PAPER www.rsc.org/softmatter | 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
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
School of Botany, University of Melbourne, Victoria 3010, Australia. composed mainly of heterogeneous polyanionic polysaccharides
E-mail: email@example.com (see ref. 12 for a review). Furthermore, studies of natural and
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
Department of Chemical and Biomolecular Engineering, University of
Melbourne, Victoria 3010, Australia
stability (see ref. 13 for a review). Consequently, recent research
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
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
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
Assay methods Table 1 Constituent atoms (atom%) of the background silica substra-
tum and of Toxarium undulatum adhesive pads on the substratum.
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.
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 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
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
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
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
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
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
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
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
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 —
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
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
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
the higher order structure of the adhesive. The AFM data
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
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
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
of loose chain entanglement but form ﬁrm colloidal gels in the
presence of divalent cations through ionic cross-linking.51 From
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
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
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
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