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Applied and Environmental Microbiology, November 1999, p. 5017-5022, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Protein-Mediated Adhesion of the Dissimilatory
Fe(III)-Reducing Bacterium Shewanella alga BrY to
Hydrous Ferric Oxide
Frank
Caccavo Jr.*
Department of Microbiology, University of New
Hampshire, Durham, New Hampshire 03824
Received 8 June 1999/Accepted 31 August 1999
 |
ABSTRACT |
The rate and extent of bacterial Fe(III) mineral reduction are
governed by molecular-scale interactions between the bacterial cell
surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the
adhesion of Shewanella alga BrY to hydrous ferric oxide
(HFO). Enzymatic degradation of cell surface polysaccharides had no
effect on cell adhesion to HFO. The proteolytic enzymes
Streptomyces griseus protease and chymotrypsin inhibited
the adhesion of S. alga BrY cells to HFO through catalytic
degradation of surface proteins. Trypsin inhibited S. alga
BrY adhesion solely through surface-coating effects. Protease and
chymotrypsin also mediated desorption of adhered S. alga
BrY cells from HFO while trypsin did not mediate cell desorption.
Protease removed a single peptide band that represented a protein with
an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide
bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HFO adhesion molecules.
S. alga BrY adhesion was inhibited by up to 46% when cells
were cultured at sub-MICs of chloramphenicol, suggesting that protein
synthesis is necessary for adhesion. Proteins extracted from the
surface of S. alga BrY cells inhibited adhesion to HFO by
up to 41%. A number of these proteins bound specifically to HFO,
suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.
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INTRODUCTION |
Dissimilatory iron-reducing bacteria
(DIRB) use ferric iron as a terminal electron acceptor for anaerobic
respiration and growth (13, 23, 24, 26, 29). This metabolism
greatly influences the geochemistry of anaerobic soils and sediments
and may also provide a mechanism for both intrinsic and engineered bioremediation of contaminated environments (25). Although
ferric iron is abundant in many nonsulfidogenic anaerobic environments, the estimated solubility product constant of Fe(III) at neutral pH is
10
38, which limits the concentration of soluble Fe(III)
to approximately 1018 M (20). The rate and
extent of dissimilatory Fe(III) reduction and the potential for
bioremediation by DIRB are thus limited by the bioavailability of
Fe(III) (22). A fundamental understanding of the
interactions between DIRB and insoluble Fe(III) minerals is requisite
to understanding the role of these organisms in geochemical cycling
within anaerobic soils and sediments and to the effective application
of this metabolism in bioremediation.
While a number of studies have suggested that DIRB cell contact with
insoluble Fe(III) minerals is necessary for Fe(III) mineral reduction
(1, 5, 6, 15, 21, 27, 28, 42), the mechanisms by which DIRB
adhere to Fe(III) minerals are not completely understood. In one study,
an Fe(III)-reducing Pseudomonas sp. that attached to and
corroded steel oil pipelines was shown to colonize the surface of the
steel coupons by means of exopolysaccharide excretion (30).
Scanning electron micrographs of Pseudomonas sp. strain 200 growing on hematite or goethite also showed that this organism
colonized the Fe(III) minerals by what appeared to be extracellular
polymer (1). Although both of these studies suggested that
extracellular polymers were involved in DIRB colonization of Fe(III)
minerals, neither study examined the initial adhesion of the DIRB
cells. A recent study that quantitatively examined the mechanisms of
Shewanella alga adhesion to hydrous ferric oxide (HFO)
suggested that the initial contact between DIRB and HFO was mediated by
hydrophobic interactions (7).
The purpose of this study was to identify the molecules that S. alga BrY uses to adhere to HFO. The results demonstrate that surface proteins play a significant role in this adhesion process.
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MATERIALS AND METHODS |
Culture conditions and cell preparation.
S. alga BrY
is a gram-negative rod that was isolated from anaerobic sediments of
the Great Bay estuary, New Hampshire (5, 34). S. alga BrY was grown aerobically in 100 ml of tryptic soy broth
(TSB; 30 g/liter; Difco Laboratories, Detroit, Mich.) at 28°C on a
rotary shaker at 150 rpm for 15 h. Cells were harvested by
centrifugation (5,520 × g, 4°C, 20 min) during the
late exponential-early stationary growth phase. Optimal Fe(III)
reductase activity is expressed at this stage of growth
(14). Cells were washed once in sodium bicarbonate buffer
(2.5 g/liter of distilled H2O, pH 7.0) that had been made
anaerobic by boiling and cooling under a stream of O2-free
gas containing 80% (vol/vol) N2 and 20% (vol/vol) CO2 (2-4). The washed cells were suspended in
bicarbonate buffer. In some experiments, cells were washed with and
suspended in PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)]
buffer (20 mM, pH 7.0) that had been made anaerobic by boiling and
cooling under a stream of 100% N2.
Adhesion assay.
The adhesion assay was conducted as
described previously (7). The final cell concentration in
adhesion assays ranged from 0.1 × 108 to 0.8 × 108 cells ml1. All adhesion assays were
performed in triplicate.
Oxidation and degradation of surface polysaccharides.
Washed
S. alga BrY cells suspended in PIPES buffer (final
concentration of 2.0 × 109 cells ml1)
were incubated with 
-glucuronidase (Sigma Chemical Co., St. Louis,
Mo.) (final concentration of 100 U ml1) for 60 min at 50 rpm and ambient temperature. The negative control was incubated in
buffer alone. Cells from each treatment were then harvested by
centrifugation (5,520 × g, 4°C, 20 min), washed once
in PIPES buffer, and suspended in PIPES buffer. These cells were used
to perform adhesion assays as described above.
Proteolytic enzyme experiments.
Protease from
Streptomyces griseus (type XI), chymotrypsin (type II), and
trypsin (type III-S) were obtained from Sigma Chemical Co. The effect
of these proteolytic enzymes on S. alga BrY adhesion to HFO
was examined in three separate experiments. The first experiment examined the effect of cell surface proteolysis on adhesion to HFO.
Washed S. alga BrY cells suspended in PIPES buffer (final concentration of 2.0 × 109 cells ml1)
were incubated with protease, chymotrypsin, and trypsin (final concentrations of 250 µg ml1) or protease and
chymotrypsin (final concentration of 250 µg ml1 for
each enzyme) for 60 min at 50 rpm and ambient temperature. The negative
control was incubated in buffer alone. Cells from each treatment were
then harvested by centrifugation (5,520 × g, 4°C, 20 min), washed once in PIPES buffer, and suspended in PIPES buffer. These
cells were used to perform adhesion assays as described above. The
second experiment examined adhesion to HFO in the presence of
proteolytic enzymes. Native enzymes or enzymes boiled for 1 h were
added to the tubes of adhesion buffer at final concentrations of 0, 5, 25, 50, 100, and 250 µg ml1. These tubes were mixed and
then used for adhesion assays as described above. The third experiment
examined the ability of the different proteolytic enzymes to remove
previously adhered cells. Cells were added to tubes of the adhesion
assay buffer and incubated for 1 h at 150 rpm and ambient
temperature to allow complete adhesion. The native or boiled enzymes
were then added to tubes of the adhesion assay buffer (final
concentration, 250 µg ml1). The number of unadhered
cells was determined in subsamples from each tube at intervals during a
1-h incubation. The Student t test was used to determine
whether the adherence inhibition curves for native and heat-denatured
enzyme treatments were statistically different. Differences were
considered significant when P was <0.05. Cell lysis induced
by proteolytic enzyme treatment was determined by incubating washed
cells with protease, chymotrypsin, or trypsin (final concentration of
2.5 mg ml1) at a cell density of 4 × 1010 to 5 × 1010 cells ml1
for 90 min. Samples were collected for direct counts as described above. Lysis was determined by comparing counts of cells treated with
proteolytic enzymes to counts of cells treated only with buffer.
Chloramphenicol experiments.
Serial twofold dilutions of
chloramphenicol (3.125- to 25-µg ml1 final
concentration) in TSB were inoculated with log-phase bacteria to
provide a final cell density of 107 cells ml1
and incubated at 28°C and 150 rpm for 18 h. The lowest
concentration of chloramphenicol that completely inhibited growth, as
determined by measuring the optical density of each culture at 600 nm,
was defined as the MIC (44).
A 15-h, exponential-growth-phase culture of S. alga BrY was
used to inoculate test tubes with 5.0 ml of TSB containing
chloramphenicol at final concentrations of 1/32, 1/16, and 1/8 MIC
(0.195, 0.391, and 0.781 µg ml1, respectively).
Controls contained no chloramphenicol and chloramphenicol at the MIC
(6.25 µg ml1). The final cell density in each culture
was 1.73 × 107 cells ml1. The cultures
were incubated at 28°C and 150 rpm for 18 h, after which the
optical density at 600 nm of each culture was determined. Washed cells
from each treatment were used in adhesion assays as described above.
Extraction of surface proteins.
A cation-exchange method was
used to obtain the cell surface extract (12). Dowex (50 by
8, 20/50 mesh, sodium form) (18 g) that had been prewashed for 1 h
in sodium bicarbonate buffer was added to 60 ml of cell suspension
(optical density of 2.0 at 600 nm) and stirred on ice for 30 min at 300 rpm. Dowex beads were then removed by centrifugation (614 × g, 4°C, 15 min), and cells were removed by a subsequent
centrifugation (15,300 × g, 4°C, 15 min). The
resulting supernatant was centrifuged again (15,300 × g, 4°C, 30 min). The supernatant resulting from this second
centrifugation was lyophilized and suspended in 3 ml of dH2O. This constituted the cell surface extract.
Aliquots of the cell surface extract were added to tubes of HFO buffer
to provide final protein concentrations of 0, 4.84,
12.4, 24.2, 36.3, and 48.4 µg ml
1. A series of negative control tubes
contained the same concentrations
of bovine serum albumin. All tubes
were incubated for 30 min at
150 rpm and ambient temperature.
S. alga BrY cells were added
to the tubes, which were then incubated
for 15 min at 150 rpm
and ambient temperature. The number of adhered
and unadhered cells
in each tube was determined after the incubation
period, as described
above.
SDS-PAGE analyses.
Aliquots (50 µl) of cell suspensions
from the first proteolysis experiment were heated at 95°C for 10 min
in 50 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) sample buffer (Bio-Rad, Hercules, Calif.). Approximately
2 × 107 cells (25 µl) were loaded into each lane of
a 12.5% polyacrylamide slab gel, and SDS-PAGE was conducted according
to the methods of Laemmli (19). Bio-Rad low-molecular-weight
standards (Mrs, 14,400, 21,500, 31,000, 42,700, 66,200, and 97,400) were used as standards. Estimates of molecular size
were made by comparing the relative mobilities of the unknown proteins
to the mobilities of the proteins of known molecular mass on SDS-PAGE gels.
SDS-PAGE analysis was also used to determine the binding of proteins in
the cell surface extract to either whole
S. alga BrY
cells
or HFO. Aliquots (0.5 ml) of washed cell suspension and
HFO buffer were
centrifuged (16,060 ×
g, 5 min, and ambient
temperature).
The supernatants were removed, and each pellet was
suspended in
0.5 ml of cell surface extract and incubated for 10 min at
150
rpm and ambient temperature. Proteins in the cell surface extract
that bound to either
S. alga BrY whole cells or HFO were
removed
by centrifugation (16,060 ×
g, 5 min, and
ambient temperature).
The resulting supernatants, containing cell
surface proteins that
did not bind to cells or HFO, were analyzed by
SDS-PAGE as described
above. A sample of the original cell surface
extract was also
analyzed as a reference. Each lane of the gel was
loaded with
1.5 µg of protein. Protein concentrations were determined
by using
the Micro Protein assay (Pierce Chemical Co., Rockford, Ill.),
with bovine serum albumin as a
standard.
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RESULTS |
Degradation of surface polysaccharides.
Previous research
demonstrated that the surface of S. alga cells contains
carbohydrates composed of uronic acids and hexose (7). These
results provided a rationale for using -glucuronidase to examine the
role of carbohydrates in adhesion to HFO. -Glucuronidase treatment
of S. alga BrY cells did not result in cell lysis. Following an adhesion assay, the number of unadhered cells treated with -glucuronidase ([2.43 ± 0.10] × 107) was not
significantly different than the number of unadhered cells that were
treated only with buffer ([2.85 ± 0.36] × 107)
(P = 0.179). -Glucuronidase treatment inhibited
(7.73 ± 0.29)% of the total cells used in the assay from
adhering. Buffer-treated cells were inhibited by (7.0 ± 0.90)%.
Influence of proteolytic enzymes on cell adhesion.
A variety
of proteolytic enzymes were used to determine if proteins mediated
S. alga BrY adhesion to HFO. The effects of surface proteolysis on cell adhesion are shown in Table
1. The numbers of cells that did not
adhere to HFO increased 2.6- and 2.9-fold relative to the untreated
control after cells were treated with protease and chymotrypsin,
respectively. Surface proteolysis with trypsin had no effect on cell
adhesion relative to the untreated control. The number of unadhered
cells increased 4.6-fold when cells were treated with both protease and
chymotrypsin simultaneously. Although surface proteolysis with protease
and/or chymotrypsin inhibited adhesion relative to the untreated
control, the percentage of unadhered cells for each of these treatments
was below 8% of the total number of cells used in each adhesion assay.
Controls for cell lysis by each of the proteolytic enzymes indicated no significant decrease in cell concentration after 1 h of incubation with final concentrations of each enzyme that were 10 times higher than
those used in this experiment (data not shown).
Preincubating HFO with protease from
S. griseus inhibited
the adhesion of
S. alga BrY cells (Fig.
1A). A linear
relationship
between the protease concentration and the number of
unadhered
cells was observed (
r2 = 0.996).
Since proteins are known to coat surfaces and change
interfacial
energies (
31), the effect of heat-denatured protease
on
S. alga BrY adhesion was also examined. A significantly
greater
number of cells failed to adhere in native enzyme treatments
than
in heat-denatured enzyme treatments (
P = 0.0009).
The highest
concentration of native protease inhibited (3.52 ± 0.25)% of the
total cells used in the assay from adhering.
Chymotrypsin also
inhibited
S. alga BrY adhesion to HFO
(Fig.
1B). A linear relationship
between the chymotrypsin concentration
and the number of unadhered
cells was observed
(
r2 = 0.991). A significantly greater
number of cells failed to adhere
in native chymotrypsin treatments than
in heat-denatured treatments
(
P = 0.003). The highest
concentration of native chymotrypsin
inhibited (4.31 ± 1.31)% of
the total cells used in the assay
from adhering. While trypsin
inhibited
S. alga adhesion and there
was a linear
relationship between the number of unadhered cells
and the trypsin
concentration (Fig.
1C,
r2 = 0.982), there
was not a significant difference between native
trypsin treatments and
heat-denatured treatments (
P = 0.885).
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FIG. 1.
Effect of pretreating HFO with various
concentrations of native ( ) or boiled ( ) S. griseus
protease (A), chymotrypsin (B), and trypsin (C) on S. alga
BrY cell adhesion. Error bars represent the standard deviations from
the means (n = 3).
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S. griseus protease removed
S. alga BrY cells
that were adhered to HFO (Fig.
2A). The
number of unadhered cells increased
with the time of exposure of the
adhered cells to native protease
but not heat-denatured protease. There
was a significant difference
between the number of unadhered cells
treated with native protease
and the number treated with denatured
protease (
P = 0.002). After
15 min of exposure to
native protease, (10.2 ± 1.85)% of the total
number of
previously adhered cells were unadhered. Chymotrypsin
also removed
S. alga BrY cells that were adhered to HFO (Fig.
2B). The
number of unadhered cells increased significantly with
the time of
exposure of the adhered cells to native chymotrypsin
relative to
exposure to heat-denatured chymotrypsin (
P = 0.00002).
After 15 min of exposure to native chymotrypsin, (3.96 ± 0.43)%
of the total number of cells previously adhered were unadhered.
Trypsin
removed (3.49 ± 0.10)% of the total cells adhered to the
HFO.
However, the number of unadhered cells did not increase with
increased
time of exposure to native trypsin, and there was no
significant
difference between the number of unadhered cells in
native trypsin
treatment and the number in heat-denatured trypsin
treatment
(
P = 0.1).
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FIG. 2.
Kinetics of detachment of S. alga BrY cells
from HFO by native () or boiled () S. griseus protease
(A), chymotrypsin (B), and trypsin (C). Error bars represent the
standard deviations from the means (n = 3).
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SDS-PAGE analyses of cells exposed to proteolytic enzymes showed that
S. griseus protease removed a single peptide band
representing
a protein with an apparent molecular mass of 50 kDa (Fig.
3, lane
3; arrow labeled SP50), relative
to the untreated control (Fig.
3, lane 2). Chymotrypsin treatment
resulted in the loss of two
peptide bands representing proteins with
apparent molecular masses
of 60 and 31 kDa (Fig.
3, lane 4; arrows
labeled CT60 and CT31,
respectively). Peptide bands that were removed
by trypsin treatment
(Fig.
3, lane 5) as well as bands removed by both
trypsin and
S. griseus protease or chymotrypsin were not
considered to be
involved in HFO adhesion.
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FIG. 3.
Coomassie brilliant blue R-250-stained SDS-PAGE analysis
showing effect of surface proteolysis on S. alga BrY cells.
Lanes: 1, molecular mass standards (molecular weights, 97,400, 66,200, 45,000, 31,000, 21,500, and 14,400, top to bottom, respectively); 2, buffer-treated control; 3, cells treated with S. griseus
protease; 4, cells treated with chymotrypsin; 5, cells treated with
trypsin.
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Influence of chloramphenicol on cell adhesion.
The MIC of
chloramphenicol for S. alga BrY was 6.25 µg
ml1. The optical densities of 15-h cultures grown with 0, 1/8, 1/16, and 1/32 MIC of chloramphenicol were 2.47, 2.12, 2.47, and
2.46, respectively. There was no difference in cell morphology between any of these cultures, as determined by phase-contrast microscopy. Cells grown in the presence of a sub-MIC of chloramphenicol were inhibited in their ability to adhere to HFO (Fig.
4). A linear relationship between the
concentration of chloramphenicol and the number of unadhered cells
existed (r2 = 0.98). The highest sub-MIC of
chloramphenicol inhibited (46 ± 5)% of the total cells used in
the assay from adhering.
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FIG. 4.
HFO adhesion of S. alga BrY cells grown in
the presence of various sub-MICs of chloramphenicol. Error bars
represent the standard deviations from the means (n = 3).
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Influence of cell surface proteins on adhesion.
Proteins
extracted from the surface of S. alga BrY cells inhibited
the adhesion of cells to HFO (Fig. 5). A
linear relationship between the amount of cell surface extract added to
HFO and the number of unadhered cells existed
(r2 = 0.90). The highest concentration of
cell surface extract inhibited (41 ± 7)% of the total cells used
in the assay from adhering.
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FIG. 5.
The influence of preincubating HFO with various
concentrations of cell surface extract () or bovine serum albumin
() on S. alga BrY cell adhesion. Error bars represent the
standard deviations from the means (n = 3).
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Inhibition by the cell surface extract could have been due to proteins
binding to either
S. alga BrY cells or HFO in the
experiments
described above. The proteins in the cell surface extract
that
did not bind to cells or HFO were thus examined by SDS-PAGE. None
of the approximately 38 proteins found in the cell surface extract
(Fig.
6, lane 1) bound to
S. alga cells (Fig.
6, lane 2). However,
all but approximately 10 of
the proteins in the cell surface extract
bound to HFO (Fig.
6, lane 3).
Interaction of the extract proteins
with HFO typically caused a
widening of the SDS-PAGE lane containing
those proteins (Fig.
6, lane
3). This resulted in a decrease in
the band intensities within this
lane after silver staining.
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FIG. 6.
Silver-stained SDS-PAGE analysis of S. alga
BrY cell surface extract proteins (lane 1), cell surface extract
proteins that did not bind to whole cells (lane 2), and cell surface
extract proteins that did not bind to HFO (lane 3). The bars on the
left represent molecular mass standards (molecular weights, 97,400, 66,200, 45,000, 31,000, 21,500, and 14,400, top to bottom,
respectively).
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DISCUSSION |
Although cell adhesion is assumed to be a crucial step in the
enzymatic reduction of insoluble ferric iron minerals, the mechanisms of adhesion are still obscure. This study presents four independent lines of evidence that indicate that proteins mediate the adhesion of
the DIRB S. alga BrY to HFO. Cell surface polysaccharide
degradation had no effect on cell adhesion. Three separate experiments
demonstrated the inhibitory effect of proteolytic enzymes on cell
adhesion to HFO. Cells grown in the presence of sub-MICs of an
antibiotic that inhibits protein synthesis showed a decreased ability
to adhere to HFO. Finally, proteins extracted from the cell surface bound specifically to HFO and competitively inhibited cell adhesion.
Previous work in this laboratory provided evidence against
polysaccharide-mediated adhesion of S. alga BrY to HFO
(7). A mutant that overproduced exopolysaccharide showed a
reduced ability to adhere to HFO relative to the wild-type S. alga strain BrY. It was postulated that this exopolysaccharide
sterically hindered the interaction of hydrophobic adhesive proteins
with HFO, resulting in weak adhesion of the mutant strain. The
experiment using -glucuronidase to examine the effect of enzymatic
cell surface polysaccharide degradation on S. alga BrY
adhesion to HFO supported these previous findings and suggested that
the initial adhesion of S. alga BrY to HFO is independent of
cell surface polysaccharide.
A number of studies have employed proteolytic enzymes to examine the
role of proteins in bacterial adhesion to a variety of surfaces
(8, 9, 11, 16, 17, 33, 38, 39, 41, 43). The surface
proteolysis experiments in this study demonstrated that preincubating
S. alga BrY cells with S. griseus protease and
chymotrypsin, but not trypsin, inhibited adhesion. SDS-PAGE analysis of
these cells demonstrated that proteolytic digestion with protease
removed a single 50-kDa protein (SP50) from the S. alga BrY
cell surface, while digestion with chymotrypsin removed proteins of 60 kDa (CT60) and 31 kDa (CT31). The inhibitory effects of protease and
chymotrypsin on adhesion and the digestion of specific cell surface
proteins by these enzymes suggest that SP50, CT60, and CT31 represent
putative HFO adhesion molecules.
Preincubation of HFO with each proteolytic enzyme also supported the
role of these proteins in HFO adhesion. The significant difference in
adhesion between native and heat-denatured S. griseus protease and chymotrypsin suggested that the primary effect of these
enzymes on adhesion was catalytic degradation of adhesion proteins on
the cell surface rather than surface-coating effects. Preincubation of
HFO with trypsin, on the other hand, resulted in inhibition solely from
surface-coating effects, since there was no significant difference
between native and heat-denatured trypsin treatments. Similar
surface-coating effects have been observed in a study examining the
adhesion of Vibrio proteolytica to hydrophilic and
hydrophobic surfaces (31).
S. alga BrY cells display a high affinity for HFO, as nearly
100% of the cells adhere immediately during HFO adhesion assays and
remain adhered indefinitely despite constant physical agitation (7). Given the strong affinity of these cells for HFO, the ability of the proteolytic enzymes S. griseus protease and
chymotrypsin to remove cells from HFO substantiates the role of SP50,
CT60, and CT31 in S. alga BrY adhesion to HFO.
Although the results of three separate proteolysis experiments each
supported the role of SP50, CT60, and CT31 in S. alga BrY
adhesion to HFO, proteolysis was not particularly efficient at
inhibiting cell adhesion. Previous studies have shown that the
efficiency of proteolytic enzymes in inhibiting protein-mediated bacterial adhesion to different surfaces is varied (8, 9, 16, 32,
33, 38). Proteolytic experiments with S. alga BrY
typically inhibited cell adhesion to HFO by less than 10%. A series of
experiments were performed to explore the possibility that adhesion is
mediated by a complement of proteins including but not limited to the
SP50, CT60, and CT31 putative adhesion molecules.
Concentrations of antibiotics below the MIC may induce changes in the
surface properties of bacteria, and a number of studies have
investigated the influence of sub-MICs of antibiotics on bacterial
adhesion (18, 40, 44). Chloramphenicol is a peptidal transferase inhibitor that binds to the 50S ribosomal subunit (31) and inhibits protein-mediated adhesion in a number of
bacterial strains (10, 31, 35-37). Inhibition of S. alga BrY protein synthesis resulted in adhesion inhibition of a
much larger proportion (46%) of S. alga cells than in the
proteolytic enzyme digestion experiments. These results support the
hypothesis that an array of proteins may play a role in S. alga BrY adhesion to HFO.
Inhibition of adhesion by preincubating HFO with the S. alga
BrY cell surface extract suggested that one or more HFO binding proteins associated with the cell surface were involved in adhesion. The inability of the control, bovine serum albumin, to inhibit adhesion
suggested that proteins in the cell surface extract specifically and
competitively inhibited adhesion by up to 41%. SDS-PAGE experiments demonstrated that 28 of the 38 proteins in the extract bound
specifically to HFO. The cell surface extract included peptide bands
with apparent molecular masses of 50, 60, and 31 kDa. Both the 60- and
31-kDa peptides bound to HFO, while the 50-kDa peptide did not.
The results of this study suggest that cell adhesion to HFO is a
complex process. This study has identified a proteinaceous, HFO-adhesion system composed of multiple proteins, including but not
limited to SP50, CT60, and CT31, that serve both iron binding and as
yet unidentified functions.
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ACKNOWLEDGMENTS |
This research was partly supported by a summer faculty fellowship
from the Graduate School of the University of New Hampshire.
I thank J. D. Coates for critically reviewing the manuscript.
| |
FOOTNOTES |
*
Mailing address: Department of Microbiology, University
of New Hampshire, Durham, NH 03824. Phone: (603) 862-2443. Fax: (603) 862-2443. E-mail: fcj{at}hopper.unh.edu.
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REFERENCES |
| 1.
|
Arnold, R. G.,
T. J. DiChristina, and M. R. Hoffman.
1988.
Reductive dissolution of Fe(III) oxides by Pseudomonas sp. 200.
Biotechnol. Bioeng.
32:1081-1096.
|
| 2.
|
Balch, W. E.,
G. E. Fox,
L. J. Magrum,
C. R. Woese, and R. S. Wolfe.
1979.
Methanogens: reevaluation of a unique biological group.
Microbiol. Rev.
43:260-296[Free Full Text].
|
| 3.
|
Balch, W. E., and R. S. Wolfe.
1976.
New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere.
Appl. Environ. Microbiol.
32:781-791[Abstract/Free Full Text].
|
| 4.
|
Bryant, M. P.
1972.
Commentary on the Hungate technique for culture of anaerobic bacteria.
Am. J. Clin. Nutr.
25:1324-1328[Free Full Text].
|
| 5.
|
Caccavo, F., Jr.,
R. P. Blakemore, and D. R. Lovley.
1992.
A hydrogen-oxidizing, Fe(III)-reducing microorganism from the Great Bay estuary, New Hampshire.
Appl. Environ. Microbiol.
58:3211-3216[Abstract/Free Full Text].
|
| 6.
|
Caccavo, F., Jr.,
B. Frølund,
F. V. Kloeke, and P. H. Nielsen.
1996.
Deflocculation of activated sludge by the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY.
Appl. Environ. Microbiol.
62:1487-1490[Abstract].
|
| 7.
|
Caccavo, F., Jr.,
P. C. Schamberger,
K. Keiding, and P. H. Nielsen.
1997.
Role of hydrophobicity in adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga to amorphous Fe(III) oxide.
Appl. Environ. Microbiol.
63:3837-3843[Abstract].
|
| 8.
|
Coconnier, M.,
T. R. Klaenhammer,
S. Kernes,
M. Bernet, and A. L. Servin.
1992.
Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.
Appl. Environ. Microbiol.
58:2034-2039[Abstract/Free Full Text].
|
| 9.
|
Conway, P. L., and S. Kjelleberg.
1989.
Protein-mediated adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium.
J. Gen. Microbiol.
135:1175-1186[Abstract/Free Full Text].
|
| 10.
|
Fletcher, M.
1980.
The question of passive versus active attachment mechanisms in non-specific bacterial adhesion, p. 197-210.
In
R. C. W. Berkeley, J. M. Lynch, J. Melling, P. R. Rutter, and B. Vincent (ed.), Microbial adhesion to surfaces. Ellis Horwood, Publisher, Ltd., Chichester, England
|
| 11.
|
Flint, S. H.,
J. D. Brooks, and P. J. Bremer.
1997.
The influence of cell surface properties of thermophilic streptococci on attachment to stainless steel.
J. Appl. Microbiol.
83:508-517[Medline].
|
| 12.
|
Frolund, B.,
R. Palmgren,
K. Keiding, and P. H. Nielsen.
1996.
Extraction of extracellular polymers from activated sludge using a cation exchange resin.
Water Res.
30:1749-1758.
|
| 13.
|
Ghiorse, W. C.
1988.
Microbial reduction of manganese and iron, p. 305-331.
In
J. B. Zehnder (ed.), Biology of anaerobic microorganisms. John Wiley & Sons, Inc., New York, N.Y
|
| 14.
|
Gorby, Y. A., and H. Bolton, Jr.
1993.
Effects of O2 on metal-reductase activity and cytochrome content in a facultative Fe(III)-reducing bacterium, abstr. Q-124, p. 37.
In
Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993. American Society for Microbiology, Washington, D.C.
|
| 15.
|
Grantham, M. C.,
P. M. Dove, and T. J. DiChristina.
1997.
Microbially catalyzed dissolution of iron and aluminium oxyhydroxide mineral surface coatings.
Geochim. Cosmochim. Acta
61:4467-4477.
|
| 16.
|
Greene, J. D., and T. R. Klaenhammer.
1994.
Factors involved in adherence of lactobacilli to human Caco-2 cells.
Appl. Environ. Microbiol.
60:4487-4494[Abstract/Free Full Text].
|
| 17.
|
Hogt, A. H.,
J. Dankert,
C. E. Hulstaert, and J. Feijen.
1986.
Cell surface characteristics of coagulase-negative staphylococci and their adherence to fluorinated poly(ethylenepropylene).
Infect. Immun.
51:294-301[Abstract/Free Full Text].
|
| 18.
|
Klainer, A. S., and R. L. Perkins.
1972.
Surface manifestations of antibiotic-induced alterations in protein synthesis in bacterial cells.
Antimicrob. Agents Chemother.
1:164-170[Abstract/Free Full Text].
|
| 19.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
|
| 20.
|
Lindsay, W. L., and A. P. Schwab.
1982.
The chemistry of iron in soils and its availability to plants.
J. Plant Nutr.
5:821-840.
|
| 21.
|
Little, B. J.,
P. A. Wagner, and Z. Lewandowski.
1997.
The role of biomineralization in microbially influenced corrosion, p. 123-159.
In
J. F. Banfield, and K. H. Nealson (ed.), Geomicrobiology: interactions between microbes and minerals. Mineralogical Society of America, Washington, D.C.
|
| 22.
|
Lovley, D. R.
1987.
Organic matter mineralization with the reduction of ferric iron: a review.
Geomicrobiol. J.
5:375-399.
|
| 23.
|
Lovley, D. R.
1991.
Dissimilatory Fe(III) and Mn(VI) reduction.
Microbiol. Rev.
55:259-287[Abstract/Free Full Text].
|
| 24.
|
Lovley, D. R.
1993.
Dissimilatory metal reduction.
Annu. Rev. Microbiol.
47:263-290[Medline].
|
| 25.
|
Lovley, D. R.
1995.
Bioremediation of organic and metal contaminants with dissimilatory metal reduction.
J. Ind. Microbiol.
14:85-93[Medline].
|
| 26.
|
Lovley, D. R.
1995.
Microbial reduction of iron, manganese, and other metals.
Adv. Agron.
54:176-217.
|
| 27.
|
Lovley, D. R., and E. J. P. Phillips.
1988.
Novel mode of microbial energy metabolism: organic carbon oxidation coupled to dissimilatory reduction of iron or manganese.
Appl. Environ. Microbiol.
54:1472-1480[Abstract/Free Full Text].
|
| 28.
|
Maurice, P.,
J. Forsythe,
L. Hersman, and G. Sposito.
1996.
Application of atomic-force microscopy to studies of microbial interactions with hydrous Fe(III)-oxides.
Chem. Geol.
132:33-43.
|
| 29.
|
Nealson, K. H., and D. Saffarini.
1994.
Iron and manganese in anaerobic respiration: environmental significance, physiology and regulation.
Annu. Rev. Microbiol.
48:311-343[Medline].
|
| 30.
|
Obuekwe, C. O.,
D. W. S. Westlake,
F. D. Cook, and J. W. Costerton.
1981.
Surface changes in mild steel coupons from the action of corrosion-causing bacteria.
Appl. Environ. Microbiol.
41:766-774[Abstract/Free Full Text].
|
| 31.
|
Paul, J. H.
1984.
Effects of antimetabolites on the adhesion of an estuarine Vibrio sp. to polystyrene.
Appl. Environ. Microbiol.
48:924-929[Abstract/Free Full Text].
|
| 32.
|
Paul, J. H., and W. H. Jeffrey.
1985.
Evidence for separate adhesion mechanisms for hydrophilic and hydrophobic surfaces in Vibrio proteolytica.
Appl. Environ. Microbiol.
50:431-437[Abstract/Free Full Text].
|
| 33.
|
Pruzzo, C.,
A. Crippa,
S. Bertone,
L. Pane, and A. Carli.
1996.
Attachment of Vibrio alginolyticus to chitin mediated by chitin-binding proteins.
Microbiology
142:2181-2186[Abstract/Free Full Text].
|
| 34.
|
Rossello-Mora, R.,
F. Caccavo, Jr.,
N. Springer,
S. Spring,
K. Osterlehner,
D. Shuler,
W. Ludwig,
R. Amann, and K. H. Schleifer.
1994.
Isolation and taxonomic characterization of a halotolerant facultatively iron-reducing bacterium.
Syst. Appl. Microbiol.
17:569-573.
|
| 35.
|
Sandberg, T.,
K. Stenqvist, and C. Svanborg-Eden.
1979.
Effects of subminimal inhibitory concentrations of ampicillin, chloramphenicol, and nitrofurantoin on the attachment of Escherichia coli to human uroepithelial cells in vitro.
Rev. Infect. Dis.
1:838-844[Medline].
|
| 36.
|
Scaglione, F.,
G. Demartini,
S. Dugnani,
F. Ferrara,
G. Maccarinelli,
C. Cocuzza, and F. Fraschini.
1994.
Effect of antibiotics on Bordetella pertussis adhering activity: hypothesis regarding mechanism of action.
Chemotherapy
40:215-220[Medline].
|
| 37.
|
Scheld, W. M.,
O. Zak,
K. Vosbeck, and M. A. Sande.
1981.
Effect of subinhibitory antibiotic concentrations on streptococcal adhesion in vitro and the development of endocarditis in rabbits.
J. Clin. Investig.
68:1381-1384.
|
| 38.
|
Smit, G.,
J. W. Kijne, and B. J. J. Lugtenberg.
1989.
Roles of flagella, lipopolysaccharide, and a Ca2+-dependent cell surface protein in attachment of Rhizobium leguminosarum biovar viciae to pea root hair tips.
J. Bacteriol.
171:569-572[Abstract/Free Full Text].
|
| 39.
|
Smit, G. J.,
T. J. J. Logman,
M. E. T. I. Boerrigter,
J. W. Kijne, and B. J. J. Lugtenberg.
1989.
Purification and partial characterization of the Rhizobium leguminosarum biovar viciae Ca2+-dependent adhesin, which mediates the first step in attachment of cells of the family Rhizobiaceae to plant root hair tips.
J. Bacteriol.
171:4054-4062[Abstract/Free Full Text].
|
| 40.
|
Sud, I. J., and D. S. Feingold.
1975.
Detection of agents that alter the bacterial cell surface.
Antimicrob. Agents Chemother.
8:34-37[Abstract/Free Full Text].
|
| 41.
|
Timmerman, C. P.,
A. Fleer,
J. M. Besnier,
L. De Graaf,
F. Cremers, and J. Verhoef.
1991.
Characterization of a proteinaceous adhesin of Staphylococcus epidermidis which mediates attachment to polystyrene.
Infect. Immun.
59:4187-4192[Abstract/Free Full Text].
|
| 42.
|
Tugel, J. B.,
M. E. Hines, and G. E. Jones.
1986.
Microbial iron reduction by enrichment cultures isolated from estuarine sediments.
Appl. Environ. Microbiol.
52:1167-1172[Abstract/Free Full Text].
|
| 43.
|
Veenstra, G. J. C.,
F. F. M. Cremers,
H. van Dijk, and A. Fleer.
1996.
Ultrastructural organization and regulation of a biomaterial adhesin of Staphylococcus epidermidis.
J. Bacteriol.
178:537-541[Abstract/Free Full Text].
|
| 44.
|
Vosbeck, K.,
H. Handschin, and E. Menge.
1979.
Effects of subminimal inhibitory concentrations of antibiotics on adhesiveness of Escherichia coli in vitro.
Rev. Infect. Dis.
1:845-851[Medline].
|
Applied and Environmental Microbiology, November 1999, p. 5017-5022, Vol. 65, No. 11
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Abstract
Introduction
