pubs.acs.org/Langmuir © 2009 American Chemical Society
Interactions of Zoospores of Ulva linza with Arginine-Rich Oligopeptide Monolayers T. Ederth,*,† M. E. Pettitt,‡ P. Nygren,† C.-X. Du,† T. Ekblad,† Y. Zhou,† M. Falk,† M. E. Callow,‡ J. A. Callow,‡ and B. Liedberg† †
Division of Molecular Physics, Department of Physics, Chemistry and Biology, Linko¨pings Universitet, Linko¨ping, Sweden, and ‡School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom Received February 26, 2009. Revised Manuscript Received April 18, 2009
We recently reported on the strong interactions of zoospores of the green alga, Ulva linza with an arginine-rich oligopeptide self-assembled monolayer (SAM) [Biofouling 2008, 24, 303-312], where the arginine-rich peptide induced not only high spore settlement, but also a form of abnormal settlement, or “pseudo-settlement”, whereby a proportion of spores do not go through the normal process of surface exploration, adhesive exocytosis, and loss of flagella. Further, it was demonstrated that both the total number of settled spores and the fraction of pseudosettled spores were related to the surface density of the arginine-rich peptide. Here we present a further investigation of the interactions of zoospores of Ulva with a set of oligomeric, de novo designed, arginine-rich peptides, specifically aimed to test the effect of peptide primary structure on the interaction. Via variations in the peptide length and by permutations in the amino acid sequences, we gain further insight into the spore-surface interactions. The interpretation of the biological assays is supported by physicochemical characterization of the SAMs using infrared spectroscopy, ellipsometry, and contact angle measurements. Results confirm the importance of arginine residues for the anomalous pseudosettlement, and we found that settlement is modulated by variations in both the total length and peptide primary structure. To elucidate the causes of the anomalous settlement and the possible relation to peptide-membrane interactions, we also compared the settlement of the “naked” zoospores of Ulva (which present a lipoprotein membrane to the exterior without a discrete polysaccharide cell wall), with the settlement of diatoms (unicellular algae that are surrounded by a silica cell wall), onto the peptide SAMs. Cationic SAMs do not notably affect settlement (attachment), adhesion strength, or viability of diatom cells, suggesting that the effect of the peptides on zoospores of Ulva is mediated via specific peptide-membrane interactions.
Introduction The colonization of surfaces by the dispersal stages of sessile marine organisms is a critical stage in their life-histories. When the surface is man-made, the consequence of colonization and subsequent growth is referred to as “biofouling”, which has adverse effects on the performance, maintenance costs, lifetime, and environmental impact of shipping, fisheries, and other industrial operations using or operating in natural waters. Recent legislative changes and international agreements restricting or banning the use of some biocides1 have stimulated research efforts aimed to increase our understanding of the influence of surface physicochemical properties on settlement and colonization by the dispersal stages of fouling organisms, such as larvae of invertebrates, or motile spores of algae. Such improved understanding may then contribute to the development of a new generation of “knowledgebased” coatings that do not rely on the use of biocides.2 Biomimetic strategies have proven successful in many branches of materials science3 and are employed also in biofouling research, for example, mimicry of naturally fouling-resistant surfaces such as shark skins4 and mollusk shells5 or the employment of chemical defense methods from marine organisms.6 Peptides are of interest *Corresponding author. E-mail:
[email protected]. (1) International Maritime Organisation Treaty 2001, implementing a ban on several biocides from 2008. (2) Rosenhahn, A.; Ederth, T.; Pettitt, M. E. Biointerphases 2008, 3, IR1–IR5. (3) Mann, S., Ed.; Biomimetic Materials Chemistry; Wiley: New York, 2006. (4) Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Schumacher, J. F.; Wilkerson, W.; Wilson, L. H.; Callow, M. E.; Callow, J. A.; Brennan, A. B. Biofouling 2006, 22, 11–21. (5) Scardino, A. J.; Guenther, J.; de Nys, R. Biofouling 2008, 24, 45–53. (6) Fusetani, N., Clare, A. S., Eds. Antifouling Compounds. Progress in Molecular and Subcellular Biology; Springer-Verlag: Berlin, 2006; Vol. 42.
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in this respect since they are involved in chemical mediation of surface colonization,7 and a number of natural peptide settlement inducers have been identified.8 Antimicrobial (or host defense) peptides are an important component of the innate immune system of both animals and plants. These peptides typically have cationic lysine or arginine residues and are effective antibiotics,9 and it has been demonstrated that surface-tethered peptides may also retain broad-spectrum antimicrobial activity.10 The bioactivity of surface-tethered peptides also raises questions about the possibilities for antifouling applications and has generated some work in this area. Peptidomimetic antifouling surfaces show promising results,11,12 and the fact that peptide chemistry, which allows great flexibility and convenience in production, in combination with automated monitoring techniques, opens possibilities for screening libraries of peptides for identification of antifoulants with selective biocidal activity.13 Chemical cues may promote or deter the settlement of fouling organisms, but despite the relevance of these to biofouling, detailed and quantitative studies of chemically mediated interactions are rare,14,15 and improved understanding of the interactions between various organisms and particular (7) Rittschof, D. J. Chem. Ecol. 1990, 16, 261–272. (8) Fusetani, N. Nat. Prod. Rep. 2004, 21, 94–104. (9) Zasloff, M. Nature 2002, 415, 389–395. (10) Haynie, S. L.; Crum, G. A.; Doele, B. A. Antimicrob. Agents Chemother. 1995, 39, 301–307. (11) Statz, A. R.; Barron, A. E.; Messersmith, P. B. Soft Matter 2008, 4, 131–139. (12) Statz, A. R.; Park, J. P.; Chongsiriwatana, N. P.; Barron, A. E.; Messersmith, P. B. Biofouling 2008, 24, 439–448. (13) Fletcher, J. T.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Ghadiri, M. R. Chem.;Eur. J. 2007, 13, 4008–4013. (14) Steinberg, P. D.; de Nys, R. J. Phycol. 2002, 38, 621–629. (15) Pawlik, J. R. Oceanogr. Mar. Biol. 1992, 30, 273–335.
Published on Web 05/21/2009
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chemistries are of great interest, as a number of broad spectrum biocides are currently being phased out from the market. A number of studies demonstrate how various properties, such as topography,16 chemical heterogeneity,17 or wetting18 may affect the settlement of zoospores of the green alga Ulva linza. “Settlement” is defined as the process whereby motile spores attach irreversibly to a surface, which involves rapid (within minutes) secretion of a glycoprotein adhesive and loss of the flagella, hence loss of motility.19 “Adhesion” is defined as the strength by which settled (attached) cells are bonded to the surface; adhesion strength is quantified by the ease of removal of cells when exposed to a calibrated shear stress. In a recent study,20 we sought to provide further insight into the molecular characteristics of spore settlement by studying their interaction with charged interfaces represented by surface-bound, cationic peptide self-assembled monolayers (SAMs). This was partly inspired by the antimicrobial properties of many cationic peptides, although our interest was rather in fundamental studies of settlement and settlement cues, rather than biocidal aspects. Briefly, settlement of spores was high on arginine-rich peptides, and a portion of the attached spore population was abnormal. The abnormal spores were described as “pseudosettled” because they had not gone through the normal and irreversible processes of adhesive exocytosis and loss of flagella. Pseudosettled spores retain the features of swimming spores, i.e., pyriform shape and four flagella and attach to the surface side-on, whereas normal settled spores are spherical and the flagella have been withdrawn inside the cell (Figure 1). It was clearly demonstrated that it is the presence of arginine in the SAMs that causes the attachment of pseudosettled spores, and that an increase in arginine content caused an increase in the total number of settled and pseudosettled spores and an increase in cell death. The abnormal effects were not observed on lysine-containing SAMs, which suggest that pseudosettlement cannot be attributed solely to electrostatic interaction between the anionic cell membrane and cationic peptides forming the SAMs. Even so, it is not clear from available data whether the strong interaction and/or anomalous settlement is a general membrane-surface interaction, or whether the peptide interacts with a specific component in the phospholipid surface membrane. We have proceeded to further investigate this interaction, and in this paper we explore the influence of arginine on attachment of zoospores of Ulva by using peptide SAMs that comprise arginine residues in different numbers and in different configurations. We also compare the settlement of spores of Ulva with that of a diatom, Navicula perminuta, a unicellular alga commonly occurring in the slimes found on immersed surfaces, especially on hydrophobic fouling-release coatings. While spores of Ulva are surrounded only by a naked lipoprotein plasma membrane, i.e., there is no discrete polysaccharide cell wall, diatoms are surrounded by a silica cell wall as well as extracellular mucilages,21 which prevents direct contact of the plasma membrane to the SAM surfaces. The peptides used in this study are divided in three subgroups (see Table 1), in which the first group consists of the peptides used (16) Schumacher, J. F.; Carman, M. L.; Estes, T. G.; Feinberg, A. W.; Wilson, L. H.; Callow, M. E.; Callow, J. A.; Finlay, J. A.; Brennan, A. B. Biofouling 2007, 23, 55–62. (17) Finlay, J. A.; Krishnan, S.; Callow, M. E.; Callow, J. A.; Dong, R. Asgill, N.; Wong, K.; Kramer, E. J.; Ober, C. K. Langmuir 2008, 24, 503–510. (18) Finlay, J. A.; Callow, M. E.; Ista, L. K.; Lopez, G. P.; Callow, J. A. Integr. Comp. Biol. 2002, 42, 1116–1122. (19) Callow, J. A.; Callow, M. E. The Ulva spore adhesive system. In Biological Adhesives; Smith, A. M., Callow, J. A., Eds.; Springer: Berlin, 2006; pp 63-78. :: (20) Ederth, T.; Nygren, P.; Pettitt, M. E.; Ostblom, M.; Du, C.-X.; Broo, K.; Callow, M. E.; Callow, J.; Liedberg, B. Biofouling 2008, 24, 303–312. (21) Molino, P. J.; Wetherbee, R. Biofouling 2008, 24, 365–379.
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Figure 1. Fluorescence images of zoospores of Ulva linza settled on (a) an acid-washed glass (AWG) surface and (b) an arginineand tyrosine-containing peptide (R(YR)3 SAM, see Table 1). Spores fluoresce red due to autofluorescence of chlorophyll contained in the chloroplast. Differences both in quantity and morphology of the spores settled on the arginine-rich SAM can be seen. Note the high proportion of pyriform (pseudosettled) spores in panel b; normal settled spores are round. Scale bars in panels a and b correspond to 20 μm. The inset in panel a shows a higher magnification image of a normal settled spore; the inset in b shows a pseudosettled spore, characterized by its pyriform shape and four flagella. Spores in the inset images were stained with fluorescein diacetate (FITC) and imaged with an epifluorescence microscope with FITC filters. The orange color corresponds to the autofluorescence of chlorophyll contained in the chloroplast at the base of the spore. Bar in both insets = 5 μm.
in a previous study,20 and the other two groups are the peptides specifically designed for the present study. In Group 2, the thickness of the peptide SAM is altered by varying the number of alternating arginine (R) and tyrosine (Y) residues, resulting in SAMs of different thicknesses, but ideally with arginine amino acids exposed at the interface. The purpose of these is to test whether the thickness of the SAM or the number of arginine residues on each peptide is important for the interaction of Ulva zoospores with the surface, thus providing information about the possibility that the interaction is a nonspecific interaction with a SAM of a particular chemistry, or whether the primary structure of the peptide is important. In the third group, the sequence of arginine and tyrosine residues in the peptide is in different permutations, as well as the total length of the peptides. Also included is a control peptide, with a single tyrosine but no arginine residue. By alternating the primary amino acid sequences, we test the sensitivity of spore settlement to structural changes and, in particular, whether spores can respond to peptides that are not terminated by an arginine residue. If arginine residues are “hidden” in the SAM, we thus effectively probe the “sensing depth” of the spore into the SAM. In our previous study on peptide SAMs,20 we also found that the SAMs were rather disordered, and that the structure was more complex than the structure of commonly used alkylthiol SAMs, Langmuir 2009, 25(16), 9375–9383
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Table 1. Ellipsometric Thicknesses and Water Contact Angles on the Peptide SAMs label
amino acid sequencea
thickness (A˚)
advancing contact angleb
Group 1c K(GK)3 K(GYK)2 R(YR)3 spacer
CGG-KGKGKGK CGG-KGYKGYK CGG-RYRYRYR CGG
16.1 21.4 26.6 14.2
35-41 39-49 36-45 41 ( 1
15.9 19.1 23.9 26.6
19 ( 1 25 ( 2 31 ( 2 39 ( 4
19.5 18.1 25.7 28.3
21 ( 1 25 ( 1 28 ( 3 34 ( 3
18.3
49 ( 1
Group 2 R R(YR)1 R(YR)2 R(YR)3
CGG-R CGG-RYR CGG-RYRYR CGG-RYRYRYR
YR RY Y3R4 R4Y3
CGG-YR CGG-RY CGG-YYYRRRR CGG-RRRRYYY
Group 3
control Y
CGG-Y a
C = cysteine, G = glycine, K = lysine, Y = tyrosine, R = arginine. All receding contact angles are 96%) of cells on all of the test surfaces were viable. Furthermore, cell death is not elevated on R(YR)3 SAMs over that seen on either the spacer control, the lysine-containing SAMs, or the AWG reference, and neither does cell death increase as a percentage of the population over 24 h.
Discussion Surface Preparation and Characterization. In view of the great variations shown for the thickness of the peptides K(GK)3, K(GYK)2, and R(YR)3, the strong correlation between thickness, the number of amino acid residues and amide I intensities in Groups 2 and 3 is perhaps somewhat surprising. This difference may reflect the more homogeneous chemical composition of these peptides, and a greater similarity in the intermolecular interactions during SAM formation. For the peptides in Group 2, it is clear from Table 1 and Figure 2 that the thicknesses of the peptides in the series varies almost linearly with the number of amino acid residues, and that this set of peptides may be used as intended: as a probe of the effect of peptide length increments with a corresponding increase in the peptide SAM thickness. This is not obvious a priori, particularly in view of the relations between the number of amino acid residues and the thicknesses for Group 1 (see also the work of Ederth et al.).20 The difficulties in determining the advancing contact angles with accuracy on the Group 1 SAMs described in ref 20 are also apparent in Groups 2 and 3, but not as pronounced. The shorter peptides and the peptides with similar amino acids grouped together suffer from these problems to a lesser extent than those in Group 1. The wetting data show that the lowest and highest contact angles are obtained on the SAMs with the shortest peptides, R and Y, with a single arginine and tyrosine residue, respectively. This observation is useful for a qualitative assessment of the order in the other peptide SAMs. That the contact angles of the longer SAMs fall between those of the Y and R peptides indicates that there is mixing of arginine and tyrosine among the exposed residues, that is, the SAMs formed from the longer peptides are disordered, and that not only the terminating residues are exposed at the interface. This is also clear from the trend in the contact angle data in Group 2, where all peptides are arginine-terminated, but the contact angle increases with increasing peptide length, indicating that a larger fraction of tyrosines is exposed at the interface as the length increases, an effect of increasing disorder with increasing peptide length. Considering the bulkiness of the longer molecules, it seems to be a fair assumption that the shortest molecules form more densely packed monolayers. This view is supported by both the ellipsometry and the IRAS data; if the packing density of all peptides were equal, the measured thicknesses and the amide I IRAS intensities would be directly proportional to the number of amino acids; although the relations are linear, the increases are not directly proportional to the number of peptides, in that the shorter peptides have both disproportionally large IR intensities and thicknesses. The linear fit of the change in thickness of the SAMs of the Group 2 peptides in Figure 2 shows that the increase is indeed linear with reasonable accuracy, but the line does not extrapolate to zero thickness at zero amino acid residues, meaning that the thickness increases more slowly than the number of residues. Also, the dotted line in Figure 2 shows the extended length of the peptides, assuming that the length per amide unit in the chain is 3.6 A˚. The observed thicknesses for the shortest peptides lie near this line, indicating that these peptides have a near-upright orientation, but those with eight and ten amino acids fall clearly below the line, and are thus either tilted or with kinks Langmuir 2009, 25(16), 9375–9383
along the chains. A very similar result is obtained if the amide I intensities are plotted versus the number of amino acid residues (not shown). This can only be accomplished if each length increment is accompanied by a decrease in packing density, though this decrease has to be small enough to permit an increase in the layer thickness. Although the intensities of the amide bands are ambiguous measures of surface density, since increased peak intensity may also be obtained via conformational changes (which may be difficult to separate from true variations in surface density), the excellent agreement between the amide I intensities and the thicknesses (Figure 4) is convincing in this case. Within Group 3, we note that the IRAS data for RY and YR are very similar, indicating a conformal similarity of these peptides. The grouping of arginine and tyrosine residues in the R4Y3 and Y3R4 peptides does affect the tyrosine ring stretching at 1515 cm-1, with the R(YR)3 as an intermediate. The reduction in intensity for R4Y3 suggests that the tyrosine rings orient to a conformation more parallel to the surface plane in this case, perhaps as a result of increasing mobility of residues at the terminal end of the peptide in the SAM. Settlement of Spores of Ulva. The data in this report support the conclusion in our previous paper20 that it is specifically the presence of the arginine amino acid in the peptides that induces the attachment of pseudosettled spores to the SAMs. We further conclude that increasing peptide length induces higher numbers of attached spores, irrespective of the peptide primary structure, within both Group 2 and Group 3. This is partly explained by increasing disorder as the peptide length increases, exposing residues further from the terminating end, but may also be attributed to a real depth sensitivity of the spores to what is beneath the outermost part of the SAM; this is supported by the increasing settlement in the series R(YR)1-R(YR)3, in which the average composition of the surface should be more or less constant, and from the higher settlement on R4Y3 than on YR, where the former is terminated by tyrosines and the latter with arginine. Considering the data in both Figure 5 and Figure 6, the fraction of pseudosettled spores is much more sensitive to the length of the peptides than is the total number of spores attached, from which we suggest that the mechanism(s) causing pseudosettlement has a higher “depth sensitivity” than the mechanism(s) attracting the spores to the surface. That the configuration of the peptides influence both the attachment of normal and pseudosettled spores is clear from the results in Group 3. When the arginine residues are located closer to the substrate and the tyrosine residues more exposed, as in RY and R4Y3, attachment of both normal and pseudosettled spores is lower than with the reverse configuration (YR and Y3R4, respectively), further demonstrating the particular effect of arginines for pseudosettlement. The smaller differences between the RY and YR peptides, as compared to the differences between R4Y3 and Y3R4, also emphasize the impact of configuration. For spores of Ulva, there is a trend toward increased settlement with increasing substrate hydrophobicity,26,27 and while it is true that the increases in settlement observed within both Groups 2 and 3 are accompanied by decreasing wettabilities, it seems unlikely that the observed variations in the attachment of normally settled spores and pseudosettled spores are related to varying wettability, since the control peptide, Y, has the lowest wettability, yet the settlement density is lowest, and the fraction of abnormal pseudosettled spores is zero. (26) Callow, M. E.; Callow, J. A.; Ista, L. K.; Coleman, S. E.; Nolasco, A. C.; Lopez, G. P. Appl. Environ. Microbiol. 2000, 66, 3249–3254. (27) Schilp, S.; Kueller, A.; Rosenhahn, A.; Grunze, M.; Pettitt, M. E.; Callow, M. E.; Callow, J. A. Biointerphases 2007, 2, 143–150.
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Settlement Dynamics of Spores of Ulva on R(YR)3 Peptide SAMs. The drop in pseudosettled spore numbers (both in absolute numbers and as a fraction of the total number of settled spores) with increasing settlement time is unlikely to result from spore lysis, (which is known to occur with prolonged exposure to the R(YR)3 peptide), as lysed spores would still be visible on the surface (albeit with nonentire margins and reduced fluorescence). It is possible that abnormally attached spores may “recover”, going through the remainder of the normal settlement stages, exuding their adhesive and rounding up. There is also the possibility that some of the attached abnormal pseudosettled spores are not damaged and are able to break free from the surface and swim away, although we consider this less likely. Spores, which are negatively charged,28 may initially be attracted to the (presumably) positively charged surface, and both of the scenarios described above would be possible if the positive charge decreased over time, thereby enabling the “attached” spores to break free and/or complete the settlement process. Since the plasma membrane surrounds the flagella as well as the spore body, “attachment” of abnormal spores via one or more flagella, allowing movement of the spore body, would be a possibility. It is hypothesized that the positive charge of the arginine-rich peptides induces the attachment of pseudosettled spores via electrostatic interaction with the negatively charged membrane, although, since the Debye length at a charged interface is less than 3 A˚ at the electrolyte concentration of ASW, the effect of the charged SAMs would be noticeable only within the nearest few nanometers from the surface, and consequently a spore must be extremely close to the surface for it to be captured via an electrostatic interaction. Data suggest that the attachment of pseudosettled spores to a surface is a very rapid process, but since the fraction of pseudosettled spores decreases with time, it means that, for longer durations of spore exposure, spore settlement data comprised of normal and pseudosettled spores may be influenced by a number of factors which remain to be investigated: • Change in surface properties of the SAM brought about by ASW “conditioning” the surface. • Recovery or detachment of pseudosettled spores. • Promotion of spore settlement by the high density of normal and pseudosettled spores already attached to the SAMs. The variation in both total settlement density and the fraction of pseudosettled spores between the data in Figure 5 and Figure 6 on one hand, and the data in Figure 7 on the other, is a normal variation between assays. Although the quantitative results vary between experiments, which is expected for “wild” material, the qualitative results and the trends for spore settlement on different SAMs remain similar. Results with Navicula. The results of the assays with Navicula suggest that cells of N. perminuta are unaffected by the presence of arginine in the SAMs; adhesion strength (cell removal on exposure to shear) is not altered, and cell death is not elevated. This is an interesting finding when considered in relation to the outcome for Ulva. Since spores of Ulva are naked, i.e., lack a cell wall, the lipoprotein cell membrane of the spore contacts the surface directly. This is not the case for diatoms, where the cell membrane is separated from the surface by a cell wall (“the frustule”, an elaborate silica covering) and extracellular polymeric substances (EPS). The membrane is therefore distanced (28) Rosenhahn, A.; Finlay, J. A.; Pettitt, M. E.; Ward, A.; Wirges, W.; Gerhard, R.; Callow, M. E.; Grunze, M.; Callow, J. A. Biointerphases 2009, 4, 7–11.
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from intimate contact with any surface to which the cell attaches. This finding lends support to the hypothesis that the effects of R(YR)3 on spores of Ulva are mediated by a specific interaction with the cell membrane. Electrophoretic mobility measurements of diatoms show that they are negatively charged at the pH of ASW, i.e., about 8.2,29,30 which makes the quantitative similarity between the removal of diatoms from AWG and from the cationic peptides an interesting observation, since the glass surface is negatively charged and the peptides are positively charged. This points to a relatively small contribution from direct electrostatic interactions to the adhesive forces between the test surfaces and the diatom cells, perhaps in favor of other interactions mediated by the EPS. The number of cells attached is markedly lower on the spacer SAM, than on the cationic peptides. As indicated above, this results from weakly adhered cells being removed during the gentle washing procedure. This is consistent with the fact that cell removal by the shear flow is highest from the spacer SAM, and thus cells of N. perminuta are the least strongly adhered to this SAM. It is not known which physicochemical characteristic(s) of this SAM make it unfavorable for diatom adhesion. Diatom attachment strength is broadly related to wettability, being lower on hydrophilic surfaces.27 However, differences in surface wettability cannot explain the lower adhesion to the spacer. The proportion of nonviable cells appears to be somewhat higher on the surface of the spacer peptide, and may arise from additional stress caused by the redistribution of cells during slide dewetting (other experiments have shown that diatom cells die following a very short exposure to air caused by dewetting of hydrophobic surfaces).31 Taken together, the results suggest that the R(YR)3 peptide, even on prolonged contact, does not cause lysis of the diatom cell in the same way it does with spores of Ulva.
Summary and Conclusions On the basis of the findings in a previous study, where we observed strong interactions between zoospores of Ulva linza and a cationic arginine-rich SAM, both in terms of the quantity and the mode of settlement, we have designed a set of peptides to further test the interaction of spores with these peptide SAMs. The peptides were designed to provide (a) a series of SAMs with increasing thickness, but with similar chemical composition at the interface, and (b) a series where the order of the amino acids had been permuted, to investigate the effect of peptide structure on settlement (attachment) of spores. Taken together, the ellipsometry, wetting, and IRAS data indicate that the set of de novo designed peptides form SAMs whose thicknesses vary in proportion to the length of the peptides, but also that the packing density and the degree of order in the SAMs decrease with increasing peptide length, although these effects do not adversely affect the usefulness of the peptides for the intended purpose. Spore settlement data on the peptide SAMs confirm the importance of arginine residues for the attachment of abnormal, pseudosettled spores: the thickness of the SAM affects both the attachment of pseudosettled spores and the settlement of normal spores, but that the former appears to be more sensitive to variations in peptide length. Further, the peptide primary structure for peptides with the same overall amino acids modulates the results, but the peptide primary structure is of relatively (29) Gelabert, A.; Pokrovsky, O. S.; Schott, J.; Boudou, A.; Feurtet-Mazel, A.; Mielczarski, J.; Mielczarski, E.; Mesmer-Dudons, N.; Spalla, O. Geochim. Cosmochim. Acta 2004, 68, 4039–4058. (30) Richmond, D. V.; Fisher, D. J. Adv. Microb. Physiol. 1973, 9, 1–29. (31) Thompson, S. E. M.; Taylor, A. R.; Brownlee, C.; Callow, M. E.; Callow, J. A. J. Phycol. 2008, 44, 967–976.
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lesser importance for the total settlement density than for the attachment of pseudosettled spores. Throughout, SAMs formed from peptides with terminating arginine residues have higher settlement of spores and a larger fraction of pseudosettled spores than peptides with the same number of amino acid residues but with the arginines farther from the surface. The kinetics of settlement of Ulva spores was studied with respect to the proportion of abnormally attached spores on one of the arginine-rich SAMs, the R(YR)3 peptide, and it was found that the proportion of pseudosettled spores decreased with increasing duration of exposure to the spore suspension, both in relative terms and in absolute numbers. We do not know the reason for this, but recovery to a normal settled morphology, and spores “breaking-free” and swimming away into the seawater from the peptide remain possibilities. The attachment (settlement) and adhesion strength of cells of Navicula in relation to different lysine- and arginine-containing
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cationic peptide SAMs are remarkably similar, and also agree with the values obtained on an AWG control, suggesting that direct electrostatic interactions play a minor role in the interaction with the substrates. Furthermore, it appears that a requirement for the strong interaction between the argininerich SAM and spores of Ulva is access to the cell membrane, which is not possible with diatoms. The reduced adhesion strength of cells of N. perminuta on the spacer (glycine-exposing) SAM is of note, although its cause and significance remain uncertain. Acknowledgment. The authors acknowledge support from the AMBIO project (NMP-CT-2005-011827) funded by the European Commission’s Sixth Framework Programme. The views expressed in this publication reflect only those of the authors, and the Commission is not liable for any use that may be made of the information contained therein. We thank Louise Stone for technical help with the biological assays.
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