhesive determined by EDXA and between carboxylate functional groups, which were detected by FTIR spectroscopy and, except for an increase in glucose (see FTIR spectroscopy and may be attributable to uronic and/or below), the constituent neutral sugars of the extract corresponded amino acids. to those of the deposited adhesive. We therefore interpret the We propose therefore that a network of ionic interactions polyanionic macromolecule extracted with EDTA as a sulfated contributes to stabilizing the sulfated glycoproteins into an glycoprotein and a fundamental organic component of the organized supramolecular structure (the ANF) which enables T. undulatum adhesive. The data showed that the sulfated glyco- the synchronized unfolding and refolding of parallel domains in protein was cross-linked by divalent cations in the native adhesive. adjacent sulfated glycoproteins. Recently, Fernandez and Approximately half the carbohydrate portion of the EDTA- colleagues provided a compelling empirical demonstration of medium extract was glucose. While it is possible that this is the synchronized unfolding of domains in a parallel protein a component of the adhesive, the high levels of glucose were homodimer using AFM.52 The homodimer comprised an engi- not present in the adherent mucilage following cell detachment. neered protein consisting of eight I27 domains (the 27th immuno- We therefore interpret this glucose as most likely arising from globulin module of titin) and a GCN4 oligomerization domain. low-molecular-mass intracellular glucans (termed ‘‘chrysolami- Stretching the covalently linked homodimer by AFM produced naran’’) released into the medium as a result of physiological a characteristic sawtooth pattern with the same interpeak perturbation of the cells during EDTA treatment.46 distances as the protein monomers but with twice the peak force The importance of divalent cations in diatom adhesion has and, when fitted with the WLC model, half the persistence been demonstrated for several biofouling diatoms. Treatment length.52 These observations corroborate our interpretations of of Amphora coffeaeformis with a range of Ca2+ channel blockers the sawtooth curves recorded for the native and reconstituted such as the drug D-600, La3+, and Ruthenium red inhibits moti- adhesive pads of T. undulatum (Figs 3A and 3D).20,22 In contrast, lity and adhesion, and these effects have largely been interpreted however, to the covalent linkage engineered into the I27-GCN4 as perturbation of intracellular Ca2+ fluxes.47,48 However, cells protein homodimer,52 the sulfated glycoproteins of T. undulatum retained the ability to adhere to the substratum if Ca2+ in the are kept in register in the ANF by a network of cross-linking growth medium was substituted with Sr2+ but not other divalent divalent cations. cations, indicating an extracellular role for the divalent cations.49 The FTIR data indicated that there was a high content of Furthermore, treatment of Amphora biofilms with 10 mM b-sheet in the protein component of the T. undulatum adhesives, EGTA caused ‘‘cohesive breaks’’ in which cells were detached raising the subject of the role of b-sheets in the structure of ANF. and adhesive material was left behind on the substratum,50 and Interestingly, AFM sawtooth patterns that could be fitted with similar effects have been reported for other diatoms, including the WLC model were recently reported for an adhesive from 818 | Soft Matter, 2008, 4, 811–820 This journal is ª The Royal Society of Chemistry 2008
  9. 9. a chlorophye alga, Prasiola linearis.53 These sawtooths were structure of the sulfated glycoproteins. The present study proposed to be derived from amyloid fibrils formed by hydro- demonstrates, however, that ionic interactions are fundamental gen-bonded intermolecular b-sheets.53 We consider this to stabilizing the supramolecular structure of T. undulatum interpretation inadequate for the T. undulatum adhesive, how- ANFs. ever, because such a model conflicts with the data in the present study, which show that the recording of sawtooth patterns is dependent upon the presence of divalent cations and that ionic Acknowledgements bonding therefore contributes significantly to the regulation of We thank Dr John Lewis at the Defence Science and Technology the nanostructure of the adhesive. Furthermore, an amyloid Organization (DSTO) of the Australian Department of Defence model for the native T. undulatum adhesive is not supported by and Mr Finlay Shanks at Monash University for valuable discus- AFM studies of amyloid fibrils formed from isolated peptides. sions and technical support. The authors gratefully acknowledge Amyloid fibrils formed by synthetic Ab1–40 or Ab25–35 funding from the Australian Research Council and our industry peptides designed from amyloid b-protein, the paradigm protein partner, Akzo Nobel, Gateshead, UK (Industry Linkage Grant for amyloid fibrils, as well as a transthyretin peptide, TTR105–115, #LP0454982), as well as financial assistance from the DSTO. give AFM force curves that are dominated by stepwise R.D. also thanks the National Science Foundation (grant decreasing plateaus attributable to the lateral unzipping of #INT0202675). b-sheets from the amyloid fibril by sequential rupture of uniformly strong hydrogen bonds.54,55 AFM sawtooths were recently recorded for amyloid fibrils formed by the synthetic References Ab1–42 peptide but these sawtooths were linear and could not be fitted with the WLC model.56 The sawtooths of Ab1–42 1 G. S. Bohlander, in Polymers in a Marine Environment, ed. D. M. Long, R. Bufton, P. Yakimiuk, and K. Williams, Institute of were rationalized as oblique versions of the standard plateau Marine Engineering, London, 1991, pp 135–138. curves recorded on amyloid fibrils offset by torsion of the 2 R. S. Alberte, S. Snyder, B. J. Zahuranec and M. Whetsone, stretched fibril during AFM probing.56 Such linear sawtooth Biofouling, 1992, 6, 91–95. 3 I. Omae, Chem. Rev., 2003, 103, 3431–3448. curves have not been observed for the native T. undulatum adhe- 4 I. Omae, Appl. Organmetal Chem., 2003, 17, 81–105. sives and contrast markedly with those that were recorded and 5 C. Anderson, M. Atlar, M. Callow, M. Candries and R. L. Townsin, could be fitted with the WLC model.20,22 J. Mar. Design Operations, 2003, 84, 11–23. We propose instead that the b-sheets in the T. undulatum 6 R. Holland, T. M. Dugdale, R. Wetherbee, A. B. Brennan, J. A. Finlay, J. A. Callow and M. E. Callow, Biofouling, 2004, 20, adhesives are predominantly intramolecular since the secondary 323–329. structure of the adhesive proteins was not appreciably altered 7 F. E. Round, R. M. Crawford, and D. G. Mann, The Diatoms, during EDTA treatment, even though the integrity of the Cambridge University Press, Cambridge, 1990. 8 G. F. Daniel, A. H. L. Chamberlain and E. B. G. Jones, Helgola ¨nder ANFs was clearly compromised. Notably, b-sheets occurring Meeresunters, 1980, 34, 123–149. as b-sandwiches in immunoglobulin-type domains also are 9 G. F. Daniel, A. H. L. Chamberlain and E. B. G. Jones, Br. Phycol. a significant component of extensible proteins with tandem J., 1987, 22, 101–118. modular domains, such as titin or tenascin.57 Native forms of 10 B. A. Wustman, M. R. Gretz and K. D. Hoagland, Plant Physiol., 1997, 113, 1059–1069. titin and tenascin and recombinant polypeptides derived from 11 B. A. Wustman, J. Lind, R. Wetherbee and M. R. Gretz, their immunoglobulin domains gave regular AFM sawtooth Plant Physiol., 1998, 116, 1431–1441. curves that were fitted with the WLC model,58–61 and these 12 K. D. Hoagland, J. R. Rosowski, M. R. Gretz and S. C. Roener, were considered models for the mechanical properties of the T. J. Phycol., 1993, 29, 537–566. 13 G. J. C. Underwood and D. M. Paterson, in Advances in Botanical undulatum ANFs.20,22 Furthermore, Sarkar et al.52 elegantly Research, ed. J. M. Callow, vol. 40, Elsevier Academic Press, demonstrated the AFM sawtooth patterns obtained by synchro- Oxford, 2003, pp 183–240. nized unfolding of domains containing antiparallel b-strands in 14 A. Chiovitti, M. J. Higgins, R. E. Harper, R. Wetherbee and A. Bacic, J. Phycol., 2003, 39, 543–554. the engineered I27-GCN4 protein homodimer. It should be 15 B. J. Bellinger, A. S. Abdullahi, M. R. Gretz and G. J. C. Underwood, noted that absorption in the region 1635–1640 cmÀ1 in the Aquat. Microbial Ecol., 2005, 38, 169–180. FTIR spectra (Fig. 2, Table 2) may be accounted for by the total 16 A. S. Abdullahi, G. J. C. Underwood and M. R. Gretz, J. Phycol., extended segments of the protein, not limited to b-sheets,33 and 2006, 42, 363–378. 17 J. L. Lind, K. Heimann, E. A. Miller, C. van Vliet, N. J. Hoogenraad such segments could contribute to the overall flexibility of the and R. Wetherbee, Planta, 1997, 203, 213–221. adhesive.20 However, we are cautious about the conformational 18 A. Chiovitti, A. Bacic, J. Burke and R. Wetherbee, Eur. J. Phycol., interpretation of the FTIR data since the native conformation of 2003, 38, 351–360. 19 A. Chiovitti, T. M. Dugdale, and R. Wetherbee, in Biological the proteins can be altered during drying, and there is evidence Adhesives, ed. A. M. Smith and J. A Callow, Springer-Verlag, some proteins, such as bovine serum albumin,62 adopt substan- Berlin, 2006, pp 79–103. tially higher levels of b-sheet structure in their solid state. 20 T. M. Dugdale, R. Dagastine, A. Chiovitti, P. Mulvaney and The size and relative complexity of the sulfated glycoproteins R. Wetherbee, Biophys. J., 2005, 89, 4252–4260. 21 W. H. C. F. Kooistra, M. De Stafano, D. G. Mann, N. Salma and also raises the possibility that cross-linking of the polymers into L. K. Medlin, J. Phycol., 2003, 39, 185–197. a supramolecular structure is assisted by specific intermolecular 22 T. M. Dugdale, R. Dagastine, A. Chiovitti and R. Wetherbee, interactions, such as interdomain interactions between adjacent Biophys. J., 2006, 90, 2987–2993. protein backbones or lectin interactions between protein back- 23 T. M. Dugdale, A. Willis and R. Wetherbee, Biophys. J., 2006, 90, L58–L60. bones and pendant glycans. Dissection of the intermolecular 24 R. A. Andersen, D. M. Jacobson, and J. P. Sexton, Provasoli-Guillard interactions will necessitate detailed characterization of the centre for culture of marine phytoplankton – catalogue of strains, This journal is ª The Royal Society of Chemistry 2008 Soft Matter, 2008, 4, 811–820 | 819
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