![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Applied and Environmental Microbiology, August 2000, p. 3249-3254, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Use of Self-Assembled Monolayers of Different
Wettabilities To Study Surface Selection and Primary Adhesion Processes
of Green Algal (Enteromorpha) Zoospores
Maureen E.
Callow,1,*
J. A.
Callow,1
Linnea K.
Ista,2
Sarah E.
Coleman,2
Aleece C.
Nolasco,2 and
Gabriel
P.
López2
School of Biosciences, The University of
Birmingham, Edgbaston, Birmingham B15 2TT, United
Kingdom,1 and Department of Chemical and
Nuclear Engineering, The University of New Mexico, Albuquerque, New
Mexico 871312
Received 30 March 2000/Accepted 16 May 2000
 |
ABSTRACT |
We investigated surface selection and adhesion of motile zoospores
of a green, macrofouling alga (Enteromorpha) to
self-assembled monolayers (SAMs) having a range of wettabilities. The
SAMs were formed from alkyl thiols terminated with methyl
(CH3) or hydroxyl (OH) groups or mixtures of
CH3- and OH-terminated alkyl thiols and were characterized
by measuring the advancing contact angles and by X-ray photoelectron
spectroscopy. There was a positive correlation between the number of
spores that attached to the SAMs and increasing contact angle
(hydrophobicity). Moreover, the sizes of the spore groups (adjacent
spores touching) were larger on the hydrophobic SAMs. Video microscopy
of a patterned arrangement of SAMs showed that more zoospores were
engaged in swimming and "searching" above the hydrophobic sectors
than above the hydrophilic sectors, suggesting that the cells were able
to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of
microorganisms to substrata having different wettabilities.
| |
INTRODUCTION |
Enteromorpha spp. are
commonly found throughout the world in the upper intertidal regions of
shores and estuaries and are the most common macroalgae that foul
man-made structures, including boats, buoys, ships, and submarines
(6, 7). Colonization of substrata occurs mainly through the
production and release into the water column of enormous numbers of
motile spores, which may be either asexual zoospores or zygotes formed
from fusion of sexual gametes (11). Zoospores are cells that
are quadriflagellate, naked (i.e., they lack a cell wall), and
pyriform, and the spore body is 7 to 10 µm long. The critical event
involved in colonization of substrata is the transition from a motile
cell to an attached, nonmotile settled cell that develops a cell wall
and germinates to produce a new plant.
Prior to permanent adhesion, a swimming spore exhibits characteristic
presettlement behavior that involves a change from random swimming to a
"searching" pattern of exploration close to the substratum
(11). During the searching phase, the spore appears to
become temporarily attached to the substratum as it spins like a top on
its apical dome, with the flagella acting as propellers. During
spinning, a pad of elastic material is sometimes extruded, and this pad
is left behind on the surface if the spore continues to search for a
suitable place on which to settle. Once a suitable area for settlement
is located, the spore commits itself to permanent adhesion through
rapid secretion of an N-linked, polydisperse, self-aggregating
glycoprotein (Mr under reducing, denaturing
conditions, 110,000) that anchors the spore to the substratum (5,
29).
A number of cues are involved in surface localization by zoospores,
including negative phototaxis (10), thigmotaxis
(18), and chemotaxis (9). The presence of a
microbial biofilm is also important in determining the number of spores
that attach to a surface (14; I. Joint, M. E. Callow, J. A. Callow, and K. R. Clark, submitted for
publication), possibly through the release of chemical signals and/or
modification of the topography or physicochemical properties of the substratum.
In this study we examined the role of surface wettability in zoospore
adhesion. Although there have been a number of reports in which the
authors have related microbial attachment to surface wettability
(1, 16-18, 28), there have been none on algae which employ
surfaces that are fully characterized and vary systematically with
respect to wettability. The substrata used in the present study were
self-assembled monolayers (SAMs) of 
-substituted alkane thiolates on
gold (3). SAM technology permits construction of surfaces
that are chemically defined and uniform with respect to surface
morphology and can present a variety of chemical functional groups.
Another aspect of our study was the use of patterned SAMs which allowed
the zoospores a "choice" of surfaces with different wettabilities.
SAMs have been used previously to study the effects of hydrophobicity
(19, 32), chemistry (19), and surface topography
(33) on bacterial attachment, as well as for a number of
studies on the effects on substratum physicochemistry on adsorption of
proteins and mammalian cells (22, 24, 25, 27). The SAMs used
in this study were formed from alkyl thiols terminated with methyl
groups (CH3) or hydroxyl groups (OH) or mixtures of CH3- and OH-terminated alkyl thiols that resulted in a
range of surface wettabilities.
| |
MATERIALS AND METHODS |
Preparation and transportation of SAMs.
SAMs were prepared
at the University of New Mexico on gold-coated coverslips (22 by 50 by
0.25 mm) or regular glass microscope slides (VWR Scientific). The glass
supports were cleaned by immersion in a solution prepared by mixing
70% (vol/vol) concentrated H2SO4 with 30%
commercial H2O2 (piranha etch) for 20 min to
1 h, thoroughly rinsed in deionized H2O, and dried
under a stream of nitrogen. Note that the piranha etch solution is a
powerful oxidizer, can react violently when it is placed in contact
with organic compounds, and should be stored in containers which
prevent pressure buildup. The samples were then placed into the chamber
of a metal evaporator. The system was evacuated to a pressure of
10
6 torr, and 10 Å of chromium and then 300 Å of gold
were deposited on the substrata. The system was then restored to room
pressure, and the samples were removed and submerged in 1 mM ethanolic
solutions of dodecane thiol (referred to below as CH3-thiol
and obtained from Aldrich Chemical), mercaptoundecanol (OH-thiol;
Aldrich Chemical), or a mixture of CH3- and OH-thiols. The
samples were immersed in the thiol solutions overnight at 4°C. The
SAMs remained in the thiol solutions at 4°C until they were shipped,
at which time they were rinsed in ethanol and dried under a stream of
N2. The resulting surfaces (i.e., the -terminated alkane
thiolates) are referred to below as CH3-SAMs and OH-SAMs.
Patterned SAMs were produced by serial electrochemical desorption and
reformation of the SAMs as previously described (31). A SAM
was formed on gold with CH3-thiol. A laser ablation system consisting of a Nikon Diaphot 300 inverted microscope adapted with a
computer-controlled, pulsed-nitrogen pumped laser (
, 390 nm; 15 µJ/pulse; 20 pulses/s) was used to cut lines in the gold film
(prepared as described above) to form electrically isolated regions in
the film. The UV laser beam was focused through a ×10 objective on the
microscope, and it ablated the gold and the SAM, which generated lines
of exposed glass that were approximately 15 µm wide. The slide was
then placed in 0.5 M ethanolic KOH, and an anode was connected to one
element. A cyclic current was then applied (
1.0 to 1.5 V versus
Ag/AgCl; 500 mV s1) to the element for six cycles.
Desorption of the CH3-SAM from the surface was monitored by
cyclic voltammetry to ensure complete removal of the SAM. The exposed
gold was then treated with a 10 mM ethanolic solution of the desired
-substituted alkane thiol for 20 min. A series of elements could
thus be addressed sequentially, which resulted in a pattern consisting
of different SAMs on a single surface.
For transportation, the SAMs were removed from the thiol solutions,
dried, and placed in plastic coverslip or slide boxes. The boxes were
then put into a plastic desiccator that was subsequently evacuated and
then flooded with N2. This cycle was repeated three times,
and after the final N2 purge, the chamber was evacuated and
sealed. All seams and orifices were then sealed with Parafilm, and the
desiccator was placed in a package. The package was sent to the
University of Birmingham, Birmingham, United Kingdom, via overnight delivery.
Surface characterization of SAMs.
Samples were tested both
before and after shipment to ensure that the integrity of the samples
was maintained during shipping. Advancing water contact angles
(
AW) were measured both immediately before packing and
upon receipt.
X-ray photoelectron spectroscopy was used to determine the surface
compositions of mixed monolayers. Samples were analyzed with a model
SSX-100 spectrometer (Surface Science Instruments, Mountain View,
Calif.) at the National ESCA and Surface Analysis Center for Biomedical
Problems at the University of Washington. Using this system, workers
analyzed an elliptical area whose short axis was adjusted so that it
was 1,000 µm long. An A1 K
1,2 monochromatized X-ray
source (h
, 1,486.6 eV) was used to stimulate photoemission. The
energy of the emitted photoelectrons was measured with a hemispherical analyzer. Survey scans for binding energies ranging from 0 to 1,000 eV
(with a pass energy of 150 eV) were performed to examine the elemental
compositions of the surfaces. At this pass energy, the transmission
function of the spectrometer can be assumed to be constant (Surface
Science Instruments). Peak areas were normalized by the number of
scans, the number of points per electron volt, the Scofield
photoemission cross sections (26), and the sampling depth.
X-ray photoelectron spectroscopy data were acquired at a photoelectron
take-off angle of 55°; the take-off angle was defined as the angle
between the surface normal and the axis of the analyzer lens.
High-resolution scans were also recorded at a pass energy of 50 eV.
Peak positions were assigned by referencing the hydrocarbon
(CHx) peak to 285.0 eV.
Plant material.
Fertile plants of Enteromorpha
linza were collected from Wembury Beach, United Kingdom
(50°18'N, 4°02'W). Zoospores were released and prepared for
attachment experiments as described previously (11).
Zoospore adhesion assays.
Zoospore suspensions were
standardized as described previously (11). The concentration
of spores was adjusted to 106 spores ml1 by
using natural seawater unless otherwise stated. Coverslips or
microscope slides were incubated with spore suspensions in the dark at
20°C. Substrata were washed in seawater before they were fixed in 2%
glutaraldehyde in seawater for 10 min and then washed as described
previously (11).
Time course of zoospore adhesion.
Glass coverslips coated
with SAMs generated by using either CH3- or OH-thiol
solutions were placed individually into 5-cm-diameter Sterilin petri
dishes to which 5-ml portions of spore suspensions were added. The
AW of the CH3- and OH-thiol surfaces were
116° and <15°, respectively. New ethanol-washed glass coverslips,
as supplied by the manufacturer (VWR), were used to compare results obtained with SAMs to the results of previous experiments
(11). Dishes were incubated for 20, 40, or 60 min before the
coverslips were removed and processed as described above. Three
replicates were used for each treatment. Attached spores were counted
in 10 fields of view, located at 1-mm intervals across the midpoint of
each of the three replicate coverslips. The mean ±95% confidence limit for 30 counts per mm2 of surface was calculated.
Zoospore adhesion to SAMs formed from mixtures of
CH3- and OH-thiols. (i) SAM-coated coverslips.
Four
coverslips containing each type of SAM were shipped as described above.
We used SAMs that were formed with different solution molar fractions
of OH-thiol (
OHsol), where
SAMs were formed from mixed thiol solutions having
OHsol of 0, 0.2, 0.45, 0.50, 0.55, 0.65, 0.70, 0.80, 0.90, and 1. Three replicate coverslips were used for the
zoospore adhesion assay. The remaining coverslip was used to determine
the contact angle. In all cases, except for the 100% OH-terminated
SAM, the contact angle was found to be ±20% of the contact angle
recorded prior to shipping. In the discussion of AW
below we refer to the measurements obtained at the University of New Mexico.
(ii) Patterned SAMs.
A microscope slide (76 by 26 mm) having
11 SAMs along the length of the long axis (each area, 5 by 15 mm) was
placed in a compartment of a polystyrene culture dish (In Vitro Systems
& Services, GmbH), and 10 ml of a spore suspension was added. After the
preparations were processed as described above, spores were counted in
20 fields of view through the midpoint of the long axis at 0.5-mm
intervals of each SAM by using analySIS software and a personal
computer connected to an Olympus model BH2 microscope equipped with a
video camera. The number of spores that were in groups (i.e., touching;
1 to 15 spores) was also recorded. Data are presented below for the
mean numbers of spores that adhered ±95% confidence limits (x = 20) and also for the percentages of total spores that were present in
groups. For clarity, the percentages of cells found in groups were
calculated by combining group sizes as follows: 1, 2 + 3, 4 + 5, 6 + 7, 8 + 9, 10 + 11, 12 + 13, and 14 + 15.
Zoospore swimming behavior assays.
In order to investigate
whether the pattern of spore swimming and searching behavior was
affected by the properties of substrata, a video analysis of zoospore
behavior was conducted by using a pattern consisting of SAMs in close
spatial proximity, which provided spores with a choice of substrata
having different wettabilities. For technical reasons, this part of the
investigation was conducted at the University of Melbourne, Melbourne,
Australia. The pattern was formed on a standard microscope slide and
consisted of a square (16 by 16 mm) that was divided into four sectors,
each of which was 8 by 8 mm. At the midpoint were corners of four
sectors bearing mixed-component SAMs formed from solutions with
OHsol values of 0.2, 0.4, 0.6, and 0.8. Adjacent SAMs were separated by approximately 15 µm of bare glass.
The contact angles of the sectors before dispatch to Australia were
100°, 96°, 64°, and 42°, respectively.
The slide was placed in a 9-cm-diameter petri dish to which 25 ml of a
spore suspension (2 × 106 spores ml1)
was added. Zoospores were released from E. linza collected
from Port Melbourne, Victoria, Australia (37°50'S, 144°55'E). The
dish was positioned on the stage of a Zeiss Universal microscope under a Plan 2.5/0.08 objective; the apparatus was set up in a darkroom to
prevent phototactic spore movements. Sequences were filmed by using a
Panasonic model WV-F250E color video camera and were recorded with a
Pioneer model V1000p rewritable video disc recorder. After 10 min the
microscope was focused on a plane just above the slide surface so that
both swimming and settled spores could be observed. Time-lapse images
were then collected every 3 min for 60 min by using a Genesis Systems
model Z84C-V1000P VDR timer controller and a Uniblitz model T132
shutter driver controller. Single images were captured on a Targa 2000 Pro video card. At the end of the filming (a total of 70 min after
spores were put into the dish), the number of spores that were firmly
attached to each sector was assessed by recording images of the slide
after unsettled spores in seawater were washed away. Finally, as a
check to ensure that the SAMs sent to Australia were performing like those sent to Birmingham, the SAM slide was fixed and processed by
using the standard cell counting procedure described above.
Spores were counted by using three consecutive video images
representing each time point (i.e., 10, 13, and 16 min after the spore
suspension was added [mean 13 min]; 37, 40, and 43 min after the
spore suspension was added) [mean, 40 min]; and 64, 67, and 70 min
after the spore suspension was added [mean, 67 min]). Spores were
counted in each sector within an area (0.45 by 0.45 mm) adjacent to the
midpoint of the four SAM sectors. The mean number of attached spores
per square millimeter ±95% confidence limits was calculated for each
group of three images. Attached spores were counted by using the single
video image after the slide was washed.
Settled spores on the fixed slide were counted in 20 fields of view
located at 100-µm intervals along the diagonal of each square,
starting at the central corner. The mean number of attached spores per
square millimeter ±95% confidence limits was calculated.
| |
RESULTS |
Surface analysis of SAMs.
Figure
1A shows AW of SAMs as a
function of the OHsol. We chose a series of
solution mole fractions so that a range of AW from 20°
to 110° was obtained with intervals of ~10° between consecutive samples. The mole fractions of OH-terminated alkyl thiolates in the
SAMs (OHsurf) were calculated by using the
O1S peak area as described elsewhere (4, 32). A
trace of contaminating oxygen was observed with the 100%
CH3-SAM (atomic percentage, 3.1). The relationship between OHsurf and OHsol
is shown in Fig. 1B. The error of analysis for the instrument used was
~10% (15).
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 1.
Surface chemical properties of mixed SAMs. (A)
AW of mixed SAMs formed from OH-thiol and
CH3-thiol. The data are averages for three test areas. The
error bars indicate one standard deviation. (B)
OHsurf in mixed SAMs as a function of the
OHsol used to form the mixed SAMs.
|
|
Time courses of spore attachment to OH- and
CH3-SAMs.
The time courses for spore attachment to OH-
and CH3-SAMs on glass showed that the rate of attachment
was highest on the CH3-SAM and lowest on the OH-SAM (Fig.
2). After 1 h of incubation there were approximately 2.5 times more spores attached to the
CH3-SAM than to the OH-SAM. The glass control, which had an
intermediate contact angle (AW, ~40°), exhibited
intermediate levels of zoospore attachment.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 2.
Time course for zoospore adhesion to glass
(AW, ~40°), a CH3-terminated (methyl)
SAM (AW, 116°), and a OH-terminated (hydroxyl) SAM
(AW, <15°). Each point is a mean based on 30 counts;
the bars indicate 95% confidence limits.
|
|
Attachment to SAMs with different proportions of surface hydroxyl
and methyl groups.
Figure 3 shows
the total number of attached spores after 1 h as a function of
OHsurf. Both the AW and the
number of spores attached decreased as the
OHsurf increased and the surface became more
wettable (i.e., hydrophilic). The most pronounced response of spore
settlement to wettability was observed for the contact angle range from
40 to 80°, corresponding to a OHsurf range
of 0.45 to 0.80.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 3.
Zoospore adhesion to and AW of SAMs
plotted versus the OHsurf in mixed SAMs. The
mean number of zoospores attached after 1 h was derived from 30 counts.
|
|
A similar relationship between the total number of spores and
wettability was observed with the patterned SAMs (Fig.
4A). Figure 4B shows that the majority of
spores were attached as single spores or in small groups (maximum
number of spores per group, 5 or 6) on the more hydrophilic SAMs
(AW, 
80°). On surfaces with AW of
>80°, the proportion of spores in larger groups increased as the
surfaces become less wettable (i.e., more hydrophobic) (Fig.
5). On the most hydrophobic surface
(AW, 110°), only 25% of the total spores were present
as single spores, and the majority of the spores were aggregated into
groups containing up to 15 spores (Fig. 4B).
View larger version (55K):
[in this window]
[in a new window]
|
FIG. 4.
Zoospore adhesion after 1 h of settling versus
AW for patterned SAMs. (A) Mean number of zoospores
attached in each sector of the pattern deposited on a microscope slide.
Each point is a mean based on 20 counts for each sector; the bars
indicate 95% confidence limits. (B) Percentages of total zoospores in
groups of various sizes.
|
|
Spore behavior on SAMs as revealed by video microscopy.
Counts
of motile spores could not be obtained until the majority of the spores
were in the same focal plane; thus, the sample was left for 10 min
before the microscope was focused on the plane of the surface. The
subsequent spore counts obtained from the video images thus represented
both swimming and settled spores. Figure
6 shows that the distribution of spores
associated with the four sectors was not random; there was a negative
correlation between spore number and increasing content of
OH-terminated groups (i.e., increasing hydrophilicity). The highest
number of spores associated with all sectors was recorded at 10 min. At
all time points the hydrophobic sectors had more spores associated with them than the hydrophilic sectors. The order of response was as follows: AW of 101° >AW of 96° > AW of 64° > AW of 42°. The numbers
of adherent spores counted in the different sectors of the slide washed
after 70 min revealed the same correlation between attachment and
wettability seen in other experiments. A comparison of the values for
the washed slide and the 67-min incubation showed that by this time
between 50 and 70% of the total spores counted were attached spores.
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 6.
Distribution of spores on patterned SAMs with
AW of 101°, 96°, 64°, and 42°. The counts
represent both cells that settled on the surface and swimming cells in
the same focal plane. The washed counts represent settled cells only,
which were recorded on video after the 70-min sample was washed.
|
|
Finally, the mean numbers of zoospores that adhered to each sector
after fixation (±95% confidence limits), as assessed by the
standard cell counting procedure were, 733 ± 48, 446 ± 50, 209 ± 59, and 173 ± 51 spores mm2 for
AW of 101°, 96°, 64°, and 42°, respectively.
Thus, the SAM slide used in this experiment performed in the same way
that the slides used in the other attachment assays performed.
| |
DISCUSSION |
Effect of wettability on spore attachment.
The surfaces used
in this study provided ranges of wettabilities and known chemical
compositions. The composition and surface properties of the SAMs are
comparable to those described previously (2, 4, 32). The
time course for spore adhesion to uncoated glass was similar to the
time course reported previously (8, 11). Settlement on the
hydrophobic CH3-SAM was most rapid, and the highest number
of attached spores was associated with this surface. A positive
correlation between the number of spores attached and a high
AW (i.e., hydrophobicity) was seen in all experiments.
The pronounced effect of wettability on spore attachment observed at
contact angles between 40° and 80° may be explained by thermodynamic models, such as the model proposed for bacterial adhesion
(1). In such models the free energy of microbial adhesion (
Fadh) is determined by the relationships of
the interfacial tensions between the organism and the substratum
(
BS), between the organism and the bulk liquid
(BL), and between the surface and the liquid (SL):
In studies such as this study, the only variable is
SL, which is directly related to the contact angle ()
by using Young's equation:
where SV and LV are the
substratum-vapor and liquid-vapor interfacial tensions, respectively.
Since in our experimental procedures, SV and
LV were presumably consistent, the differences in
surface energy depended only on cos. When the data from Fig. 3, for
example, are plotted as spore attachment versus cosAW, the regression of the resulting plot is nearly linear, and the regression coefficient (R2) is 0.912. Thus, the
results obtained are consistent with thermodynamic models. However,
such models were originally developed for equilibrium situations with
inert, colloidal particles, and their limitations when they are applied
to living cells that exhibit complex attachment biology have been
extensively discussed (17). Nevertheless, the model is
consistent with the results and suggests that spore attachment may be
dominated by the same forces which control adhesion of colloidal
particles and that hydrophobic interactions are important. The
favorable effects of hydrophobic surfaces on promoting spore attachment
are also demonstrated by an analysis of group size. Gregarious
settlement of spores onto glass was observed previously when high
concentrations of spores were employed (11) or when settlement occurred in the presence of detritus associated with a
microbial biofilm (10). However, in the experiments
described here, the spore concentration employed was relatively low
(106 spores ml1). On glass substrata, this
concentration would not have produced the extensive level of gregarious
settlement that was associated with the more hydrophobic SAMs (contact
angles greater than approximately 80°).
At this stage it is not possible to identify in cell biological terms
the precise point(s) in the whole spore attachment process at which
hydrophobic interactions could be important. As determined by other
researchers working on aquatic adhesion systems (reviewed in reference
16), hydrophobic interactions assist in the
displacement of water molecules from interfaces and therefore
facilitate the substratum-adhesive bonding process. On this basis,
spores which committed themselves to attachment to a hydrophilic
surface presumably were not able to form an adhesive bond and, thus,
under the experimental conditions used, could be readily detached by
the slide rinsing procedure and would not be detected as settled cells.
However, hydrophobic interactions could also be important in the
preadhesion, surface selection phase of attachment, during which
swimming spores actively probe the surface before engaging in a
spinning behavior, in which a spore rotates on its apical papilla
through a small elastic pad consisting of temporary adhesive. The spore
may then commit itself to permanent adhesion, which involves discharge
of a permanent adhesive, or it may detach and move to another site.
Displacement of water between the apical papilla and the substratum may
be assisted by hydrophobic interactions, which thus allows closer
proximity between the plasma membrane of the spore and the surface and
which may facilitate the transfer of any signals required by the spore
to trigger the release of the permanent adhesive (11).
Effect of substratum wettability on swimming spore behavior.
Support for the hypothesis that surface wettability had an effect on
the surface selection phase of settlement was obtained from the video
time-lapse studies on the accumulation of swimming spores over the
different SAM sectors. The results suggest that exploration was not
random. Between 10 and 16 min after a spore suspension was introduced
above a 2-by-2 pattern of SAMs, there were approximately three times
more spores (swimming and settled) associated with the most hydrophobic
sector than with the most hydrophilic sector. The number of spores that
settled in the first 16 min was probably no more than approximately
10% of the total (Fig. 2), so the total spore count at this time
mainly represented swimming spores. An unusual feature of these results
is that the total numbers of spores associated with the four sectors
all decreased over time. The observed decreases may have been due to a
number of factors, including a change in the surface properties of the SAMs (i.e., the hydrophobic surfaces became more hydrophilic with time
and vice versa) and the possibility that the flashes of light necessary
for time-lapse video recording caused some spores to swim away from the
area being observed as this area was subjected to the most intense illumination.
Although the results described above are consistent with the hypothesis
that surface wettability may influence attachment at the preadhesion,
surface selection stage, they could also be explained by other
mechanisms. It is known that Enteromorpha spores respond
positively to signals from previously settled spores during gregarious
settlement behavior (11), possibly because of diffusible chemical signals. Other observations have also shown that zoospores exhibit chemoattractive behavior (9, 10). It is possible, therefore, that on the more hydrophobic SAMs the attached spores provide a ready source of diffusible signals that attract more spores
to the interface compared with hydrophilic surfaces. This explanation
is also supported by the data which showed that gregarious settlement
was greater on hydrophobic surfaces.
It is becoming increasingly apparent that in aquatic bacterial systems
attachment to surfaces having different hydrophobicities proceeds by
seemingly different cellular mechanisms. Enzymatic and detergent
treatment of Vibrio proteolytica, for example, leads to
differential attachment to hydrophobic surfaces but not hydrophilic surfaces (23). Observations of attachment of the marine
bacteria Pseudomonas sp. strain NCIMB 2021 (32,
33) and Halomonas marina ATCC 25374 (L. K. Ista,
unpublished data) to SAMs have revealed that these bacteria attach to
CH3-SAMs by their cell bodies and to OH-SAMs in the polar
region. Furthermore, studies performed with H. marina and
surfaces whose hydrophobicity can be switched in response to an
environmental cue have shown that there are probably different
mechanisms for attachment to hydrophobic and hydrophilic surfaces
(20). The hydrophobicity of the substratum can even alter
the way in which cells of an individual bacterial species arrange
themselves on a surface. Dalton and colleagues have shown that the
marine organism strain SW5 aggregates as tightly packed layers on
hydrophobic surfaces and forms chains on hydrophilic surfaces
(13).
The present study, for the first time, allowed us to investigate the
effect of surface wettability on Enteromorpha zoospore settlement and primary adhesion independent of other surface
characteristics and in situations in which the spores have a choice of
surfaces. The results of several other studies have indicated that in
general, the strength of adhesion of both micro- and macroorganisms is lower on hydrophobic surfaces with low surface free energy (12, 21, 30). This property is being exploited commercially as coatings with low surface energies, such as silicone elastomers, are
now being employed to control biofouling.
| |
ACKNOWLEDGMENTS |
This work was supported by Office of Naval Research grant
N00014-96-1-0373 to J.A.C. and M.E.C. by ONR grant N00014-95-1-0901 to
G.P.L., and by NIGMS grant 5S06GM5276-04 supporting A.C.N. The National
ESCA and Surface Analysis Center for Biomedical Problems at the
University of Washington is funded through NIH grant RR01296.
We thank Ruth Perry (University of Birmingham) for technical
assistance, J. A. Finlay (University of Birmingham) for contact angle measurements, Tim Spurck (University of Melbourne, Melbourne, Australia) for assistance with video microscopy, Deborah
Leach-Scampavia (University of Washington) for providing the X-ray
photoelectron spectroscopy data, and Víctor Pérez-Luna
(University of New Mexico) for discussions concerning data interpretation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biosciences, The University of Birmingham, Edgbaston, Birmingham B15
2TT, United Kingdom. Phone: 44 121 414 5579. Fax: 44 121 414 5925. E-mail: m.e.callow{at}bham.ac.uk.
| |
REFERENCES |
| 1.
|
Absolom, D. R.,
F. V. Lamberti,
Z. Policova,
W. Zingg,
C. J. van Oss, and A. W. Neumann.
1983.
Surface thermodynamics of bacterial adhesion.
Appl. Environ. Microbiol.
46:90-97[Abstract/Free Full Text].
|
| 2.
|
Bain, C. D.,
J. Evall, and G. M. Whitesides.
1989.
Formation of monolayers by the coabsorption of thiols on gold: variations in the head group, tail group, and solvent.
J. Am. Chem. Soc.
111:7155-7164[CrossRef].
|
| 3.
|
Bain, C. D.,
E. B. Troughton,
Y.-T. Tao,
J. Evall, and G. M. Whitesides.
1989.
Formation of monolayer films by the spontaneous assembly of organic thiols from solution onto gold.
J. Am. Chem. Soc.
111:321-335.
|
| 4.
|
Bain, C. D., and G. M. Whitesides.
1988.
Formation of two-component surfaces by the spontaneous assembly of monolayers on gold from solutions containing mixtures of organic thiols.
J. Am. Chem. Soc.
110:6560-6561[CrossRef].
|
| 5.
| Callow, J. A., M. S. Stanley, R. Wetherbee,
and M. E. Callow. Cellular and molecular approaches to
understanding primary adhesion in Enteromorpha. Biofouling,
in press.
|
| 6.
|
Callow, M. E.
1986.
Fouling algae from "in-service" ships.
Bot. Mar.
29:351-357.
|
| 7.
|
Callow, M. E.
1996.
Ship-fouling: the problem and method of control.
Biodeterior. Abstr.
10:411-421.
|
| 8.
|
Callow, M. E., and J. A. Callow.
1998.
Attachment of zoospores of the fouling alga Enteromorpha in the presence of zosteric acid.
Biofouling
13:87-95.
|
| 9.
|
Callow, M. E., and J. A. Callow.
1998.
Enhanced adhesion and chemoattraction of zoospores of the fouling alga Enteromorpha to some foul-release silicone elastomers.
Biofouling
13:157-172.
|
| 10.
| Callow, M. E., and J. A. Callow.
Substratum location and zoopspore behaviour in the fouling alga,
Enteromorpha. Biofouling, in press.
|
| 11.
|
Callow, M. E.,
J. A. Callow,
J. D. Pickett-Heaps, and R. Wetherbee.
1997.
Primary adhesion of Enteromorpha (Chlorophyta, Ulvales) propagules: quantitative settlement studies and video microscopy.
J. Phycol.
33:938-947[CrossRef].
|
| 12.
|
Callow, M. E., and R. L. Fletcher.
1994.
The influence of low surface energy materials on bioadhesion: a review.
Int. Biodeterior. Biodegrad.
34:333-343[CrossRef].
|
| 13.
|
Dalton, H. M.,
L. K. Poulsen,
P. Halasz,
M. L. Angles,
A. E. Goodman, and K. C. Marshall.
1994.
Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.
J. Bacteriol.
176:6900-6902[Abstract/Free Full Text].
|
| 14.
|
Dillon, P. S.,
J. S. Maki, and R. Mitchell.
1989.
Adhesion of Enteromorpha swarmers to microbial films.
Microb. Ecol.
17:39-47.
|
| 15.
|
Favio, P.,
V. H. Perez-Luna,
T. Boland,
D. G. Castner, and B. D. Ratner.
1996.
Surface chemical composition and fibrinogen adsorption-retention of fluoropolymer films deposited from and RF glow discharge.
Plasmas Polymers
1:299-326.
|
| 16.
|
Fletcher, M.
1996.
Bacterial attachment in aquatic environments: a diversity of surfaces and adhesion strategies, p. 1-24.
In
M. Fletcher (ed.), Bacterial adhesion: molecular and ecological diversity. Wiley-Liss, New York, N.Y.
|
| 17.
|
Fletcher, M., and J. H. Pringle.
1985.
The effect of surface free energy and medium surface tension on bacterial attachment to solid surfaces.
J. Colloid Interf. Sci.
104:5-14[CrossRef].
|
| 18.
|
Fletcher, R. L., and M. E. Callow.
1992.
The settlement, attachment and establishment of marine algal spores.
Br. Phycol. J.
27:303-329.
|
| 19.
|
Ista, L. K.,
H. Fan,
O. Baca, and G. P. López.
1996.
Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment.
FEMS Microb. Lett.
142:59-63[CrossRef][Medline].
|
| 20.
|
Ista, L. K.,
V. H. Pérez-Luna, and G. P. López.
1999.
Surface-grafted, environmentally sensitive polymers for biofilm release.
Appl. Environ. Microbiol.
65:1603-1609[Abstract/Free Full Text].
|
| 21.
|
Lindner, E.
1992.
A low surface energy approach in the control of marine biofouling.
Biofouling
6:193-205.
|
| 22.
|
López, G. P.,
M. W. Albers,
S. L. Schreiber,
R. Carroll,
E. Peralta, and G. M. Whitesides.
1993.
Convenient methods for patterning the adhesion of mammalian cells to surfaces using self-assembled monolayers of alkanethiolates on gold.
J. Am. Chem. Soc.
115:5877-5878[CrossRef].
|
| 23.
|
Paul, J. H., and W. H. Jeffery.
1985.
Evidence for separate adhesion mechanisms for hydrophilic and hydrophobic surfaces in Vibrio proteolytica.
Appl. Environ. Microbiol.
50:431-437[Abstract/Free Full Text].
|
| 24.
|
Prime, K. L., and G. M. Whitesides.
1991.
Self-assembled organic monolayers: model systems for studying adsorption of proteins at surfaces.
Science
252:1164-1167[CrossRef][Medline].
|
| 25.
|
Roberts, C.,
C. S. Chen,
M. Mrksich,
V. Martichonok,
D. E. Ingber, and G. M. Whitesides.
1998.
Using mixed self-assembled monolayers presenting RGD and (EG3)OH groups to characterize long-term attachment of bovine capillary endothelium cells to surfaces.
J. Am. Chem. Soc.
120:6548-6555[CrossRef].
|
| 26.
|
Scofield, J. H.
1976.
Hartree-Slater subshell photoionization cross sections at 1254 and 1487 eV.
J. Electron Spectroscop. Relat. Phenom.
8:129-137[CrossRef].
|
| 27.
|
Sigal, G. B.,
M. Mrksich, and G. M. Whitesides.
1998.
Effect of surface wettability on the adsorption of proteins and detergents.
J. Am. Chem. Soc.
120:3464-3473[CrossRef].
|
| 28.
|
Sorongon, M. L.,
R. G. Bloodgood, and R. F. Burchard.
1991.
Hydrophobicity, adhesion, and surface exposed proteins of gliding bacteria.
Appl. Environ. Microbiol.
57:3193-3199[Abstract/Free Full Text].
|
| 29.
|
Stanley, M. S.,
M. E. Callow, and J. A. Callow.
1999.
Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha.
Planta
210:61-71[CrossRef][Medline].
|
| 30.
|
Swain, G. W.,
W. G. Nelson, and S. Preedeekanit.
1998.
The influence of biofouling adhesion and biotic disturbance on the development of fouling communities on non-toxic surfaces.
Biofouling
12:257-269.
|
| 31.
|
Tender, L. M.,
R. L. Worley,
H. Y. Fan, and G. P. López.
1996.
Electrochemical patterning of self-assembled monolayers onto microscopic arrays of gold electrodes fabricated by laser-ablation.
Langmuir
12:5515-5518[CrossRef].
|
| 32.
|
Weincek, K. M., and M. Fletcher.
1995.
Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers.
J. Bacteriol.
177:1959-1966[Abstract/Free Full Text].
|
| 33.
|
Weincek, K. M., and M. Fletcher.
1997.
Effects of substratum wettability and molecular topography on the initial adhesion of bacteria to chemically defined substrata.
Biofouling
11:293-311.
|
Applied and Environmental Microbiology, August 2000, p. 3249-3254, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Ista, L. K., Callow, M. E., Finlay, J. A., Coleman, S. E., Nolasco, A. C., Simons, R. H., Callow, J. A., Lopez, G. P.
(2004). Effect of Substratum Surface Chemistry and Surface Energy on Attachment of Marine Bacteria and Algal Spores. Appl. Environ. Microbiol.
70: 4151-4157
[Abstract]
[Full Text]
-
Finlay, J. A., Callow, M. E., Ista, L. K., Lopez, G. P., Callow, J. A.
(2002). The Influence of Surface Wettability on the Adhesion Strength of Settled Spores of the Green Alga Enteromorpha and the Diatom Amphora. Integr. Comp. Biol.
42: 1116-1122
[Abstract]
[Full Text]
-
Blomster, J., Back, S., Fewer, D. P., Kiirikki, M., Lehvo, A., Maggs, C. A., Stanhope, M. J.
(2002). Novel morphology in Enteromorpha (Ulvophyceae) forming green tides. Am. J. Bot.
89: 1756-1763
[Abstract]
[Full Text]
Top
Abstract
Introduction
![&khgr;<SUB><UP>OH</UP></SUB><SUP><UP>sol</UP></SUP>=<FR><NU>[<UP>OH − thiol</UP>]</NU><DE>[<UP>OH − thiol</UP>]+[<UP>CH</UP><SUB>3</SUB><UP> − thiol</UP>]</DE></FR>](/content/vol66/issue8/fulltext/3249/img001.gif)
